# Shipboard Trials of Hyde ‘Guardian’ system in Carib bean

Sea and Western Pacific Ocean, April 5 th 

# - October 7 th 

# , 2008.

Final Report to Hyde Marine and Lamor Corp.

by


## David A. Wright, University of Maryland Center for Environm ental 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 followin g ballasting/treatment 7 Examination of treated and untreated water following resi dence 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 5 th 

### -18 th 

### , 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 30 th 

### – July 13 th 

### , 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 17 th 

### -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 testin g (Trial 3) showing mold counts not included in culturable heterotrophic bac teria 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 Servic es Division. 121

### 3



## Shipboard Trials of Hyde ‘Guardian’ system in Caribbean S ea and Western

Pacific Ocean, April 5 th 

## - October 6 th 

## , 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 cm 2 

### for treatment of ballast water at flow rates up to

250 m 3 

### h -1 

### . Trials took place during the vessel’s regular spring sche dule in the Caribbean Sea, the
summer schedule in the N.W. Pacific Ocean between Whi ttier, Alaska and Vancouver, Canada, and during the repositioning cruise from the western Pacif ic 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 untreat ed ballast water samples, with reference to both IMO G8 and the U.S. Coast Guard Shipboard Technolo gy 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 de signed to document system performance under normal seagoing conditions and under dif ferent geographical and seasonal conditions, with the objective of determining the degree o f 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 toxi city

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 ha bitats (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 bee n 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 glo bal 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 Balla st 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 w ater either through an empty-refill procedure or a pass-through procedure involving 3x the volume of t he tank (or less if the 95% exchange is satisfactorily met). ‘Successful treatment ’ was defined as the discharge of less than 10 viable organisms/m 3 

### greater than or equal to 50 μm in minimum dimension and less than 10 viable organisms/ml less than 50 μm in minimum dimension a nd 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, Se ction 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 i n January 2008 by the state of California, essentially representing the complete elim ination of plankton in the >50 μm (minimum dimension) size class and a live density of 0.01or ganisms/ml. in the >10 - <50 μm (minimum dimension) size class. California ballast wat er legislation also includes standards for total live bacteria post treatment, (less than 1,000 bacte ria per 100 ml.) and viruses (less than 10,000 viruses per 100 ml.) as well as more rigorous standards rel ating to indicator bacteria, i.e.
concentrations of microbes that are less than 126 colo ny forming units/100 ml. of Escherichia

coli ; 33 colony forming units per 100 ml. of intestinal enterococc i and 1 colony forming unit per 100 ml. or 1 colony forming unit per gram of wet weight of z oological 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 trea t 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 th at only approved technologies may be used on specified vessels to discharge treated ballast in Washington waters. For appr oval, technologies must meet one of the following criteria:

 

### Previously approved by Washington Department of Fisheries and Wi ldlife for use in WA waters
 

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

### Enrolled in U.S. Coast Guard Shipboard Technology Evaluat ion 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 authoriz ed by the U.S. State Department and U.S. Coast Guard for use in nationa l waters.

Table 1 provides a summary of current legislation, both n ational 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 fo r 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 des igned 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 sy stem aboard the M/V Coral

Princess essentially followed 3 ‘in tank’ replicate x 3 time per iod (during the de-ballasting operation) x 2 treatment (i.e. treated/untreated) x 3 tr ial matrix design. It should be noted that, for the Guardian system, a ‘treatment’ consists of [filtr ation + UV irradiation] during the ballasting cycle PLUS [UV irradiation] during the de-ballasting cyc le. Therefore, a treatment is not deemed complete until after the water passes through the UV sy stem 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 foll owing a residence time (4 days in Trial 1, 5 days in Trial 2 and 10 days in Trial 3) in the ballast ta nks. 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 biologi cal examination.

Examination of ‘challenge water’ immediately followin g ballasting/treatment :

In order to obtain information on the effect of tank re sidence time on biota, a series of samples (5 in Trial 1, 9 in Trials 2 and 3) were obtaine d from treated and untreated (control) tanks immediately following the initial ballasting/treat ment 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 resi dence time in the tanks.
Following the residence time in the tank, samples of tr eated 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 wi thin 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 bacteri al counts were unfiltered, although phytoplankton samples were further concentrated by filtr ation through 10 μm filters before growout and examination. These procedures are described below an d 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 pl anktonic 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 manua l transport up to the cabin space where samples were turned over to microscopists for examinati on. On receiving samples from the collection team, microscopists further concentrated s amples through 10μm mesh filters to produce volumes suitable for microscopy, usually 10-20ml. Concentra ted samples were transferred via Stempel pipets to counting wheels mounted on compound micro scope stages for microscopical examination. For each sample, the appropriate multiplie r 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 Appendi x A for taxonomic breakdown of zooplankton from each cruise), and records kept of the dim ensions of each group of organisms, including maximum and minimum dimensions (μm). Dimensions of organisms were measures using reticule eye-pieces calibrated against National Ins titute of Standards and Technology certifies beads from 10 - 200μm in diameter. Sizing was al so 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 supplem ented by the use of the vital stain Neutral

Red.

### 10

Figure 1. Schematic of Shipboard Trial sampling scheme des ign for Coral Princess Trials,
2008

>4day residence time

UV UV UV UV filter 

### Sea Chest

400L

Bacteria

10

- 5 0 /

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 U3 U2 U1

Filter Mesocosm Sample Ballast Tank (count ASAP) 500L 500L

72hr grow-out period samples Bacteria 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 s ome taxonomic groups, such as dinoflagellates, are clearly motile, many have vegetati ve 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 growt h conditions. The grow-out period for phytoplankton usually consists of a 24h (or 48h) peri od of irradiation under fluorescent lighting and non-limiting nutrient conditions through the addition of f/2 growth medium. Phytoplankton growth may be conveniently assesse d by measuring in vivo chlorophyll a concentration before and after the grow-out period. W hile 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 (gr owth rate) etc. Also, chlorophyll a data cannot be interpreted in terms of published standards.

Unfiltered 1L samples for phytoplankton analyses were take n from the same sample stream as that used for zooplankton sampling. Each sample w as concentrated to a volume <10ml. for examination/grow-out, using a 10μm Nitex screen. Micr oscopic 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 wi th ‘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 phytoplankto n taxa before and after a 24h grow-out period under fluorescent lights in ambient seawat er supplemented with f/2 growth medium. Following initial examination of phytoplankton (b efore and after grow-out) to determine their general appearance, observations of chlor oplast integrity and the activity of motile forms, samples were preserved in Lugol's Solution f or 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 m ade on the basis of (a) chlorophyll a analysis, (b) vital staining techniques and (c) ce ll counts before and after grow-out.

### 12

Bacteria

. Samples for bacterial analyses were be taken direct ly from the unfiltered discharge from the sampling port, and stored at temperatures just above fre ezing (1-4 O 

### C) prior to and during transport to the University of Maryland Center for Environm ental Science, Chesapeake Biological Laboratory, although in Trial 3 some culturi ng/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 abo ard this vessel. The following bactericidal endpoints were employed:

• 

### Plate counts of cultural heterotrophic bacteria. It is em phasized that only approximately 1% of marine bacteria will culture successf ully, and that this figure may vary according to geographical area. This, therefore, represents only an approximate measure of the efficacy of BWT technology, an d is somewhat variable according to geographical location.

• 

### Primary focus was on taxonomic groups specified in recent ly published IMO standards: colony-forming units of E.Coli, Enterococcus and Vibrio Cholera (with specific emphasis on virulent serotypes). Fluorescence-b ased 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 de ionized 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 count s following 18h incubation at 35 O 

### C.
Fluorescence counting of Enterococcus was determined by IDEX X Laboratories Inc. (ME) Enterolert Quantitray 2000 counts following 24h incubatio n at 41 O 

### C.
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 sample s transported to the University of

### 13

Maryland Center for Environmental Science, Chesapeake Bi ological Laboratory, Molecular Biology Laboratory, directed by Dr. Carys Mitchelmore.

Water Quality Measurement.
The following water quality parameters were analyzed: te mperature, salinity, pH, dissolved oxygen, total suspended solids, total particulate ca rbon, 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 wat er, enabling raw counts to be appropriately multiplied to compute numbers of organisms per ton of wate r. This file is included in this report as Appendix A. Results from biological analyses from t hese trials were compared with published standards. Additionally, zooplankton mortalities in trea ted samples will expressed in terms of percentage removal relative to untreated controls. An as sessment 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 ‘enviro nmental acceptability of water treated by this technology, WET tests were conducted on s tandard test organisms exposed to treated and untreated water from Trial 3 to provide empiri cal information on possible residual toxicity on ballast discharge, resulting from chemical c hanges induced by UV irradiation during treatment. Details of these tests are provided on page 59.

### 14



## TRIAL 1, April 5 th 

## -18 th 

## , 2008

Sampling Timetable
Ballasting took place close to the port of Aruba on April 8 th 

### , 2008, day 4 of a ten day cruise. Collection of samples 1-18 (see Materials and M ethods) commenced immediately after the ballast pump began retrieving water from each of the respe ctive tanks, following a residence time
in the tanks of 96h. Ballast water was be taken on/trea ted after the vessel left Aruba on April 9 th



### , 2008) and was discharged/sampled/analyzed four days later, afte r the ship departed Ocho Rios, Jamaica on April 13 th 

### . Samples for bacterial examination were kept on ice until their return to the Chesapeake Biological Laboratory on April 15 th 

### incubation and grow-out of these samples continued until April 18 th 

### , 2008.

Results and Discussion.

