# 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



## January 2009

### 1 

## Table of Contents

Page .

INTRODUCTION AND PROJECT DESCRIPTION 3 Background. 3
MATERIALS AND METHODS 6 Disposition of treated and untreated ballast water. 6 Sample collection. 6 Examination of ‘challenge water’ immediately 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 movin g 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 mad e 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

Treated (UV + filter during ballasting) → (UV at de-ballasting) Start de-ballast (N=3) 0 125±21

Treated (UV + filter during ballasting) → (UV at de-ballasting) Start de-ballast (N=3) 0 131±99

Treated (UV + filter during ballasting) → (UV at de-ballasting) Start de-ballast (N=3) 0 75±72

Phytoplankton.
Phytoplankton counts from treated and untreated (control ) samples from T=0 and T=96h are shown in tables 3-5. Based on microscopic examination, 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/m

3 ‘Live’ Diatoms. Density/m

3 Total ‘Live’ phytoplankton. Density/m

3 Dead Dinoflagellates. Density/m

3 Dead Diatoms. Density/m

3 Total Dead phytoplankton. Density/m

3 Untreated Controls, T=0 Rep. C1, T=0 9,821 17 3,661 183,182 0 0 0 Rep. C1, T=0 + 48h growout 16,111 31,944 48,105 556 556 1,112

Rep. C2 T=0 4,018 62,946 66,964 0 0 0 Rep. C2 T=0 + 48h growout 4,722 15,277 20,000 0 833 833

Rep. C3 T=0 23,661 127,679 151,340 0 0 0 Rep. C3 T=0 + 48h growout 2,500 63 ,333 65,833 278 556 834

Mean ‘Live’ Phytoplankton. Control samples, T=0 (after grow-out) 133,829± 60 ,0 5 5

(44,646±23,111) Treated, T=0 Rep. T1, T=0 9,583 71,667 81,250 0 0 0 Rep. T1, T=0 + 48h growout 4,444 5,000 9,444 833 278 1,111

Rep. T2 T=0 7, 917 75,833 83,800 2,083 417 2,500 Rep. T2 T=0 + 48h growout 3,056 6,111 9,167 0 0 0

Rep. T3 T=0 3,333 69,583 72,916 417 0 417 Rep. T3 T=0 + 48h growout 3,056 5,278 8,334 0 0 0

Mean ‘Live’ Phytoplankton. Treated samples, T=0 (after grow-out) 79,322± 5, 692

(8,982±578)

### 17

Table 4. Summary of phytoplankton results (>10<50μm minimum 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) ‘Liv e’



### Dinoflagellates. Density/m 3 ‘Live’



### Diatoms. Density/m 3 

### Total ‘Live’



### phytoplankton. Density/m 3 Dead



### Dinoflagellates. Density/m 3 Dead



### Diatoms. Density/m 3 

### Total Dead



### phytoplankton. Density/m 3 

### Untreated Controls, T=96hStart Deballast. Rep. C1a, T=96 278 5,278 5,556 0 0 0

Rep. C1b T=96 278 5,000 5,278 0 556 556 Rep. C1c T=96 0 2,500 2,500 0 0 0 Mean C1, T = 96h samples 4,445±1,690

Mid Deballast. Rep. C2a, T=96 278 18,889 19,167 0 3,333 3,333

Rep. C2b T=96 833 14,444 15,277 0 2,500 2,500 Rep. C2c T=0 1,111 8,889 10,000 0 3,889 3,889 Mean C2, T = 96h samples 14,815±4,601

End Deballast. Rep. C3a, T=96 1,111 14,722 15,883 556 5,278 5,834

Rep. C3b T=96 833 16,389 17,222 278 3,889 4,167 Rep. C3c T=96 556 20,556 21,112 0 3,889 3,889 Mean C3, T = 96h samples 18,072±2,716

Mean Live Phytoplankton 12,444±6,770 (n=9)

### 18 Table 5 Summary of phytoplankton results (>10<50μm minimum 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/m 3 ‘Live’



### Diatoms. Density/m 3 

### Total ‘Live’



### phytoplankton. Density/m 3 Dead



### Dinoflagellates. Density/m 3 Dead



### Diatoms. Density/m 3 

### Total Dead



### phytoplankton Density/m 3 

### Treated, T=96hStart Deballast. Rep. T1a, T=96 556 3,611 4,167 278 0 278

Rep. T1a, T=96 + 48h growout 0 1,667 1,667 0 556 556

Rep. T1b T=96 278 6,944 7,222 0 556 556 Rep. T1b T=96 + 48h growout 0 833 833 0 278 278

Rep. T1c T=96 0 6,111 6,111 556 0 556 Rep. T1c T=96 + 48h growout 0 1,389 1,389 0 0 0

Mean T1, T=96h samples(after growout) 5,833±1,546 (1,296±425)

Mid Deballast. Rep. T2a, T=96 278 7,222 7,500 278 278 556

Rep. T2a, T=96 + 48h growout 0 1,389 1,389 0 0 0

Rep. T2b T=96 0 6,389 6,389 0 0 0 Rep. T2b T=96 + 48h growout 0 4,444 4,444 0 278 278

Rep. T2c T=96 278 5,278 5,556 278 0 278 Rep. T2c T=96 + 48h growout 556 556 1,112 0 3,056 3,056

Mean T2, T = 96h samples(after growout) 6,482±975 (2,315±1,849)

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

### 19 ballast. Rep. T3a, T=96 Rep. T3a, T=96 + 48h growout 0 2,222 2,222 0 1,944 1,944

Rep. T3b T=96 278 6,944 7,222 0 1,389 1,389 Rep. T3b T=96 + 48h growout 278 4,722 5,000 0 556 556

Rep. T3c T=96 556 8,056 8,612 0 0 0 Rep. T3c T=96 + 48h growout 556 1,389 1,945 0 278 278

Mean T3, T = 96h samples(after growout) 6,667±2,274 (3,055±1,690)

Mean Live Phytoplankton(after growout) 6,327±1,421 (n=9) (2,222±1,483) (n=9)

vital stain Neutral Red. Based solely on these criteria , initial treatment (filter + UV during ballasting) resulted in an immediate 41% reduction in live cell numbers relative to untreated samples at T=0. However, following a 96h residence time in the tank, untreated ‘live’ cell numbers had fallen to 7% of the initial, untreated T=0 density, and treated ‘live’ cell numbers had fallen to 4.7% of that initial concentration (i.e. 95.3 % removal). Under such circumstances a comparison between treated and control ‘live’ densities at 96h probably has little meaning as it is clear that the ballast tank provides an inhospitable 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 after grow-out (table 6). While some positive growth was seen in untreated control samples at

### 20

T=0, 2/3 control samples exhibited a small amount of growth after 96h, while no growth relative to intake water was recorded from treated samples at 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

. Ch lorophyll a concentrations from measurement of in vivo fluorescence

. (all values are means of 3 determinations)

T=0 T=0, Pre grow

- out chlorophyll a conc. (μg
L -1 ) chlorophyll a conc . (μg L - 1 ) after 24h grow-out (T=0 + 24) chlorophyll a conc . (μg
L -1 ) after 48h grow-out (T=0 + 48)
Control 1 start

ballasting 2.75 4.33 4.3

Control 2 mid

ballasting 1.81 1.95 1.98

Control 3 end

ballasting 7.78 8.58 8.76

Treated 1 start

ballasting 1.31 0.92 0.88

Treated 2 mid

ballasting 1 0.98 0.82

Treated 3 end

ballasting 1.13 1 0.94

T=96h T=96h, Pre grow

- out Chlorophyll a conc. (μg
L -1 ) 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

Control 2 mid de

ballasting 1.43 1.86 1.77

### 21

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

### 22

Control T=96h C3a 0 0 0 0 0 0 0 Control T=96h C3b 0 0 0 0 0 0 0 Control T=96h C3c 0 0 0 0 0 0 0 Treated T=96h T1a 0 0 0 0 0 0 0 Treated T=96h T1b 0 0 0 0 0 0 0 Treated T=96h T1c 0 0 0 0 0 0 0 Treated T=96h T2a 0 0 0 0 0 0 0 Treated T=96h T2b 0 0 0 0 0 0 0 Treated T=96h T2c 0 0 0 0 0 0 0 Treated T=96h T3a 0 0 0 0 0 0 0 Treated T=96h T3b 0 0 0 0 0 0 0 Treated T=96h T3c 0 0 0 0 0 0 0

### 23

Table 8. Trial 1. Cultural bacteria in treated and 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±291 0

Control (untreated) Mid de-ballast (N=3) 437±624 1,123±572

Control (untreated) End de-ballast (N=3) 43±74 971±654

332±431 698±683 Treated (UV + filter during ballasting) → (UV at de-ballasting) Start de-ballast (N=3) 0 75±16

Treated (UV + filter during ballasting) → (UV at de-ballasting) Mid de-ballast (N=3) 0 144±194

Treated (UV + filter during ballasting) → (UV at de-ballasting) End de-ballast (N=3) 0 224±89

Counts indicated live residuals of smaller zooplankton, the great majority of which were marine nematodes, in both treated and untreated samples taken at 114h, although these did not fall into the >50μm size category. Live/dead nematode 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=114hControl (untreated). Start deballast (N=3) 0 0

Control (untreated) Mid deballast (N=3) 0 0

Control (untreated) End deballast (N=3) 0 11±15

Treated (UV + filter during ballasting) → (UV at deballasting). Start de-ballast (N=3) 0 0

Treated (UV + filter during ballasting) → (UV at deballasting) Mid de-ballast (N=3) 27±27 69±98

Treated (UV + filter during ballasting) → (UV at deballasting) End de-ballast (N=3) 80±39 160±48

Phytoplankton.
Phytoplankton counts from treated and untreated (control ) samples from T=0 and T=114h are shown in tables 12-15. Based on microscopic examination, 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.

### 30

Following a 114h residence time in the tank, untreated ‘live ’ cell numbers had fallen to 30% of the initial, untreated T=0 density (compared with 7% in 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) Untreated Controls, T=0 ‘Live’

Dinoflagellates. Density/m

3 ‘Live’ Diatoms. Density/m

3 Total Live’ phytoplankton. Density/m

3 Dead

Dinoflagellates. Density/m

3 Dead Diatoms. Density/m

3 Dead

Rep. C1 a , T=0 2 59
, 7 00 12
, 045
, 5 00 12
, 305
, 2 00 0 0 0 Rep. C1 a , T=0 + 42h growout 774,200 6,483,900 7 , 258
, 1 00 0 354,800 354,800 Rep. C 1b , T=0 4 49
, 4 00 19
, 887
, 6 00 20
, 337
, 0 00 0 0 0 Rep. C 1b T=0 + 42h growout 483.800 11,838,700 12
, 322
, 5 00 32,300 32300 64600 Rep. C 1c , T=0 1 93
, 5 00 17
, 451
, 6 00 17
, 645
, 1 00 0 0 0 Rep. C 1c T=0 + 42h growout 354.800 12,709,700 13
, 064
, 5 00 96,800 129,000 225,800
Mean C1 samples (after grow-out) 16,762,433±4,088,004 10,881,700±3,159,984
Rep. C 2a T=0 3 92

. 2 00 19
, 779 , 4 00 20,1 71
, 6 00 0 0 0 Rep. C 2a T=0 + 42h growout 4 51

. 4 00 29
, 236
, 1 00 29
, 687
, 5 00 0 34
, 7 00 34
, 7 00

Rep. C 2b , T=0 64
, 5 00 3
, 548
, 4 00 3
, 612
, 9 00 0 64
, 5 00 64
, 5 00 Rep. C 2b T=0 + 42h growout 2 90
, 3 00 6 , 838
, 7 00 7 , 129
, 0 00 0 129
, 0 00 129
, 0 00
Rep. C 2c , T= 0 7 41
, 9 00 4
, 838
, 7 00 5
, 580
, 6 00 0 0 0 Rep. C 2c T=0 + 42h growout 1 29
, 0 00 3
, 709
, 7 00 3
, 838
, 7 00 96 , 7 00 129
, 0 00 225
, 8 00