Zooplankton. Results of zooplankton counts are summarized in table 2. Th ese indicated a 98.6% mortality of zooplankton (>50μm minimum dimension) immedi ately 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 cont rast, 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 tho se 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 dimens ion) for Trial 1 of Hyde ‘Guardian’ BWT system aboard M/V Coral Princess , April 5th-18th, 2008.

Zooplankton Alive (>50μm). Density/m 3 

### Dead (>50μm)

Density/m 3


### 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, cel ls were scored as ‘live’ based on morphological characteristics such as chloroplast inte grity and the ability to concentrate the

### 16

Table 3. Summary of phytoplankton results (>10<50μm minimum di mension) 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 0.0 16 0.0 3 2 0.0 48 0.000 6 0.0006 0.00 1

Rep. C2 T=0 0.00 4 0.0 62 0.07 0 0 0 Rep. C2 T=0 + 48h growout 0.005 0.0 15 0.02 0 0.000 8 0.0008

Rep. C3 T=0 0.024 0. 1 3 0. 15 0 0 0 Rep. C3 T=0 + 48h growout 0.003 0.063 0.066 0.0003 0.000 6 0.000 8

Mean ‘Live’ Phytoplankton. Control samples, T=0 (after grow-out) 0. 133± 0.0 6 1

(0.45±0.023) Treated, T=0 Rep. T1, T=0 0.01 0.072 0.0 81 0 0 0 Rep. T1, T=0 + 48h growout 0.004 0.005 0.00 9 0.000 8 0.0003 0.00 1

Rep. T2 T=0 0.008 0.076 0.084 0.00 2 0.000 4 0.00 25 Rep. T2 T=0 + 48h growout 0.003 0.006 0.009 0 0 0

Rep. T3 T=0 0.003 0.070 0.0 72 0.000 4 0 0.000 4 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 ± 0.00 6

(0.009±0.0006)

### 17

Table 4. Summary of phytoplankton results (>10<50μm minimum di mension) 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 dim ension) 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 concentrat ions.

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)

### 19End 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 envir onment 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. Conce ntrations of treated cells following grow-out, shown in red in table 5, clearly indicate a fa ilure to grow, based on the fact that they
represent a mean reduction in cell numbers of 65% relative to the corresponding sa mples 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 untr eated (control) samples after 96h in the tank). This assessment is reinforced by measurement of chl orophyll 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 eit her T=0 or T=96h. If ‘live’ cell counts based on morphological examinatio n 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 standa rd of 10 7 

### live cells per m 3 

### (10 live cells/mL) the U.S. Coast Guard Standard standard of 10 5 

### live cells per m 3 

### (0.1 live cells/mL), and the much more rigorous standard of 10 4 

### live cells per m 3 

### (0.01 live cells/mL) currently employed by the state of California. We conclude that ce ll 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 l ow in challenge water samples, and we observe that, in this trial, even untreated sa mples would comply with IMO and U.S. Coast Guard standards at discharge, but not the more rigoro us 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

Cont rol 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 ) C hlorophyll a conc . (μg L - 1 ) after 24h grow-out (T=96 + 24) C hlorophyll a conc . (μg

L -1 ) after 48h grow-out (T=96 + 48)
Control 1 start de

ballasting 1.2 1.29 1.4

### 21

Control 2 mid de

ballasting 1.43 1.86 1.77

Control 3 end de

ballasting 1.26 1.45 1.22

Treated 1 start de

ballasting 1.34 1.26 1.22

Treated 2 mid de

ballasting 1.31 1.23 1.15

Treated 3 end de

ballasting 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 ball ast water samples from this trial and only

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

cholerae cells, live or dead, were found in any of the samples e xamined.
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 m ean 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 untreat ed (control) samples at T=0 and T=96h. (10ml. samples were filtered through 0.45μm. Plates wer e read after 72h incubation at 25 o



### 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 Ha rbor on April 8 th 

### , 2008. The salinity of challenge water (untreated water at T=0) varied betwee n 36.4 – 37.1 PSU (mean 36.8), water temperatures ranged from 25.2 – 29.0 o 

### 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 determ ined to be 95% in untreated water at T=0 Nutrient levels in challenge water extremely low, rang ing 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 challeng e water and treated ballast water at time of ballasting (T=0) near Aruba, April 8 th , 2008. (DO = Dissolved O2; TSS = Total Suspended Solids; POC – Particulate Organic Carbon; DOC = Dissolved Organic Carbon; NO 3 = Nitrate; PO 4 = Phosphate)

pH DO (mg L -1 ) Salinity

(PSU) Temp(

O C) TSS (mg L -1 ) POC

(mg L -1 ) DOC

(mg L -1 ) NO 3

(μgN L -1 ) PO 4

(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 oligotro phic 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 guidel ines. Based on results obtained from the first trial, the Hyde ‘Guardian’ syst em would comply with those portions of IMO Regulation D-2 and current California regulations rel ating 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. a nd California regulations. Only 1 cfu

### 25

each of coliforms intestinal Enterococci were detect ed 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 untre ated 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 an d 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 30 th 

## – July 13 th 

## , 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 1 st 

### , 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 sample s, water followed the same path as the treated samples, except that the filter was by-pa ssed 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 obtain ed 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 1 st 

### , 2008, and was discharged/sampled/analyzed 4.5days later, afte r the ship departed Ketchekan on July 6 th 

### . Samples for bacteriological analysis were kept on i ce following collection and accompanied the scientific team to Dr. Mitchelmore’s laboratory at the University of Maryland Center for Environmental Science, Chesapeake Bi ological Laboratory, Solomons, Maryland. Culture and analysis of these samples contin ued until July 13 th 

### 2008.
Preparation of samples for biological examination foll owed procedures described for Trial 1 (see Figure 1.) Methods for sample examination an d analysis were as previously

described.

### 27

Results and Discussion

Zooplankton. Results of zooplankton counts are summarized in table 10. R esults indicated a 99.99% mortality/removal of zooplankton (>50μm minimum dimension ) immediately following treatment on ballasting relative to untreated samples c ollected 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 wer e found in this size range in treated samples, indicating that the large majority were remo ved 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 organis ms >50μm in the challenge water showed a 35-fold increase relative to the much more oligotrophic co nditions encountered in Caribbean waters during the first, April 2008, trial. Unlike trial 1, there was a dramatic (98.4%) decline in over all numbers of live organisms in the untreated (control) tank after the 114h residenc e period. Both treated and untreated samples retrieved from the tanks after 114h were quite di fferent in character from the samples examined at T=0. In treated samples, after 114h in the tan k, and following UV irradiation on deballasting, numerous zooplankters in the >50μm minimum dim ension 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 see n 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 dimensi on) for Trial 2 of Hyde ‘Guardian’ BWT system aboard M/V Coral Princess , June 30 th 

### – July 8 th 

### , 2008.

Zooplankton Alive (>50μm). Density/m

3 Dead (>50μm) Density/m

3

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) 4 64 ± 6 70 1,1 23 ± 5 7 2

Control (untreated) End de-ballast (N=3) 43± 74 9 7 1± 654

Mean control (untreated) No. at de-ballast 3 37 ± 441 698±683

Treated (UV + filter during ballasting) → (UVat de-ballasting) Start deballast (N=3) 0 75± 20

Treated (UV + filter during ballasting) → (UVat de-ballasting) Mid deballast (N=3) 0 256 ± 374

Treated (UV + filter during ballasting) → (UVat de-ballasting) End deballast (N=3) 0 533 ± 9

Mean treated No. at de

ballasting 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 numb ers are shown in table 11. Nematode numbers were almost completely absent from t he T=0 samples, and were seen to increase in T=114h samples from the beginning to the end o f the de-ballasting cycle, reflecting a probable difference in plankton densities throughout the w ater column in the tanks. The most likely explanation for this is that, as the tank approache s 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 cy cle, 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 u ntreated samples.
Trial 2. June 30 th 

### -July 11 th 

### 2008.

Nematode densities (concentrations per m 3 )

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, ce lls were scored as ‘live’ based on morphological characteristics such as chloroplast inte grity and the ability to concentrate the vital stain Neutral Red. Unlike trial 1, initial treatment (filt er + 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 tr ial 1). This, despite the presence of exceptionally high numbers of apparently viable diatoms in t he 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, i t is clear that the ballast tank provides a poor
environment for treated and untreated cells alike. Concen trations 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 fr om treated samples at either T=0 or

T=114h. If ‘live’ cell counts based on morphological examinatio n are used as the basis for regulatory compliance, the ‘live’ phytoplankton count of 189,500 per m 3 

### (0.189 live cells/mL) recorded for treated samples at 114h (table 15) would comply with current IMO standard of 10 7


### live cells per m

3 

### , (10 live cells/mL) but not the 2008 U.S. Coast Guard regulatio ns, 10 5 

### live cells

per m 3 

### (0.1 live cells/mL) or the much more rigorous standard of 10 4 

### live cells per m 3 

### (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 m 3 

### (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 b e 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 concentr ations 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 (tabl e 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 di mension) for Trial 2 of Hyde ‘Guardian’ BWT system aboard M/V Coral Princess , June 30 th -July 8 th , 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. C1 a , T=0 0. 2 6 1

. 2 12

. 3 0 0 0

After 4 2 h growout 0. 7 7 6 .5 7 .3 0 0. 3 0 0. 3 0

Rep. C 1b , T=0 0. 4 5 19 .9 20 .3 0 0 0
After42h growout 0. 4 8 11

. 8 12

. 3 0.0 3 0 .03 0.06

Rep. C 1c , T=0 0. 1 9 17

. 4 17

. 6 0 0 0

After42h growout 0. 3 6 12

. 7 13 .1 0.1 0. 1 3 0.23

Mean C1 samples (after grow-out) 16 .8 ±4 .1 (10.9±3.2)

Rep. C 2a T=0 0. 3 9 19 .8 20 .2 0 0 0

After42h growout 0. 4 5 29

. 2 30.0 0 0.03 0.03

Re p. C 2b , T=0 0. 64 3

. 5 3.6 0 0.06 0.06 After42h growout 0. 2 9 6

. 8 7.1 0 0.13 0.13

Rep. C 2c , T=0 0. 7 4 4

. 8 5.6 0 0 0 After42h growout 0. 1 3 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. C 3a , T=0 0. 9 7 2

. 5 2.6 0 0 0 After 42h growout 0. 2 2 7 .4 7.6 0 0.40 0.40

Rep. C 3b , T=0 0. 12 2 .3 2.4 0 0 0 After42h growout 0. 2 1 0. 88 1.1 0 0.06 0.06

Rep. C 3c , T=0 0. 14 2 .7 2.8 0 0.02 0.02 After42h growout 0. 1 7 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 30 th -July 8 th , 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. T1 a , T=0 0.13 0.06 0.19 0 0 0 

### After 42h growout 0.03 0.10 0.13 0.06 64
, 5 0 0.06 0 0.13

Rep. T 1b , T=0 0.02 0.05 0.07 0 0 0 

### After 42h growout 0.10 0.03 0.13 0.03 0 0.03

Rep. T 1c , T=0 0.15 0 0.15 0 0 0 

### After 42h 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. T 2a , T=0 0.13 0.22 0.35 0 0 0 

### After 42h growout 0.29 0.23 0.52 0.14 0 0.14

Rep. T 2b , T=0 0.08 0.22 0.30 0.03 0.08 0.11 

### After 42h growout 0.32 0 .45 0.77 0.03 0.10 0.13

Rep. T 2c , T=0 0.13 0.26 0.39 0 0 0 

### After 42h 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. T 3a , T=0 0.19 0.06 0.26 0 0 0 

### After 42h growout 0.16 0.21 0.36 0.13 0.03 0.16

Rep. T 3b , T=0 0.32 0.32 0.64 0 0.05 0.05 

### After 42h growout 0.19 0.14 0.33 0.14 0.09 0.23

Rep. T 3c , T=0 0.07 0.61 0.61 0.02 0.10 0.12 

### After 42h growout 0.10 0.03 0.13 0.03 0.03 0.06

### 33

Table 14. Summary of phytoplankton results (>10<50μm minimum di mension) for Trial 2 of Hyde ‘Guardian’ BWT system aboard M/V Coral Princess , June 30 th -July 8 th , 2008. T=114h untreated (control) samples. Due to time constraints, dead cell nu mbers were not recorded from C3 samples following grow-out.

Phytoplankton. (>10μm- <50μm). Untreated, T=114h ‘Live’

Dinoflagellates. Density/mL ‘Live’

Diatoms. Density/ mL Tota l ‘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 grow

out 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 grow

out 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 grow

out 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 grow

out 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 grow

out 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 grow

out 0.88 1.24 1.33

Rep. C3b T=114h 0.17 1.10 1.30 0 0.02 0.23

After 42h grow

out 0.14 1.26 1.40

Rep. C3c T=114h 0.09 0.72 0. 8 1 0 0.12 0.12

After 42h grow

out 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 di mension) for Trial 2 of Hyde ‘Guardian’ BWT system aboard M/V Coral Princess , June 30 th -July 8 th , 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



### After 42h 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



### After 42h 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



### After 42h 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



### After 42h growout 0 0.02 0.02 0 0.02 0.02

Rep. T2b, T=114h 0 0.16 0.16 0 0 0



### After 42h growout 0 0.09 0.09 0 0 0

Rep. T2c T=114h 0 0.09 0.09 0 0.05 0.05



### After 42h 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



### After 42h growout 0.03 0.04 0.07 0 0 0

Rep. T3b, T=114h 0 0.18 0.18 0 0 0



### After 42h growout 0 0.12 0.12 0 0 0

Rep. T3c, T=114h 0 0.28 0.28 0 0 0



### After 42h 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 concentrati ons 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 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 L 1 ) 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 im mediately 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 re trieved 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 sys tem 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 i n T=0 controls, which did not differ significantly from treated samples examined at T=0. Afte r a 144h residence time in the ballast tanks, both treated and untreated samples showed elevate d counts, although this was particularly evident in treated samples where, in all but one (the l ast) sample investigators had to estimate the cfus as either >100 or >200 (table 18).

Table 18. Trial 2. Culturable bacteria counts from treated an d 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 1 st 

### , 2008. The salinity of challenge water was 30.8 – 31.9 PSU, water temperatures r anged from 12.1 = 14.0 o 

### C and pH from 8.25 – 8.36. Dissolved oxygen (DO) concentrations ranged fr om 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 cy cle. A similar pattern was seen in Particulate Organic Carbon (POC) levels, which ranged f rom 3.62 – 7.28 mg L -1 

### . UV Transmittance was high (94%). Nutrient levels in challenge water were significantly hi gher 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 1 st 2008. (DO = Dissolved O2; TSS = Total Suspended Solids; POC – Particulate Organic Carbon; DOC = Dissolve d Organic Carbon; NO 3 = Nitrate; PO 4 = Phosphate)

pH DO (mg L -1 ) Salinity

(PSU) Temp

( O C) TSS (mg L -1 ) POC

(mg L -1 ) DOC

(mg L -1 ) NO 3

(μgN L -1 ) PO 4

(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 hi gher densities of organisms compared with trial 1. However, zooplankton densities in un treated challenge water did exceed the value of 10 x D-2 required by IMO G8 guidelines. Based thes e results, the Hyde Guardian system would comply with those portions of IMO Regulatio n 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 we re detected in T=0 samples. We therefore interpret the appearance of these bacteria at de-ballas ting as the result of their association with planktonic organisms present in the tanks before the tria l took place. As with the culturable heterotrophic bacteria, treatment was actually associate d with an increase in bacterial flora

relative to controls. Mean numbers of Enterococci wer e 10 cfus per 100ml. and 36 cfus per 100ml. for untreated and treated samples respectively. The se data are consistent with the unusual nature of the samples collected during de-ballasting. Samp les were characterized by a large amount of flocculated material, particularly in the tre ated tank, including several specimens of decaying zooplankton, some >>1000μm in the smallest dimension

.

### 42



## TRIAL 3. September 17 th 

## -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 othe r (5P) for untreated water. The scientific team joined the vessel in Long Beach, CA on September 1 7 th 

### . Ballasting took place on September 19 th 

### , 2008, day 3 of a seventeen day cruise. As with Trials 1 a nd 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 ball asting 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 tan ks immediately following the initial ballasting/treatment cycle. In the third trial a resi dence time of 10 days elapsed before samples were retrieved from the tanks in Aruba Harbor on Septem ber 29 th 

### , 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 Ente rococcus assays, and culturable heterotrophic bacterial assays for T=0 and T=10 Day sampl es in an air-conditioned passenger cabin. Other samples were carried over ice to the Univers ity of Maryland Center for Environmental Science, Chesapeake Biological Laboratory , Solomons, Maryland for further analysis. Culture and analysis of these samples continue d until October 6 th 

### , 2008.
Otherwise, preparation of samples for biological exami nation followed procedures described for previous trials (see Figure 1.) Methods for sam ple 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) immedi ately 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 so me 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 b allast tank was nearly empty (Table 21).

Table 20. Summary of zooplankton results (>50μm minimum dimensi on) for Trial 3 of Hyde ‘Guardian’ BWT system aboard M/V Coral Princess , September 17 th 

### -October 6 th


### , 2008.

Zooplankton Alive (>50μm). Density/m 3 

### Dead (>50μm)

Density/m 3



### 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 un treated samples. Trial 3. September 17 th 

### -October 6 th 

### , 2008.

Nematode densities (concentrations per m 3 

### )

Live Dead
T=0h
Control 6±5 0

Treated 0 0 T= 10 DaysControl (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 untre ated samples collected at ballasting (table 22). Only three treated samples at T=0 were subjected to gr ow-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 sample s at T=0 treated samples show a 77% reduction in live cell counts relative to untreated sampl es. ‘Live’ cell numbers following grow-out had fallen to 2,469,400/m 3 

### (2.4 cells/mL) and 1,181,737/m 3 

### (1.2 cells/mL) in control and treated samples respectively. Following a 10 day resi dence period in the tank both treated and untreated samples showed a >50% drop in live cell number s 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 untrea ted and treated samples respectively. Based on grow-out data the number of viable c ells 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/m 3 

### (0.103 cells/mL) vs. 100,000 live cells/m 3 

### (0.100 cells/mL). However, this number represents an 83% decreas e 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 gro wth 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. Aft er 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 in dicated negligible chlorophyll a after 10 days in the tank.