Mean C2 samples (after grow-out) 9,788,367±9,045,806 13,548,733±14,065,331
Rep. C 3a , T=0 96 , 8 00 2
, 549
, 7 00 2
, 646
, 6 00 0 0 0 Rep. C 3a T= 0 + 42h growout 2 20
, 6 00 7 , 389
, 7 00 7 , 610
, 3 00 0 404
, 4 00 404
, 4 00
Rep. C 3b , T=0 122
, 5 00 2
, 279
, 4 00 2
, 402
, 0 00 0 0 0 Rep. C 3b T=0 + 42h growout 206
, 6 00 867
, 8 00 1,0 74
, 4 00 0 62
, 0 00 62
, 0 00
Rep. C 3c , T=0 140
, 7 00 2
, 696 , 1 00 2
, 836
, 8 00 0 24
, 5 00 24
, 5 00 Rep. C 3c T=0 + 42h growout 1 65
, 6 00 2
, 450
, 3 00 2
, 615
, 9 00 66 , 2 00 99 , 3 00 165
, 5 00

Mean C3 samples (after grow-out) 2,628,467±217,966 3,766,867±3,416,583

### 32

Table 13. Summary of phytoplankton results (>10<50μm minimum dimension) for Trial 2 of Hyde ‘Guardian’ BWT system aboard M/V Coral Princess , June 30 th -July 8 th , 2008. Treated (control) T=0 samples, including live grow-out cell concentrations (in red). Mean ‘Live’ Phytoplankton (Nos. in red are after grow-out) 9 , 726 , 4 00 ±7 , 8 80 , 6 00 2,936,800±6,608,000

Phytoplank ton (10μm-50μm) Treated, T=0 ‘Live’

Dinoflagellates. Density/m

3 ‘Live’ Diatoms. Density/m

3 Total ‘Live’ phytoplankton. Density/m

3 Dead Dinoflagellates. Density/m

3 Dead Diatoms. Density/m

3 Total Dead phytoplankton. Density/m

3 Rep. T1 a , T=0 129
, 0 00 6 4
, 5 00 193
, 5 00 0 0 0 Rep. T1 a , T=0+42hr 32
, 3 00 96 , 8 00 129
, 1 00 64
, 5 00 64
, 5 00 129
, 0 00
Rep. T 1b , T=0 24
, 5 00 49
, 0 00 73
, 5 00 0 0 0 Rep. T 1b , T=0+42hr 96 , 7 00 32
, 2 00 129
, 0 00 32
, 2 00 0 32
, 2 00
Rep. T 1c , T=0 147
, 1 00 0 147
, 1 00 0 0 0 Rep. T 1c , T=0+42hr 57
, 5 00 459
,, 8 00 517
, 2 00 0 57
, 4 00 57
, 4 00

138,033 ± 60,512 258,433±224,099 Rep. T 2a , T=0 135
, 1 00 216
, 2 00 351
, 3 00 0 0 0 Rep. T 2a , T=0+42hr 287
, 4 00 229
, 9 00 517
, 3 00 143
, 7 00 0 143
, 7 00
Rep. T 2b , T=0 82
, 0 00 218
, 6 00 300
, 6 00 27
, 3 00 82
, 0 00 109
, 3 00 Rep. T 2b , T=0+42hr 32 2
, 6 00 451
, 6 00 774
, 2 00 32
, 3 00 96 , 8 00 129
, 1 00
Rep. T 2c , T=0 129
, 0 00 258
, 1 00 387
, 1 00 0 0 0 Rep. T 2c , T=0+42hr 85
, 5 00 128
, 2 00 213
, 7 00 0 0 0

346,333 ± 43,463 501,733±280,574 Rep. T 3a , T=0 193
, 5 00 64
, 5 00 258
, 0 00 0 0 0 Rep. T 3a , T=0+42hr 155
, 4 00 207
, 2 00 362
, 7 00 129
, 5 00 25
, 9 00 155
, 4 00
Rep. T 3b , T=0 318
, 6 00 318
, 6 00 637
, 3 00 0 49
, 0 00 49
, 0 00 Rep. T 3b , T=0+42hr 188
, 7 00 141
, 5 00 330
, 2 00 141
, 5 00 94
, 3 00 235
, 8 00
Rep. T 3c , T=0 73
, 5 00 612
, 7 00 686
, 3 00 24
, 5 00 98
, 0 00 122
, 5 00 Rep. T 3c , T=0+42hr 96 , 8 00 32
, 3 00 129
, 1 00 3 2
, 3 00 32
, 3 00 64
, 6 00
527,200 ± 234,418 274,000±126,535

### 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/m

3 ‘Live’ Diatoms. Density/m

3 Tota l ‘Live’ phytoplankton. Density/m

3 Dead

Dinoflagellates. Density/m

3 Dead Diatoms. Density/m

3 Total Dead phytoplankton. Density/m

3

Start De

- ballast. Rep. C1a, T=114h 1
, 200
, 5 00 18
, 1 17
, 3 00 19
, 317
, 8 00 81
, 8 00 381
, 9 00 463
, 8 00

Start De

- ballast. Rep. C1a, T=114h + 42h 988,500 17,823,400 18,811,900 101,200 299, 600 400,800

Rep. C1b T=114h 25
, 0 00 650
, 4 00 675
, 4 00 0 25
, 0 00 25
, 0 00

Rep. C1b T=114h + 42h 0 349,900 349,900 46,000 54,600 100,600

Rep. C1c T=114h 77 , 8 00 596
, 6 00 6 74
, 4 00 0 25
, 9 00 25
, 9 00

Rep. C1c T=114h + 42h 36,800 602,700 639,500 0 36,400 36,400

Mean C1 , T= 114h samples After grow-out 6 , 889 , 2 00 ± 10 , 763 , 5 00
6,600,433±8,635,620

Mid De

- ballast. Rep. C2a, T=114h 52
, 3 00 890
, 6 00 9 4 2,9 00 0 78
, 5 00 78
, 5 00