### 46

Table 22. Summary of phytoplankton results (>10<50μm minimum dime nsion) for Trial 3 of Hyde ‘Guardian’ BWT system aboard M/V Coral Princess , September 15 th 

### -October 6 th


### , 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 B allast. Rep. C1a, T=0 0.55 5.32 5.87 0.10 0.55 0 .65

After 48h grow

out 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 grow

out 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 grow

out 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 grow

out 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 grow

out 1.2 2.3 3.5 0.1 0.5 0.6

Rep. C2 c T= 0 0.6 3.8 4.4 0.21 0.42 0.63 After 48h grow

out 0.72 4.44 5.16 0.11 0.45 0.56

Mean C 2, 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 grow

out 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 grow

out 0.48 5.0 5.48 0.20 0.45 0.65

Mean C 3, 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 dime nsion) for Trial 3 of Hyde ‘Guardian’ BWT system aboard M/V Coral Princess , September 15 th 

### -October 6 th


### , 2008. Treated T=0 data. Grow-outs were recorded for one sample e ach 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. T1 a , T=0 1.80 0.12 1.92 0 0.08 0.08 After 48h grow

out ND

Rep. T 1b , T=0 1.32 0.2 3 1.56 0 0 0 After 48h grow

out 2.48 0 2.48 0 0.16 0.16

Rep. T 1c , T=0 2.48 0 2.48 0 0.16 0.16 After 48h grow

out ND

Mean T1 T=0 samples 1.98 ± 0.46

Rep. T 2a , T=0 0.81 0.26 1.06 0 0 0 After 48h grow

out ND

Rep. T 2b , T=0 1.61 0.18 1.79 0 0 0 After 48h grow

out 0 0.33 0.33 0 0 0

Rep. T 2c , T=0 1.73 0.23 1.97 After 48h grow

out ND

1.61 ± 0.48 Rep. T 3a , T=0 1.44 0 1.44 0 0 0 After 48h grow

out ND

Rep. T 3b , T=0 2.15 0 2.15 0 0 0 After 48h grow

out 0.73 0 0.73 0 0 0

Rep . T 3c , T=0 0.85 0 0.85 After 48h grow

out 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 dime nsion) for Trial 3 of Hyde ‘Guardian’ BWT system aboard M/V Coral Princess , September 15 th 

### -October 6 th


### , 2008. Untreated (control) T=10 Days data, including live grow-out cel l 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 grow

out 

### 0.39 2.16 2.55

Rep. C1b, T=10 Days 0.73 1.47 2.20 0.20 0 0.20

After 48h grow

out 

### 0.66 0.50 1.16

Rep. C1c, T= 10 Days 0.54 4.90 5.44 0.15 0 0.15

After 48h grow

out 

### 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 grow

out 

### ND

Rep. C2b, T= 10 Days 0.44 1.29 1.73 0 0 0

After 48h grow

out 

### 0.83 0 0.83
Rep. C2c, T= 10 Days 0.83 3.88 4.71 0 0.16 0.16

After 48h grow out 

### 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 grow

out 

### ND
Rep. C3b, T= 10 Days 0 0.66 0.66 0 0 0

### 50 After 48h grow

out 

### 0 0.33 0.33
Rep. C3c, T= 10 Days 0 3.15 3.15 0 0.33 0.33

After 48h grow

out 

### 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 dime nsion) for Trial 3 of Hyde ‘Guardian’ BWT system aboard M/V Coral Princess , September 15 th 

### -October 6 th


### , 2008. Treated T=10 Days data, including live grow-out cell concentrat ions (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 grow

out 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 grow

out 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 grow

out 0.14 0.10 0.25 0 0.50 0.50

Mean T 1 , 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 grow

out 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 grow

out 0.16 0.1 1 0.26 0.12 0 0.12

Rep. T2c T= 10 Days 0 0 0 0 0.42 0.42

After 48h grow

out 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

A fter 48h grow

out 0 0.10 0.10

Rep. T3b, T= 10 Days 0 0.24 0.24 0.15 0 0.15

After 48h grow

out 0 0.10 0.10

Rep. T3c, T= 10 Days 0 0.14 0.14 0.14 0 0.14

After 48h grow

out 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 L 1 ) 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 L 1 ) 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 l ater (Table 27). No coliforms or
E. Coli were found in any treated samples at de-ballasting, and n o 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 approximat ely 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 sh owed 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 Beac h, S. California on September

19 th 

### , 2008. The salinity of challenge water was 33.3 PSU, wate r temperatures ranged from 23.7 –

24.3 o 

### C and pH from 7.5 - 7.82. Dissolved oxygen (DO) concentratio ns 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 thro ughout 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 be tween 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 sa mples 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 sl ightly 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 19 th , 2008. (DO = Dissolved O2; TSS = Total Suspended Solids; POC – Particulate Organic Carbon; DOC = Dissolved Organic Carbon; NO 3 = Nitrate; NO 2 = Nitrite; PO 4 = Phosphate)

pH DO (mg L -1 ) Salinity

(PSU) Temp

( O C) TSS (mg L -1 ) POC

(mg L -1 ) DOC

(mg L -1 ) NO 3

(μgN L -1 ) NO 2

(μgN L -1 ) PO 4

(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 i nitially only about 15% of concentrations seen in trial 2, but still exceed G-8 D2 re gulations for challenge water. As in trial 2, zooplankton mortalities (>50 μm) were 100% in treated sampl es at T=0 in trial 3, and numbers of untreated organisms in this size category declined precip itously over the period of residence in

the ballast tanks.

### 58

Phytoplankton cell numbers in trial 3 challenge water, 6.5 x 10 6 

### cells m 3 

### (6.5 cells/mL) were approximately 2/3 of those seen in trial 2, 9.7 x 10 6 

### cells m 3 

### (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 mo rphological characteristics, live cell numbers at trial 3 de-ballasting, 2.81 x 10 6 

### cells m 3 

### (2.8 cells/mL) were comparable to the
numbers seen in trial 2 after 114h residence time 2.88 x 10 6 

### cells m 3 

### (2.9 cells/mL). Based on morphological characteristics, cell numbers in treat ed vs. untreated samples at de-ballasting had further decreased by respectively 93.4% and 78.2% in trials 2 an d 3. If grow-out is considered as a criterion for phytoplankton cell viability, the viable c ell count in treated samples is further reduced to 103,000 cells per m 3 

### (0.103 cells/mL) , just exceeding the U.S. Coast Guard standard of 0.100 cells/mL) for cells in the >10 - <50μm size categor y. The IMO D-2 standard is easily met for this phytoplankton population.

All indicator bacteria were within national and intern ational 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 des cribed 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 r esult 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 assessm ents 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 Subs tances (G9), as revised, (resolution MEPC.169(57))”

While, to date, no potentially toxic chemical changes re sulting from UV irradiation have been identified, this resolution adopts an approach requiri ng 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 t esting to be incorporated into landbased testing of the system, which ended in July, 2008. Hen ce, 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 e xamination 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 an d de-ballasting) the definitive comparison is regarded as that between treated and untreated water on f inal discharge (T=10 Days). Nevertheless, from an experimental standpoint, T=0 samp les, untreated vs. treated, also represent a valid comparison, and provide a useful means of controlli ng for any possible toxic agents that might be introduced into the water as a result of prolonge d storage in the ballast tanks.

Sample collection and storage.

For WET tests 20L of each water sample to be tested wa s collected in the middle of each ballasting/deballasting operation. Thus, treated and untrea ted T=0 samples were collected during sequence T2a-T2c for treated samples and between sequence C 2a-C2c for untreated samples. The logistics of being at sea during ballasting and de-ball asting 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 o ver ice, with the temperature not to
exceed 6 O 

### C. In order to preserve the integrity of the samples t o the greatest degree possible it was therefore decided to freeze the samples as soon as po ssible after collection, pending toxicity bioassays. On landing at the destination port of Fort La uderdale on October 2 nd 

### , 2008 frozen water samples in plastic ‘Cubitainers’ were transporte d overnight to the respective bioassay laboratories, where samples were thawed and testing com menced (October 3 rd 

### ,2008 for larval fish and mysid shrimp assays; October 4 th 

### , 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) bio assays:

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 to psmelt, Atherinops affinis (Method 2006.0)

Phytoplankton.

96-hour Chronic Static Non-renewal Definitive Test using t he marine brown alga (diatom) Isochrysis galbana , strain T. Iso. (adapted from Method 1003.0)

The invertebrate and vertebrate tests were carried ou t at the University of Maryland Center for Environmental Science, Chesapeake Biologica l 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 dilutio n steps with clean, reconstituted seawater matched to the salinity of the treatment wate r, 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 rene wal 48 h and 96 h LC 50 

### 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 acclima ted (30PSU) Isochrysis galbana (Tahitian strain T. Iso) was obtained from the Provasol i-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 a t 33PSU and 21 O 

### C. Tests were conducted

at 21 O 

### C 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 res ulted in any significant toxicity to either species. > 97% mysid shrimp survival was recorded from all sam ples, both treated and untreated, retrieved from the tanks at the time of de-ballasting. To psmelt survival in undiluted treated and untreated water at time of discharge was 100% and 95.2% respe ctively with no noticeable toxicity associated with other treatments in the dilut ion 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) 0.00 3 <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 inte rval) 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 inte rval) 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 Treatmen t 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 Sta tistics 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 inte rval) 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 inte rval) 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 a mmonia (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 inte rval) 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 Dilut ion 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 % surv ival 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 inte rval) 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) 0.00 4 <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 inte rval) 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 inte rval) 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 s ummarized in table 30. Results indicate variable growth in T=0 control (untreated) sampl es, with no clear relationship to the dilution series, and a slight positive relationship betw een degree of dilution and growth rate in treated T=0 samples. Definitive samples taken during de-ba llasting indicate some growth inhibition, although this appears not to be related to UV i rradiation.

### 70

Table 30. Summary data from Phytoplankton WET bioassay. T=0 samples collected September 19 th 

### , 2008. T=10 Days samples collected September 29 th 

### , 2008. Test begum
October 4 th 

### , 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 ± 2 53,418 793 124.22 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 6.2 5% 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 se veral different requirements and interests that guided the testing timetable and other proce dures. 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 or der to provide a seasonal dimension to the testing regime. Under U.S. Coast Guard Shipboard Technolo gy 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 conduc ting these three trials between April

5 th 

### and October 6 th 

### , 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 ret ention time of ballast water in the tanks in order to investigate any possible latent effect s 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 th e discretion of the officers and crew of the vessel, according to their schedule and other logis tics. In all three trials the numbers of live zooplankton >50μm (narrowest dimension) complied with all published ballast water treatment regulat ions, in that no live zooplankton in
this size class were seen at the time of ballast wat er 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 li ve organisms in the microscope field of view. Trial 2 was characterized by a large amount of detr itus 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 seve ral 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 a s much as three orders of magnitude among jurisdictions (table 1). Natural populations of phytoplan kton may have an even greater

### 72

concentration range among different water bodies, and c ell densities encountered during these
trials demonstrated 100-fold differences between Caribbea n waters and Alaskan waters. Alaskan water (trial 2) showed the highest densities encountered throughout these trials (ca. 10 8 

### cells/m 3 

### ), although very much higher cell concentrations would not be unusual in Pacific waters. We, therefore, categorize, phytoplankton densities throughout t his 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 o f this issue exist (e.g. Wright 2007, Veldhuis et al. 2001, Veldhuis and Brussaard 2006). Further probl ems 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 cat egory as was the case in the current

trials. In all of these trials phytoplankton numbers following t reatment (at de-ballasting) were
well below the standard of 10 7 

### live cells/m 3 

### set by IMO under G-8 guidelines. When compared to
the U.S. Coast Guard standard of 10 5 

### live cells/m 3 

### (=100,000 live cells/m 3 

### ), the grand mean from all three trials (2,222, 75,667, 103,300/3 = 60,398 live cells/m 3 

### ) complies with the standard, although the individual value from trial 3 (103,300 live cells/m 3 

### ) is marginally higher than the 100,000 live cells/m 3 

### U.S. Coast Guard standard. Another way of looking at th ese data takes note of the fact that cell densities following grow-out repre sent 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 untr eated ballast water. In untreated samples 27 out of 43 genera for which data were available sho wed positive growth rate (average increase after grow-out 1.25), whereas no treated sample s exhibited positive growth of any phytoplankton genus (average decline of 75% in cell numbers aft er grow-out). Several analytical techniques are currently available to differentiate betw een dead and live, but non-motile cells (Bruussard et al 2001, Veldhuis et al. 2001, Veldhuis and Bruussa rd 2006), although quantification is difficult within the context of curren t published standards

Bacterial counts illustrate some of the difficulties involved with conducting trials aboard

### 73

working ships where, unlike land-based trials, there is limit ed ability to control some test parameters. A summary of bacterial counts from all th ree trials is shown in table 31. As judged by indicator bacteria, water examined from trial s 1 and 3 indicate relatively pristine conditions. Trial 1 showed very low densities o f 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 bact eria in all samples. Although they are not regulated by IMO or the U.S. Coast Guard, January 2008 Cali fornia regulations set a standard of 1000cfu/100ml for this group (Table 1). Cfus for cultur al heterotrophic bacteria nominally comply with this standard in all three trial s, 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 a t 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 c ulturable 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 indica tor 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. N o indicator bacteria were reported from any treated samples from trial 3. Cultural bacteria wer e 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), particularl y in treated samples.

Bacterial counts from trial 2 differed from the other t wo trials, and were characterized by higher counts of cultural bacteria at the time of disch arge (de-ballasting) and the appearance of

intestinal enterococci in both treated and untreated s amples at time of de-ballasting, despite the
fact that no cfus from this taxa were recorded in the r espective 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 hig her 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 correspon ding 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 sa mples 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 detrit us loads and large zooplankton specimens that were not seen in T=0 samples. These incl uded several harpacticoid copepod adults >1000μm (some >2000μm), and indicated a quite different po pulation 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 var ying states of decay. It must be borne in mind that the filtered was turned off at de-ballasting as pe r normal de-ballasting protocol, and would not have filtered out large organisms during the disc harge cycle.

It seems reasonable to conclude that the appearance of En terococci in samples withdrawn from the tank at discharge would result from the decay/disintegration of these planktonic organisms with the concomitant release of en dogenous 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 o f large planktonic organisms identified at discharge but not previously seen:

### 75 1. An earlier ballasting operation (before trial 2) may n ot have properly employed the filter, or

2. Small eggs and/or juvenile stages (<50μm) may have passed t hrough a correctly functioning filter rated for a 50μm cut-off, then subsequent ly grew and formed a live population within the tank. A similar situation has been observed by this author in pr evious shipboard trials (Wright et al. 2007 and unpublished). A drawback of conducting shipboard tests, the refore, relates to the fact that it is impossible to know if residual flora/fauna i nhabit the tank prior to the onset of a trial. If such a situation exists, there may be significant, extr aneous, qualitative and quantitative differences between the discharged water and that chara cterized 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 si milar problem relates to the flushing of the, often lengthy, piping and associated dead-space that co nstitutes the ballasting system. Current IMO shipboard testing protocols simply require a co mparison between treated and untreated water in shipboard trials following a cert ain defined residence time in the tanks, although there is an additional requirement to character ize 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 illu strate the importance of inline sampling and analysis of a representative number of trea ted samples as well as untreated samples at T=0, particularly where a filter is involved. Results of Whole Effluent Toxicity (WET) bioassays con ducted during trial 3 indicated no significant differences between the toxicity of tr eated vs. untreated water samples at the time of discharge from the vessel. In invertebrate and verteb rate larval assays undiluted water from both treated and untreated tanks appeared not to show any si gnificant 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 f ollowing the (10 day) residence time, although the toxicity did not differ significantly betwee n treated and untreated water. It might be concluded from those data that any toxic element present i n discharged water did not result from UV irradiation. Such results have been supported by an ext ensive literature search that has

### 76

revealed no evidence of residual chemical toxicity in wate r resulting from prior UV irradiation of that water. While some changes in nutrient levels appear ed to be associated with water treatment it was not possible to definitively conclude that these c hanges directly resulted from UV irradiation. For example, both phosphate and nitrate leve ls 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 increas e in the degradation of planktonic organisms resulting from the treatment process, rather t han UV irradiation directly. Nitrite levels were also higher in treated samples 10 days after treatme nt. While UV photolysis of nitrate to nitrite has been demonstrated in UV-irradiated drinking wa ter (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 negligi ble relative to these standards and pose no toxicological threat assoc iated with discharge. Table 32 provides a summary of compliance/non-compliance o f the Hyde ‘Guardian’ BWT system with current published national and internatio nal standards. Under the conditions encountered during these trials, the system is seen to c omply 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 (En terococcus) 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 lar ge 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 r elated to the test. Another concerns the relative lack of flexibility in certain cases in deter mining 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 ba llast water treatment system under ‘real world conditions’. In this case, under the ambient condi tions and for the flow rates encountered throughout these trials, the Hyde Marine ‘Guardian’ syste m appears to be a highly effective means of treating ballast water to remove potentially i nvasive 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. N T = 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 √ √ √ NT Trial 2 Phytoplankton √ √ X 2 

### Trial 2 Bacteria √ √ √ Trial 3 Zooplankton √ √ √ √ Trial 3 Phytoplankton √ X 1 

### X 2 

### 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% follo wing 83% reduction in cell concentration after grow-out. Such a reduction in cell c ount 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 o n morphological characteristics their lack of growth potential determined through cell counts befor e and after grow-out can be interpreted as ‘non-viable’, in which case California regul ations 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 infecti on. Aquatic Microbial Ecology 26, 157 – 166.