Mid De

- ballast. Rep. C2a, T=114h + 42h 54,600 1,237,900 1,292,500 0 36,800 36,800

Rep. C2b T=114h 0 623
, 9 00 623
, 9 00 0 23
, 1 00 23
, 1 00 Mean ‘Live’ Phytoplankton (Nos. in red are after grow-out) 298,400±229,700 125,600±143,800

### 34 Rep. C2b T=114h + 42h 0 789,100 789,100 0 37,000 37,000

Rep. C2c T=114h 0 654
, 8 00 654
, 8 00 0 78
, 5 0 0 78
, 5 00

Rep. C2c T=114h + 42h 0 1,046,700 1,046,700 0 46,000 46,000

Mean C2 , T= 114h samples After grow-out 740 , 6 00 ± 176 , 0 00
1,042,767±205,531

End De

- ballast. Rep. C3a, T=114h 125
, 0 00 800
, 5 00 925
, 5 00 0 125
, 0 00 125
, 0 00

End De

- ballast. Rep. C3a + 42h 87,900 1,238,600 1,326,500

Rep. C3b T=114h 168
, 7 00 1
, 097
, 0 00 1
, 265 ,7 00 0 22
, 0 00 225
, 0 00

Rep. C3b T=114h +42h 137,700 1,260,800 1,398,500

Rep. C3c T=114h 96 , 2 00 722
, 0 00 8 18
, 2 00 0 1203 00 1203 00

Rep. C3c T=114h +42h 102,200 902,300 1,004,500

Mean C3, T= 114h samples After grow-out 1 , 003 , 2 00 ±233 , 7 00
1,243,167±171,304

Mean Live Phytoplankton After grow-out 2 , 877 , 6 00 ±6 , 168 , 4 00 2,962,122±5,613,127

### 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/m

3 ‘Live’ Diatoms. Density/m

3 Total ‘Live’ phytoplankton. Density/m

3 Dead

Dinoflagellat es Density/m 3 Dead

Diatoms. Density/m

3 Total Dead phytoplankton. Density/m

3
Start De

- ballast. Rep. T1a, T=114h 49
, 0 00 98
, 0 00 147
, 0 00 0 49
, 0 00 49 ,00 0

Start De

- ballast. Rep. T1a, T=114h + 42h 19,900 37,000 56,900 0 56,800 56,800

Rep. T1b, T=114h 25
, 9 00 207
, 5 00 233
, 4 00 0 51
, 8 00 51
, 8 00

Rep. T1b, T=114h + 42h 20,500 28,000 48,500 22,000 67,600 89,600

Rep. T1c, T=114h 50
, 0 00 200
, 1 00 250
, 1 00 0 0 0

Rep. T1c, T=114h + 42h 0 40,500 40 ,500 0 22,000 22,000

Mean T1 , T= 114h samples After grow-out 2 10 , 2 00 ±55 , 3 00

48,633±6,696
Mid De

- ballast. Rep. T2a, T=114h 23
, 6 00 236
, 8 00 2 60
, 5 00 0 23
, 6 00 23
, 6 00

Mid De

- ballast. Rep. T2a, T=114h + 42h 0 23.600 23,600 0 23,600 23,600

Rep. T2b, T=114h 0 156
, 8 00 156
, 8 00 0 0 0

Rep. T2b, T=114h + 42h 0 92,000 92,000 0 0 0

Rep. T2c T=114h 0 94
, 7 00 94
, 7 00 0 47
, 3 00 47
, 3 00

Rep. T2c T=114h + 42h 0 56,900 56,900 0 64,900 64,900

Mean T1, T= 114h samples After grow-out 170 , 7 00 ±83 , 8 00

57,500±27,927

### 36 E nd De

- ballast. Rep. T3a, T=114h 32
, 9 00 65
, 8 00 98
, 7 00 0 0 0

End De

- ballast. Rep. T3a, T=114h + 42h 30,600 44,600 75,200 0 0 0

Rep. T3b, T=114h 0 1 84
, 4 00 184
, 4 00 0 0 0

Rep. T3b, T=114h + 42h 0 123,000 123,000 0 0 0

Rep. T3c, T=114h 0 2 80
, 1 00 280
, 1 00 0 0 0

Rep. T3c, T=114h + 42h 0 164,400 164,400 0 0 0

Mean T3, T= 114h samples (After grow-out) 187 , 7 00 ±90 , 7 00

(120,867±36,447)
Mean Live Phytoplankton ( After grow-out) 189 , 5 00 ±69 , 8 00 (75,667±41,861)

### 37

Table 16. Trial 2. Chlorophyll a concentrations from measurement of in vivo fluorescence. (all values are means of 3 determinations)

T=0 T=0, Pre grow-out chlorophyll a conc. (μg L -1 ) chlorophyll a conc. (μg
L -1 ) after 24h grow-out (T=0 + 24) chlorophyll a conc. (μg 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) N=9 1,391±918 192±233 Treated (UV + filter during ballasting) N=7 0 11±4

Control (untreated) Start de-ballast (N=3) 24±0 32±13

Control (untreated) Mid de-ballast (N=3) 8±7 13±19

Control (untreated) End de-ballast (N=3) 16±17 13±10

Treated (UV + filter during ballasting) → (UV at de-ballasting) Start de-ballast (N=3) 0 0

### 44 Treated (UV + filter during ballasting) → (UV at de-ballasting) Mid de-ballast (N=3) 0 3±4

Treated (UV + filter during ballasting) → (UV at de-ballasting) End de-ballast (N=3) 0 3±4

Table 21. Densities of living nematode worms in treated and 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/m

3 ‘Live’ Diatoms. Density/m

3 Dead Dead Dinoflagellates. Density/m

3 Dead Diatoms. Density/m

3 Dead

Start De

- ballast. Rep. C1a, T=0 546,720 5,323,400 5,870,120 100,800 551,400 652,200

Rep. C1a, T= 0 + 48h 250,500 123,500 37 4,000 186,000 448,300 634,300

Rep. C1b T= 0 489,600 7,833,600 8,323,200 0 816,000 816,000 Rep. C1b T= 0 + 48h 184,600 848,700 1,033,300 0 93,200 93,200

Rep. C1c T= 0 488,889 3,422,222 3,911,111 244,444 244,444 488,888 Rep. C1c T= 0 + 48h 384,500 336.800 721,300 0 36,800 36,800

Mean C1 , T= 0 samples (After grow-out) 508,403 4,499,741 6,034,810 ± 2,210,650 (709,533± 329,807) 115,096 53 7,297 652, 393

Mid De

- ballast. Rep. C2a, T=0 408,000 8,976,000 9,384,000 0 408,000 408,000

Mid De

- ballast. Rep. C2a, T=0 + 48h 296,700 1,968,000 2,264,700 0 212,100 212,100

Rep. C2b T= 0 2,760,000 10,304,000 13,064,000 368,000 552,000 920,000 Rep. C2b T= 0 + 48h 1,176,300 2,349,600 3,525,900 102,000 464,000 566,000

Rep. C2c T= 0 633,333 3,800,000 4,433,333 211,111 422,222 63 3,333 Rep. C2c T= 0 + 48h 720,900 4,444,000 5,164,999 118,000 446,000 564,000

Mean C 2, T=0 samples (After grow-out) 1, 267,111 7,693,333 8,960,444 ± 4,330,895 (3,651,866± 1,454,247) 193,037 460,741 653,778

End De

- ballast. Rep. C3a, T=0 998,045 500,122 1,49 8,167 97,618 384,067 481,685

Rep. C3a, T= 0 + 48h 1,021,400 686,700 1,708,100 59,000 302,300 361,300

### 47 Rep. C3b T= 0 1,339,1 00 3,252,1 00 4,591,3 00 0 382,60 0 382,60 0 Rep. C3b T= 0 + 48h 989,000 960,800 1,949,800 0 188,000 188,000

Rep. C3c T= 0 785,7 00 6,678 ,5 00 7,464,2 00 196,4 00 392,8 00 589,2 00 Rep. C3c T= 0 + 48h 484,600 4,998,000 5,482,600 202,400 447,600 650,000

Mean C 3, T=0 samples (After grow-out) 1,062,422 3,476,956 4,517, 88 9 ± 2,983,694 (3,046,833± 2,112,895) 98, 016 386,511 484,527

Mean Live Phytoplankton

(After grow-out) 938,845 4,910,804 6,504,381 ±3,458,482 (2,469,400 ±1,866,202(n=9)) 13 5,383 4 60,811 59 6 , 899

### 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/m 3 ‘Live’



### Diatoms. Density/m 3 

### Total ‘Live’



### phytoplankton. Density/m 3 Dead



### Dinoflagellates. Density/m 3 Dead



### Diatoms. Density/m 3 

### Total Dead



### phytoplankton. Density/m 3 Rep. T1 a , T=0 1,804,152 115,805 1,919,957 0 82,459 82,459 Rep. T 1a , T=0+48hr ND
Rep. T 1b , T=0 1,323,529 2 34,118 1,557,647 0 0 0 Rep. T 1b , T=0+48hr 2,479,339 0 2,479,339 0 165,289 165,289
Rep. T 1c , T=0 2,47 6 , 546 0 2,47 6,546 0 16 4
, 300 16 4
, 300 Rep. T 1c , T=0+48hr ND

Mean T1 T=0 samples 1,984,717 ± 462,860
Rep. T 2a , T=0 807,259 256,780 1,064,039 0 0 0 Rep. T 2a , T=0+48hr ND
Rep. T 2b , T=0 1,614,350 179,372 1,793,722 0 0 0 Rep. T2b, T=0+48hr 0 330,579 330,579 0 0 0
Rep. T 2c , T=0 1,735,296 234,118 1,969,414 Rep. T 2c , T=0+48hr ND
1,609,058 ± 480,106 Rep. T 3a , T=0 1,438.090 0 1,438,09 0 0 0 0 Rep. T 3a , T=0+48hr ND
Rep. T 3b , T=0 2,152,466 0 2,152,466 0 0 0 Rep. T3b, T=0+48hr 735,294 0 735,294 0 0 0
Rep. T 3c , T=0 848,400 0 848,400 Rep. T 3c , T=0+48hr ND
1,479,652 ± 653,026 Mean ‘Live’ Phytoplankton(After growout) 1,372,720 124,073 1, 511,770 ±719,434 (n=9) (1,181,737± 1,141,830 (n=3)) 0 27,528 27,528

### 49

Table 24. Summary of phytoplankton results (>10<50μm minimum 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/m 3 ‘Live’



### Diatoms. Density/m 3 Dead Dead 

### Dinoflagellates. Density/m 3 Dead



### Diatoms. Density/m 3 Dead



### Rep. C1a, T=10 Days 170,940 1,538,462 1,709,002 0 683,761 683,761

Rep. C1a, T=10 Days + 48h 392,157 2,156,863 2,549,020

Rep. C1b, T=10 Days 735,294 1,470,588 2,205,882 196,078 0 196,078

Rep. C1b, T=10 Days + 48h 661,157 495,868 1,157,025

Rep. C1c, T= 10 Days 540,300 4,898,270 5,438,570 147,059 0 147,059

Rep. C1c, T= 10 Days ND

Mean C1, T=10 Days 3,117,818± 2,025,127 Rep. C2a T= 10 Days 1,661,157 2,495,868 3,157,025 0 165,289 165,289

Rep. C2a T= 10 Days + 48h ND

Rep. C2b, T= 10 Days 441,177 1,294,118 1,735,295 0 0 0
Rep. C2b, T= 10 Days + 48h 826,446 0 826,446
Rep. C2c, T= 10 Days 826,446 3,878,460 4,704,906 0 165,289 165,289
Rep. C2c, T= 10 Days + 48h ND
Mean C2, T=10 Days 3,199,075± 1,485,252 Rep. C3a, T= 400,969 2,180,200 2,581,169 0 211,007 211,007