Carlton, J.T. and Geller, J.B. (1993). Ecological roulet te: The global transport of nonindigenous marine organisms. Science 261 , 78-82.

Cohen, A.N. and Carlton, J.T. (1998). Accelerated invasio n 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 Associ ated with Aquatic Invasive Species. Proceedings of the Aquatic Invaders of the Delawa re Estuary Symposium, Malvern, Pennsylvania, May 20, 2003, pp. 3-5.

Raaymakers, S. (2002). The Ballast Water Problem:Global Ec ological, 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: me chanisms, 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 Americ a: 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 shi ps. Nature 408, 49-50.

Sharpless, C.M. and Linden K.G. (2001). UV photolysis of nit rate. Effects of natural organic matter and dissolved inorganic carbon and implications f or UV water disinfection. Environmental Science and Technology, 29 , 2949-2955.

### 79

Veldhuis M.J.W. and Brussaard, C.P.D. (2006) Harmful alga e 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 pe rmeability, photosynthetic activity, pigmentation and growth. European Journal of P hycology 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. Oran o-Dawson (2005). Shipboard trials of ballast water management options: Part 2. Efficacy of b allast 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 M oesel, 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 contai ner 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 5-1 5 2008 (updated 10/08/2008) CONSOLIDATED SPECIES LIS T

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 SE A ) APR 5-15 2008 (updated 10/08/2008)

Num. Num. Total Total Grand Event Treatment Rep Group Alive Dead Tota l alive dead total

--------------------------------------------------- -------------------------

T0 CONTROL 1 COPEPOD 328 136 46 4 1 COPEPOD NAUP 72 0 7 2 1 EGGS 312 0 31 2 1 NEMATODE 8 0 8 1 POLYCHAETE 48 0 4 8 768 136 904

2 CLADOCERAN 16 0 1 6 2 COPEPOD 128 24 15 2 2 COPEPOD NAUP 168 8 17 6 2 EGGS 224 104 32 8 2 POLYCHAETE 80 0 8 0 616 136 752

3 BIVALVE 16 0 1 6 3 COPEPOD 104 40 14 4 3 COPEPOD NAUP 24 0 2 4 3 TINTINNID 96 0 9 6 240 40 280

4 COPEPOD 32 0 3 2 4 COPEPOD NAUP 64 0 6 4 4 EGGS 88 0 8 8 4 POLYCHAETE 16 0 1 6 4 TINTINNID 112 0 11 2 312 0 312

5 BIVALVE 32 8 4 0 5 COPEPOD 40 0 4 0 5 COPEPOD NAUP 80 8 8 8 5 EGGS 368 0 36 8 5 NEMATODE 8 0 8 5 POLYCHAETE 24 0 2 4 552 16 568

TREATED 1 COPEPOD 16 0 1 6 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 1 6 16 0 16

T96 CONTROL 1 BIVALVE 8 0 8 1 COPEPOD 40 104 14 4 1 COPEPOD NAUP 24 8 3 2 1 EGGS 384 0 38 4 1 POLYCHAETE 8 0 8 464 112 576

2 BIVALVE 8 8 1 6 2 COPEPOD 48 72 12 0 2 EGGS 16 0 1 6 72 80 152

3 BIVALVE 16 8 2 4

### 82

CORAL PRINCESS BALLAST WATER TRIAL #1 (CARIBBEAN SE A ) APR 5-15 2008 (updated 10/08/2008)

Num. Num. Total Total Grand Event Treatment Rep Group Alive Dead Tota l alive dead total

--------------------------------------------------- -------------------------

T96 3 COPEPOD 24 272 29 6 3 COPEPOD NAUP 24 0 2 4 3 EGGS 624 240 86 4 3 POLYCHAETE 64 0 6 4 3 PROTOZOAN 8 0 8 3 TINTINNID 16 0 1 6 776 520 1296

4 BIVALVE 8 8 1 6 4 COPEPOD 8 88 9 6 4 EGGS 32 0 3 2 48 96 144

5 BIVALVE 16 8 2 4 5 COPEPOD 128 80 20 8 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 32 8 6 COPEPOD NAUP 680 344 102 4 6 FORAMINIFERA 8 0 8 6 POLYCHAETE 24 0 2 4 6 TINTINNID 0 24 2 4 712 704 1416

7 CHAETOGNATH 16 8 2 4 7 COPEPOD 0 344 34 4 7 COPEPOD NAUP 584 440 102 4 7 EGGS 0 8 8 7 NEMATODE 24 0 2 4 7 POLYCHAETE 16 0 1 6 7 TINTINNID 0 8 8 640 808 1448

8 BIVALVE 8 8 1 6 8 CHAETOGNATH 24 8 3 2 8 COPEPOD 80 648 72 8 8 COPEPOD NAUP 552 752 130 4 8 EGGS 0 40 4 0 8 FORAMINIFERA 16 0 1 6 8 POLYCHAETE 32 8 4 0 8 TINTINNID 0 32 3 2 712 1496 2208

9 CHAETOGNATH 8 0 8 9 COPEPOD 8 672 68 0 9 COPEPOD NAUP 856 568 142 4 9 EGGS 0 8 8 9 FORAMINIFERA 16 0 1 6 9 POLYCHAETE 48 16 6 4 9 TINTINNID 0 16 1 6 936 1280 2216

### 83

CORAL PRINCESS BALLAST WATER TRIAL #1 (CARIBBEAN SE A ) APR 5-15 2008 (updated 10/08/2008)

Num. Num. Total Total Grand Event Treatment Rep Group Alive Dead Tota l alive dead total

--------------------------------------------------- ------------------------- T96 TREATED 1 ACTINULA 0 16 1 6 1 BIVALVE 0 8 8 1 COPEPOD 0 104 10 4 1 GASTROPOD 0 8 8 1 NEMATODE 0 16 1 6 0 152 152

2 ACTINULA 0 16 1 6 2 COPEPOD 0 80 8 0 0 96 96

3 COPEPOD 0 128 12 8 3 COPEPOD NAUP 0 8 8 3 TINTINNID 0 8 8 0 144 144

4 COPEPOD 0 176 17 6 4 COPEPOD NAUP 0 8 8 4 FORAMINIFERA 0 8 8 0 192 192

5 COPEPOD 0 184 18 4 0 184 184

6 COPEPOD 0 16 1 6 0 16 16

7 COPEPOD 0 64 6 4 7 COPEPOD NAUP 0 16 1 6 0 80 80

8 COPEPOD 0 144 14 4 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 LIS T

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 COA ST 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 COA ST 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 COA ST 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 COA ST 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 COA ST 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 19-29 200 8 (updated 10/08/2008) CONSOLIDATED SPECIES LIS T

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 COA ST 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 COA ST 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 S EP 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 COA ST 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 : 19 th



### 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 i n 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 sa mples (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: 19 th



### September 2008 samples collected Storage: N/A; samples processed immediately Time: 15.40-18.10; processed Treated first (9 samples T1-T9), then c ontrol 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 late r (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 35 O 

### C (19 th 

### Sept 2008) Control samples placed in incubator at 18.10pm at 35 O 

### C (19 th 

### Sept 2008) Read times: Treated samples read at 11.15 (20 th 

### Sept 2008) – 18 hours Control samples read at 12.00 (20 th 

### 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, 14 S) (but this was at 28 hours!)

### 97

(2) Idexx Tests: (2) Enterobacteriacae (Enterolert)

Sample ID: Trial 3, T=0 Date: 19 th



### September 2008; sample collection Storage: N/A; processed immediately Time: 17.40-18.50; processed Treated first (9 samples T1-T9), then c ontrol 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 late r (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 (19 th 

### Sept 2008) Control samples placed in incubator at 18.55pm at 41C (19 th 

### Sept 2008) Read times: Treated samples read at 20.10pm (20 th 

### Sept 2008) ~ 25 hours Control samples read at 20.30pm (20 th 

### 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: 19 th



### September 2008 (sample collection) Storage: Samples stored at 4C until processing (on September 20 th 

### 2008). Time: 16.00-17.40; processed Treated first (9 samples T1-T9), then c ontrol 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. Sto red at -20 O 

### C until processing. Filters at 4 O 

### C until freezing on September 21 st 

### 2008.

### 99

(4) Aerobic (culturable bacteria) sample tests:

Sample ID: Trial 3, T=0 Date: 19 th



### September 2008 (sample collection) Storage: Samples stored at 4 O 

### C until processing (on September 20 th 

### 2008). Time: 11.40-14.40; processed Treated first (9 samples T1-T9), then c ontrol 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). Bl ackfield 0.45um filters placed on vacuum manifold and 25ml or 10ml samples slowly filtered on to filters. Filters placed in 47mm sterile plates, with pads containing (soaked in) 2ml of st erile 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, als o make note of fungi. Score mid-large colonies very tiny ones (which do not grow at longer incu bations) do not score, but make note. Incubation times: Treated samples placed in incubator at 13.00 at 35 O 