### 50 10 Days Rep. C3a, T= 10 Days + 48h ND
Rep. C3b, T= 10 Days 0 661,157 661,157 0 0 0
Rep. C3b, T= 10 Days + 48h 0 330,579 330,579
Rep. C3c, T= 10 Days 0 3,150,787 3,150,787 0 330,579 330,579
Rep. C3c, T= 10 Days + 48h ND
Mean C3, T=10 Days 668,115 1,997,381 2,220,086± 1,358,517 Mean ‘Live’ Phytoplankton (After grow-out) 

### 170,940 2,396,445 2,815,977± 1,505,752 (n=9) (1,271,768± 951,515(n=4)) 42,892 172,881 211,007

### 51

Table 25. Summary of phytoplankton results (>10<50μm minimum 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/m

3 ‘Live’ 

### Viable Diatoms. Density/m

3 Total ‘Live’ phytoplankton. Density/m

3 Dead Dinoflagellates Density/m

3 Dead Diatoms. Density/m

3 Total Dead phytoplankton. Density/m

3

Start De

- ballast. Rep. T1a, T=10 Days 431,304 646,957 1,078,261 215,6 00 215,6 00 431, 200

Start De

- ballast. Rep. T1a, T=10 Days + 48h 98,000 64 ,900 162,900 199,500 170,000 369,500

Rep. T1b, T= 10 Days 0 0 0 2,160,000 540,000 2,700,000

Rep. T1b, T= 10 Days + 48h 0 0 0 1,700,000 226,000 1,926,000

Rep. T1c, T= 10 Days 652,174 434,783 1,086,957 0 652,174 652,174

Rep. T1c, T= 10 Days + 48h 144,300 1 02 ,400 2 46,700 0 459,000 459,000

Mean T 1 , T=10 Day samples (After grow-out) 361,159 360,580 721,739 ±625,060 (136,533 ±125,446) 791,884 469,275 1,261,159

Mid De

- ballast. Rep. T2a, T=10 Days 173,333 0 173,333 173,333 866,667 1,040,000

Mid De

- ballast. Rep. T2a, T=10 Days + 48h 58,000 0 58,000 160,000 430,000 590,000

Rep. T2b, T= 10 Days 891,429 1,089,524 1,980,953 198,095 0 198,095

Rep. T2b, T= 10 Days + 48h 1 52,800 1 06,600 2 59,400 122,200 0 122,200

Rep. T2c T= 10 Days 0 0 0 0 417,331 417,331

Rep. T2c T= 10 Days + 48h 0 0 0 0 326,000 326,000

Mean T2, T=10 354,921 363,175 718,09 5± 123,809 427,999 551,809

### 52 Day samples

(After grow-out) 1,097,095

( 1 05,800 ±136,146)
End De

- ballast. Rep. T3a, T=10 Days 0 842,857 842,857 168,571 0 168,571

End De

- ballast. Rep. T3a, T=10 Days + 48h 0 102,000 102,000

Rep. T3b, T= 10 Days 0 238,597 238,597 146,996 0 146,996

Rep. T3b, T= 10 Days + 48h 0 100,700 100,700

Rep. T3c, T= 10 Days 0 137,500 137,500 137,500 0 137,500

Rep. T3c, T= 10 Days + 48h 0 0

Mean T3, T=10 Day samples (After grow-out) 0 490,179 406,318±381,418

(67,567±58,518) 153,036 0 153,036

Mean Live Phytoplankton (After grow-out) 238,582 376,690 614,273±677,711 (103,300 ±101,585) 355,239 299,092 654,663

### 53

Table 26. Trial 3. Chlorophyll a concentrations from measurement of in vivo fluorescence. (all values are means of 3 determinations)

T=0 T=0, Pre grow-out chlorophyll a conc. (μg L -1 ) chlorophyll a conc. (μg
L -1 ) after 24h grow-out (T=0 + 24) chlorophyll a conc. (μg 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

### 55

Treated T =0 T2c 0 0 0 0 0 0 0 Treated T =0 T3a 0 0 0 0 0 0 0 Treated T =0 T3b 0 0 0 0 0 0 0 Treated T =0 T3c 0 0 0 0 0 0 0 Control T=10 Days C1a 0 1 0 1 0 0 0 Control T=10 Days C1b 0 1 0 1 0 0 0 Control T=10 Days C1c 0 1 0 0 0 0 0 Control T=10 Days C2a 0 0 0 0 0 0 0 Control T=10 Days C2b 0 1 0 0 0 0 0 Control T=10 Days C2c 0 0 0 0 0 0 0 Control T=10 Days C3a 0 0 0 0 0 0 0 Control T=10 Days C3b 0 0 0 0 0 0 0 Control T=10 Days C3c 0 1 0 1 0 0 0 Treated T=10 Days T1a 0 0 0 0 0 0 0 Treated T=10 Days T1b 0 0 0 0 0 0 0 Treated T=10 Days T1c 0 0 0 0 0 0 0 Treated T=10 Days T2a 0 0 0 0 0 0 0 Treated T=10 Days T2b 0 0 0 0 0 0 0 Treated T=10 Days T2c 0 0 0 0 0 0 0 Treated T=10 Days T3a 0 0 0 0 0 0 0 Treated T=10 Days T3b 0 0 0 0 0 0 0 Treated T=10 Days T3c 0 0 0 0 0 0 0

Table 28. Trial 3. Culturable heterotrophic bacteria counts from treated and untreated (control) samples at T=0 and T=10 Days.