### C (20 th 

### Sept 2008) Control samples placed in incubator at 14.40 at 35 O 

### C (20 th 

### Sept 2008) Read times: Treated samples read at 14.05 (22 nd 

### Sept 2008) ~ 49 hours Control samples read at 14.50pm (22 nd 

### 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 1C5 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 (22 nd 

### Sept 2008) ~ 58 hours Control samples read at 23.30pm (22 nd 

### 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 (23 rd 

### Sept 2008) ~ 67 hours Control samples read at 09.30 (23 rd 

### 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 (23 rd 

### Sept 2008) ~ 75 hours Control samples read at 17.50 (23 rd 

### 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 M arine ‘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 M aryland 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 Wat er Treatment System (BWTS) was conducted by a team led by Dr. David Wright, Pro fessor of Environmental Toxicology at the University of Maryland Center for Envi ronmental 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, bel ow:
Table 1. Shipboard testing team and associates for G-8 shipboar d 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 micr obiology 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 t he biological effectiveness of shipboard filtration/separation and ultraviolet radiati on equipment as a ballast water treatment method. The sampling protocol was designed to satisfy IMO G 8 requirements, with the objective of comparing biological endpoints to those published under regul ation 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 te sting, 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 t aken from a customized sampling port mounted downstream of the BWTS in the machinery spac e of the vessel. These samples were compared in order to test the efficacy of the sys tem to IMO and other standards (table 2) standards under normal working conditions. The BWTS syst em consists of a primary disc filter manufactured by Arkal Inc., Tel Aviv, Israel mounted in se ries with a medium pressure UV irradiation system rated by the manufacturer/vendor at 200 mJ cm 2 

### for treatment of ballast water at flow rates up to 250 m 3 

### h -1 

### . Trials took place during the vessel’s regular spring sche dule 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 consist ed of determination of water quality parameters and a comparison of biological endpoints in t reated and untreated ballast water samples, with reference to both IMO G8 and the U.S. Coas t Guard Shipboard Technology Evaluation Program (STEP). Sampling procedures and endpoint determinations followed IMO G8 guidelines for shipboard trials and the exercise was desi gned to supplement land-based trials being conducted concomitantly at NIOZ, Texel, Netherland s to determine the efficacy of the BWT system under varying water quality conditions. Shipbo ard trials were designed to document system performance under normal seagoing conditi ons and under different geographical and seasonal conditions, with the objective of determining the degree of compliance with IMO and STEP requirements. Whole Efflue nt Toxicity (WET) tests were also conducted as part of the third and last trial to determine wh ether any significant chemical changes in ballast water after exposure to UV irradiat ion, 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 proto cols were employed in order to comply with IMO G-8 guidelines and produce results that wer e 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 s hipboard 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 thre e 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 recogni zed standards/chain of custody procedures. While standard water quality parameters were me asured aboard ship, nutrient analyses were conducted by the University of Maryland Analy tical Services Division, which has been an independent, nationally and internationally reco gnized 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 be ginning, 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 filter 

### Sea Chest

400L

Bacteria

10

- 5 0 /

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 U3 U2 U1

Filter Mesocosm Sample Ballast Tank (count ASAP) 500L 500L

72hr grow-out period samples Bacteria Bacteria (count ASAP)

### 108

Sampling Strategy.

Triplicate samples were taken at the beginning, middle a nd end of a collection cycle. It must be emphasized that treatment by the Hyde ‘Guardian’ s ystem is only complete following a second ‘pass’ through the UV system at the time on disch arge from the ship. Therefore, definitively treated samples can only be collected and ex amined 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 cle an ‘rinsed’ tanks. It is, therefore of particular importance to define the nature of the intake (challenge) w ater as well as discharged water, in order to identify important differences between the biolog y 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 sampli ng port located downstream from the Ballast Water Treatment System (BWTS) in t he 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 or ganisms). 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 bott le that forms the `cod-end' of the net also had 20μm mesh `windows' to facilitate the filtratio n process. Separate nets were used to sample from treated and untreated samples, and nets wer e rinsed with hot tap-water between sampling cycles. They were also examined for tears, lea ks and imperfections and any repairs made. In the machinery space the contents of each mesoco sm 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 tra nsferred via Stempel pipets to counting wheels mounted on compound microscope stages for m icroscopical examination. Using these procedures, it was possible to begin microscopic ex amination 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 var iability 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 through out these trials. Organism sizes were recorded as the minimum dimension of the cel l/organism, using calibrated reticule eyepieces mounted in the eyepiece lens of each microsco pe. However, a particularly useful means of cross-calibration involved the ‘seeding’ of count ing slides with glass beads of specific sizes (10 μm, 20 μm, 50 μm, 100 μm, 200 μm). These glass beads ar e 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 cros s-calibration tool among microscopists and has been referenced in recent shipboard studies (Wri ght et al. 2009). In order to address precision associated with counting a majority of plankto n 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 cal ibration.

QA/QC considerations for shipboard sampling include rigoro us washing of the sampling nets with freshwater and examination of nets and other s ampling 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 possibl e 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 sys tem of the shipping before sampling commences. An important consideration in this respect i s that the collection of treated samples always precedes the collection of untreated samples. Thi s minimizes the potential of carry-over of live individuals (i.e. Type 1 error) that might have be en trapped in dead-spaces. Conversely, carry-over of dead organisms (Type 2 error) through a ‘treat ed 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 ot her 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 pas sed through a 200μm mesh net to remove any large predatory zooplankton. Experience with shi pboard trials has indicated that grazing can be a factor, even within the context of ball ast tank residence time and sample holding time prior to examination. Each sample was concentrate d to a volume <10ml. for examination/grow-out, using a 10μm Nitex screen. This enabl ed 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 afte r grow-out. Use of grow-out technique. Live-dead status of phytoplankton for the determination of b allast water treatment efficacy remains problematic to the extent that, while s ome taxonomic groups, such as dinoflagellates, are clearly motile, many have vegetati ve 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 growt h 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 w as determined that growth potential represents a robust indicator of phytoplankton viability w ithin the constraints of shipboard analyses, although this endpoint remains difficult to quanti fy 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 conditi ons through the addition of f/2 growth medium. This medium had the following constituents: FeCl 3 

### .6H 2 

### O, 1.45 mg L -1 

### ; KNO 3 

### 200 mg L -1 

### ; NaH 2 

### PO 4 

### .2H 2 

### 0, 34.8 mg L -1 

### ; H 3 

### BO 3 

### , 34.2 mg L -1 

### ; EDTA.2H 2 

### 0, 30 mg L -1 

### ; MnCl 2 

### .4H 2 

### 0, 4.3 mg L -1 

### ; CoCl 2 

### .6H 2 

### 0, 0.13 mg L -1 

### ; ZnSO 4 . 

### 7H 2 

### 0, 0.364 mg L -1 

### ; H 2 

### SeO 3 

### , 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 taxonom ic 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 (us eful for several but not all taxonomic groups), numbers of viable cells determined in this way ofte n declined rather than increased following ‘grow-out’. Under such circumstances it may b e 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 w as 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 co mpared with ‘splits’ of the same sample that were stained with Neutral Red. Samples fol lowing 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 preserve d in Lugol's Solution for more intensive taxonomy and determination of cell sizes. Chlorophyll a Phytoplankton growth may be conveniently assessed by measur ing in vivo chlorophyll a concentration before and after the grow-out period. Whi le 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 (gr owth 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 com munities may include taxa which differ in volume by more than two orders of magnitude. Un der such circumstances chlorophyll a levels would be biased towards larger cells. We, neverthe less find that useful information can be obtained from chlorophyll a analyses, particularly with r espect 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 Desi gns 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 d irectly from the unfiltered discharge from the sampling port, and stored at temperatures just abo ve freezing (1-4 O 

### C) prior to and during transport to the University of Maryland Center for En vironmental Science, Chesapeake Biological Laboratory, although in Trial 3 some culturi ng/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 abo ard this vessel. The following bactericidal endpoints were employed:

• Cultural bacteria. For total culturable heterotrophic bacteria, samples we re

### 111 serially diluted three to four orders of magnitude. 20 ml. f ull 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 Pe tri dish that contained a sterile absorbent pad soaked in 2ml of autoclaved marine broth (Di fcoTM Marine Broth 2216). The filter paper was placed so as to expose the side i noculated with bacteria. The Petri dishes were then closed and place d upside down in a 25 ±0.5

o 

### C 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 bact eria 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 publishe d 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 L aboatories.) were employed to quantify coliforms, E. Coli and Enterococcus in treated and untreated ballast water.