Colonyforming Units Colony-forming

Units Colonyforming Units 25% 10% 1% Treatment Time Replicate

Control t=0 C1a 51 28 5 Control t=0 C1b >100 17 4 Control t=0 C1c 30 17 3 Control t=0 C2a >100 22 1 Control t=0 C2b 9 27 7 Control t=0 C2c >100 88 15 Control t=0 C3a 2 2 0 Control t=0 C3b 0 97 24 Control t=0 C3c 6 9 1 Treated t=0 T1a >100 10 2 Treated t=0 T1b 9 5 1

### 56 Treated t=0 T1c >100 9 6 Treated t=0 T2a 4 2 1 Treated t=0 T2b >100 22 4 Treated t=0 T2c 3 4 0 Treated t=0 T3a 3 0 0 Treated t=0 T3b 23 10 1 Treated t=0 T3c >100 2 0 Control T=10 Days C1a 8 0 0 Control T=10 Days C1b 2 0 0 Control T=10 Days C1c 4 0 0 Control T=10 Days C2a 0 0 0 Control T=10 Days C2b 8 0 0 Control T=10 Days C2c 23 4 0 Control T=10 Days C3a 12 4 0 Control T=10 Days C3b 4 1 1 Control T=10 Days C3c 0 0 0 Treated T=10 Days T1a 0 0 0 Treated T=10 Days T1b 0 0 0 Treated T=10 Days T1c 0 0 0 Treated T=10 Days T2a 0 0 0 Treated T=10 Days T2b 0 0 0 Treated T=10 Days T2c 0 0 0 Treated T=10 Days T3a 18 0 0 Treated T=10 Days T3b 0 0 0 Treated T=10 Days T3c 0 0 0

Water Chemistry. Ballast water during trial 3 was taken south of Long 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,

### 57

treated samples at T=10 days contained much higher nitrate levels, possibly due to an increase in nutrient release from dying/dead organisms in these samples (table 29). Nitrite levels, although much lower, also showed a 3-4 fold increase in treated 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

### 58

of untreated organisms in this size category declined precip itously over the period of residence in
the ballast tanks. 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-@ 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 a approach requiring tests for residual toxicity that essentially follow IMO G-9 guidelines, even for systems not involving the addition of active substances. The resolution was adopted too late for such 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) 0.0 0 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 ammoni a (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 w ater 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

Crit eria 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 Treatment Waters pH (SU) 7.93

- 8.25 Salinity (ppt) 29

- 33 Total ammonia (mg / L) 0.003

- 0.014 Temperature: 21.1

- 22.3 Dissolved oxygen (DO mg /L): 6.30

– 7.62



### Survival of Americamysis bahia at test termination (48 hours)

% test water % survival 48

- h Statistics 0 100 LC50: N/A 6.25 97.5 LOEL: N/A 12.5 100 NOEL: N/A 25 100 50 97.5 100 100 

### 1: LC50 = median-lethal concentration (95% confidence 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 Tem perature: 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 ammonia (mg / L) 0.003

- 0.006 Temperature: 20.0

- 22.3 Dissolved oxygen (DO mg /L): 6.40

– 7.62



### Survival of Atherinops affinis at test termination (96 hours)

% t est 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 Dilution water Final pH (SU) 8.00 8.15 Salinity (ppt) 33 33 Total ammonia (mg / L) <0.003 <0.003 Temperature: 19.6 21.6 Dissolved oxygen (DO mg /L): 5.71 7.27 

### SU = standard units ppt = parts per thousand
Ranges of water quality tests for all treatments

Criteria Treatment Waters pH (SU) 7.93

- 8.18 Salinity (ppt) 31

- 33 Total ammonia (mg / L) <0.003 Temperature: 20.0

- 22.3 Dissolved oxygen (DO mg /L): 6.40

– 7.62



### Survival of Atherinops affinis at test termination (96 hours)

% test water % survival 48

- h Statistics 0 95 LC50: N/A 6.25 100 LOEL: N/A 12.5 90 NOEL: N/A 25 80 50 75 100 100 

### 1: LC50 = median-lethal concentration (95% confidence 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 ammo nia (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.

Untreated, T=0h Phytoplankton Cell Counts

BeforeGrowout 72h After 96h Grow-out

Mean ± S.D. 5 reps % T. Iso @ 96h/0h % growth

relative to controls @ 96h 6.25% 251,697 1066547 1,227,844±315,040 488 72.21 12.50% 249,546 1195623 1,597,287±366,670 640 93.94 25% 255,761 1369875 1,511,954±379,578 591 88.92 50% 249,546 1647627 2,207,671±349,221 885 129.84 100% 270,820 1015395 1,486,712±243,092 549 87.44
Treated, T=0h 6.25% 266,517 1698904 2,112,251±253,418 793 124.22 12.50% 268,908 1726984 2,171,482±267,665 808 127.71 25% 259,107 1497038 1,631,086±230,473 630 95.93 50% 248,112 953725 1,173,489±235,826 473 69.01 100% 245,722 1026390 1,127,643±176,926 459 66.32
Untreated, T=10 Days 6.25% 244,527 1111246 2,057,322±324,840 841 120.99 12.50% 256,956 1164310 1,961,950±144,565 764 115.38 25% 253,371 1318245 1,908,216±301,344 753 112.22 50% 248,351 1135627 1,429,680±470,361 576 84.08 100% 266,995 924086 1,028,255±84,855 385 60.47
Treated, T=10 Days 6.25% 261,498 1377285 1,861,175±337,341 712 109.46 12.50% 248,829 1357684 1,973,662±128,668 793 116.07 25% 268,430 1109333 1,596,570±290,540 595 93.9 50% 263,171 1105270 1,553,019±223,125 590 91.33 100% 247,634 998663 1,363,134±55,263 550 80.17

Lab Controls 256,275 1214745 1,700,356 675

### 71

SUMMARY CONCLUSIONS.
In conducting these trials investigators were aware of several different requirements and interests that guided the testing timetabl e and other procedures. For example, under IMO G-8 guidelines there is a requirement to perform th ree tests, the first and last of which should be separated by a period of not less than si x months, in order to provide a seasonal dimension to the testing regime. Under U.S. Coast Guard S hipboard Technology Evaluation Program (STEP) requirements, two tests should be conducted in two different water bodies in which the vessel normally operates. These requirements were met by conducting these three trials between April 5 th



### and October 6 th 

### , and by testing water picked up respectively in the Caribbean Sea, the North West Pacific off Alaska, an d the Western Pacific off Long Beach. The distributors of the system, Hyde Marine Inc. expressed a n interesting in varying the retention time of ballast water in the tanks in order to investig ate any possible latent effects of UV irradiation. This was accommodated by varying the retention times by as much as 6 days: 96h in Trial 1, 114h in Trial 2 and 10 days in Trial 3. Actual ballas ting times were at the discretion of the officers and crew of the vessel, according to thei r schedule and other logistics. 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 concentration range among different water bodies, and c ell densities encountered during these

### 72

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

### 73

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 100cfu/100ml for this group (Table 1). While cfus for cultural bacteria exceed this standard in trial 1 at T=0, both treated and untreated numb ers declined at the time of discharge to levels below the California standard. At de-ballasting, cfus in treated samples were as low as one

Table 31 . Summary of Bacterial endpoints for Trial 1 (April ), Trial 2 (July) and Trial 3 (September). Numbers are reported as cfu/100ml.