Samples were diluted an order of magnitude with sterile de ionized 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 detec tion media into the Quantitray and sealed and incubated at 37 o 

### C for 18 hours. The analytical method is a multi-well Most Probable Number (MPN) method invol ving exposure to a mixture of o -nitrophenol- β -D -galactopyranoside (ONPG) and 4-methylumbelliferyl-

β -D -gluronide (MUG). Coliform bacteria produce a yellow col or 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) a s a result of the action of β -glucuronidase. Yellow and fluorescent squares were counted a nd 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 fluor escent end-product. Like the coliform method, the IDEXX Laborator ies Inc. Enterolert Quantitray 2000 (ASTM method # D6503-99) relies on a MPN endpoint, following 24h incubation at 41 o 

### C. 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 sample s transported to the University of Maryland Center for Environmental Science, Chesapeake Bi ological 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-c alibrated via chlorophyll a standard measurements conducted both aboard ship and at the UMCES Ana lytical Services Division, Solomons MD. pH, dissolved oxygen (DO), salinity, temperat ure were all measured using a Yellow Springs Instruments ‘Multimeter’, which was cal ibrated against laboratory-based instrumentation before and after each trial, with pH/s alinity 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 perf ormed by the UMCES Analytical Services Division, Chesapeake Biological Labor atory, Solomons, MD, U.S.A. Standard Operating Procedures from this Laboratory have bee n 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 summa rized below

Total Suspended Solids (TSS). Total Suspended Solids (TSS) and Particulate Organic Carbo n (POC) were measured using GF/C filters that were initially dried to (102-105 O 

### C), pre-weighed (gm.) to 4 decimal places and brought aboard the vessel in individual sealed P etri dishes. Filter volumes varied between 500ml. -1-5 L. Filters were rinsed with freshwat er to remove salt and brought back to the laboratory over ice. Each filter was then dried at 60 O 

### C for 24h and re-weighed (APHA 1975, USEPA 1979a). Total Suspended Solids (TSS) were calculated fro m the difference in between this weight and that of the pre-weighed filter. For POC measurement filters were then combusted in a 500 O 

### C 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. Sa mples for DOC analysis are treated with hydrochloric acid and sparged with ultra pure carrier gr ade 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 (CO 2 

### ). The CO 2 

### is carried by ultra pure air to a nondispersive infrared detector (NDIR) where CO 2 

### is detected (Sugimura and Suzuki 1988).

Nitrite + Nitrate Filtered samples are passed through a granulated copper-cadm ium column to reduce nitrate to nitrite. The nitrite (originally present plus reduced nitra te) then is determined by diazotizing with sulfanilamide and coupling with N-1- naphthylethylenediamine di hydrochloride to from a colored azo dye. Nitrate concentration is obtained by s ubtracting 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-moly bdate complex which is reduced to an intensely blue-colored complex by ascorbic acid. Colo r 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 am monium 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 prec ipitation of hydroxides of calcium and magnesium (Technicon 1986, Kerouel and Aminot 1987).

University of Maryland Analytical Services Division - Statemen t of Quality Assurance/Quality Control Procedures.

The analytical services division of the University of M aryland Center for Environmental Science (UMCES) is a nationally and internationally rec ognized resource for aquatic chemical analyses, which for over thirty years has provided this s ervice 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 Servi ces laboratory is assuring the quality of data generated by the procedures presented in this ma nual. Further, indication of data quality is accomplished by analyzing duplicates, spikes, sta ndards-as-samples, standard reference materials and participating in cross-calibration exercis es.
Laboratory Duplicates Approximately 5% of the total number of samples analyzed c onsist of laboratory duplicates. For dissolved analytes, after a sample is a nalyzed, the same sample container is placed farther along in the automatic sampler and re-anal yzed. The mean of the two values is reported as the concentration for that sample. If a di fference of >10% is observed between replicates, then all of the replicates for that part icular analytical run are carefully reviewed. If only one of the duplicate pairs is in question, then only t hat sample is re-analyzed. If all show a similar trend, then instrumentation/reagent problems are s uspected and the analytical run is halted until such time as the problem is resolved. This pro cedure is practiced for all dissolved analytes that are not consumed completely in the ana lytical procedure. For those that are completely consumed and for particulate analytes, duplicat e 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 separa te QA/QC data file along with the sample number, sample collection date and analysi s date. The mean concentration and standard deviation of the replicates are calculated in thi s 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 suffici ent quality assurance data to compensate for the omission of laboratory spikes for th ese 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 de termining accuracy of results. Examples of such cross calibration exercises include th e Chesapeake Bay Quarterly Split Sample Program, US EPA Method Validation Studies and Internationa l Council for the Exploration of the Sea Intercomparison Exercise for Nutrients in Sea Water.

Sample Custody Upon arrival at the laboratory, samples are counted, o bserved for potential problems (melting, broken containers, etc.) and placed in a freez er until analysis. Sample information and date of arrival are recorded on a log sheet.

Instrument Maintenance Analytical instruments are maintained on a regular basi s 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 an d 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 i s 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 existi ng instrumentation are preformed. No predetermined number of data pairs are used to make the a ssessment 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 determini ng the number of pairs that must be addressed before bringing an instrument on-line and in determining ins trument comparability.

The analyst who performs these comparisons should be expe rienced, open-minded and impartial. This person can give an evaluation of ease of instrument operation and a very important general statement of comparability. This stat ement on comparability must then be substantiated via statistical analysis of the data. As pr eviously mentioned, these data must encompass the entire concentration range, matrix inte rferences, percent recovery, results of standard reference material analyses, etc. The data int erpretation must support comparability. The analyst and laboratory QA/QC officer must concur an d finally, some sort of presentation

### 115

(written or verbal) must be given to the contractor ex plaining 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 a re kept on a monthly basis. Thermometers used in this equipment are calibrated agains t a certified NBS thermometer.

Laboratory Spikes Approximately 5% of the total number of samples analyzed co nsist 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 co ncentration of the sample. This concentrated standard is used to minimize any possible chan ge 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 expe cted value. These concentrations (original, expected and actual) are recorded in a separate Q A/QC data file along with sample number, sample collection date, analysis date and the am ount 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 percen tage 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 ana lytical 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 s o-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 anal ysis is out of control and requires immediate attention.

Standard as Sample Standards are analyzed as samples throughout the analyti cal run. This is an excellent means of evaluating instrument performance during the course o f an analytical run. Standards are analyzed every 12 - 20 samples, depending on the instrument an d 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 paramete rs 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 Cana da. It is certified by the Council for carbon content, gives a non-certified range of resul ts for phosphorus, but no information for nitrogen. We have analyzed this sediment for many years an d maintain a substantial database for nitrogen and phosphorus, as well as carbon values. We an alyze this sediment quarterly and compare these results to the certified value, non-cert ified range of values and our historical values. Dissolved Analytes and Hardness: Standard refer ence materials for ammonium, nitrite + nitrate, nitrite, orthophosphate, dissolved nitrogen, disso lved 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 concen trations 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 fr ozen 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 EP A 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, respecti vely.

Data Management. In general, Standard Methods (APHA, 1998), section 9020B.10-11, guided t he procedures for documentation, recordkeeping and data handling. D ata 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 in tegral 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 ma y affect quality and report such problems to the project director. A team approach is adopte d in order to correct observed deviations for which there is an obvious solution during th e routine implementation of the sampling procedures. During data validation and assessmen t, deviations in the project data are corrected as appropriate. If data are determined to be inva lid, 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 deviatio ns 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 com pletely reflect the raw data of the studies. Responsible persons for zooplankton, phytoplankton and micr obial 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 perf ormed;

2. Methods/SOPs followed for the analysis;

3. Brief analytical assessment of the results highlightin g 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, 14 th 

### Edition. American Public Health Association. Washington, D.C. 1193pp.

American Public Health Association (APHA), American W ater Works Association, Water Environment Federation. 1998. Standard Methods for the exam ination of water and wastewater (20 th 

### edition). Clesceri, LS., A.E. Greenberg and A.D. Eat on, eds. American Public Health Association.Washington DC. Froelich, P.N. and M.E.Q. Pilson. 1978. Systematic absor bance error with Technicon AutoAnalyzer II colorimeter. Water Res. 12:599-603.

IMO (International Maritime Organization) 2004. Internat ional convention for the control and management of ships’ ballast water and sediment. Availa ble 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 Wastew ater, 20th Edition (1998) under sections 10-20 and 10-21, “Biological Examination” Sugimura, Y. and Y. Suzuki. 1988. A high temperature catalytic o xidation 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. Technico n Industrial Systems. Tarrytown, New York, 10591.

Technicon Industrial Method No. 158-71 W/A† Tentative. 1977. Tec hnical Industrial Systems. Tarrytown, New York, 10591.

Technicon Industrial Method No. 804-86T. August 1986. Technicon In dustrial Systems. Tarrytown, New York, 10591.

United State Environmental Protection Agency 1979a. Method No. 160.2 (with slight modification) in Methods for chemical analysis of wat er and wastes. United States Environmental Protection Agency, Office of Research and D evelopment. 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 E nvironmental Protection Agency, Office of Research and Development. Cincinnati, Ohio. Report No. EP A-600/4-79-020 March 1979. 460pp.

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United State Environmental Protection Agency 1979c. Method No. 365.1 in Methods for chemical analysis of water and wastes. United States E nvironmental Protection Agency, Office of Research and Development. Cincinnati, Ohio. Report No. EP A-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. M organ 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. Envi ron. (C4) 3-12.

Wright, D.A. (2007) Problems associated with performance co mpliance 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 abo ard a working tanker in Pacific waters. Environ. Technol. (in review)

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## Appendix E.

Standard Operating Procedures for UMCES Analytical Service s Division.

### Filename: PCL Final Reportcorrected April 2009.doc

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