Total Culturable Coliforms E. Coli Enterrococci Vibrio cholerae Treatment Time Trial 

### Control t=0 1 122±129 0.2±0 ND 0.2±0 0 

### Treated t=0 1 194±104 ND ND ND 0 

### Control t=96h 1 50±57 ND ND ND 0 

### Treated t=96h 1 10±17 ND ND ND 0 

### Control t=0 2 522±707 ND ND ND 0 

### Treated t=0 2 211±262 ND ND ND 0 

### Control t=114h 2 1156±1201 ND ND 2±1 0 

### Treated t=114h 2 1978±881 ND ND 36±27 0 

### Control t=0 3 341±342 ND ND ND 0 

### Treated t=0 3 71±67 ND ND ND 0 

### Control t=10 days 3 27±29 6±5 3±5 ND 0 

### Treated t=10 days 3 8±23 ND ND ND 0



### tenth of that standard.

Samples from trial 3 were similar to trial 1. No indica tor bacteria were reported in either treated or untreated samples at T=0, although low numbers of coliforms, including E. Coli
, appeared in untreated samples at the time of discharge. 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.

### 74

Bacterial counts from trial 2 differed markedly from the other two trials, and were characterized by:
1. High counts of cultural bacteria at the time of disch arge (de-ballasting)
2. Appearance of intestinal enterococci in both treated a nd untreated samples at time of de-ballasting, despite the fact that no cfus from this t axa were recorded in the respective T=0 samples.

While IMO and the U.S. Coast Guard have no standards appli cable to culturable heterotrophic bacteria, the numbers of cfu in these sampl es clearly exceeded the California standards. No coliforms were recorded from any samples i n this trial. With respect to Enterococci, numbers of cfus in treated samples at de-ballasting, 36±27cfu/100ml (table 31), were noticeably higher than in untreated samples (2±1cfu/100 ml.). The figure of
36±27cfu/100ml is below the standard of 100cfu/100ml set for this group by IMO and, therefore, complies with IMO regulations. However it marginally fails the corresponding standard of 33 cfu/100ml set jointly by U.S. Coast Guard and the state o f California (table 1). These anomalous figures should be viewed, however, within the context of th e zooplankton samples, which had high detritus loads and large zooplankton specimens that were not seen in T=0 samples. These included several harpacticoid copepod adults >1000μm (some >2000μm), a nd indicated a quite different population that that sampled at T=0. Despite t he presence of these organisms at the time of de-ballasting, all specimens in the treated samples were dead; some recently, others in varying states of decay. It must be borne in mind that the fil tered was turned off at de-ballasting as per normal de-ballasting protocol, and would not have filtered out large organisms during the discharge cycle.

It seems reasonable to conclude that the appearance of 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 3 

### Trial 2 Bacteria √ X 1 

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

### X 3 

### Trial 3 Bacteria √ √ √

1


### – Intestinal enterococci appeared at de-ballasting despite n ot being seen in T=0 samples, thought to be associated with zooplankton present in the t reated tank prior to the conduct of the test.

2


### – ‘Live’ phytoplankton present in treated samples after 10 days in tank + UV irradiation on deballasting. Residuals exceed U.S, C.G. standard by 3% 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).

3


### – 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.
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). Springer-

### 79

Verlag, 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

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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

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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 me ter

<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)

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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.

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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 

### ;

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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.

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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.

### 120

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)

### 121 

## Appendix E.

Standard Operating Procedures for UMCES Analytical Service s Division.

## NUTRI ENT ANAL YTI CAL S ERVI CES L ABO RATO RY

STANDA RD OP ERATING P ROC EDUR ES

C a r o lyn W . K e e fe K i m ber l y L. B l odni k ar Walter R . B oy nton C he ry l A

. C la rk Je r om e M

. Fr ank N ancy L. K aumey er Ma rg ar et M. We ir K at hr y n V

. W ood C ar l F. Zi m m er m ann



### Sp e c i a l Pu b l i c a t i o n Se r i e s No. SS

- 8 0

- 0 4

- CBL C ent er for E nvi r onm ent al and Est uar i ne St udi es T he U ni ver s i t y of M ar y l and Sy s t em

C hes apeake B i ol ogi cal L aborat ory

P. O

. B ox 38 Sol om ons , M ar y l and 20688- 0038 ( 410) 326- 7252

w w w

. cbl. um ces. edu /

na sl

Febr uar y , 2004

## TABLE OF CONTENTS

I N T R O D U C T I O N

... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 1

D I S S O L V E D I

N O R G A N I C A N A L Y T E S

... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 3

O r t h o p h o s p h a t e

... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... . . 7 A m m o n i u m

... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... . . 11 N i t r i t e

... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 14 N i t r i t e + N i t r a t e..

... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 17 S i l i c a t e

... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... . . 21 S u l f a t e , C h l o r i d e a n d

N i t r a t e

... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 24

O R G A N I C A N A L Y T E S

... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... . 27

T o t a l D i s s o l v e d

N i t r o g e n a n d

P h o s p h o r u s

... ... ... ... ... ... ... ... ... ... ... ... ... . . 29 T o t a l a n d D i s s o l v e d

O r g a n i c C a r

b o n

... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 35

P A R T I C U L A T E A

N A L Y T E S

... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... . . 38

P a r t i c u l a t e C a r

b o n a n d N i t r o g e n

... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 39 Particulate Ph osph orus and Particulate I norg anic P hosp horus

. . . . . . . . . . . . . . . . . . . . . . . . . . 44 P a r t i c u l a t e B i o g e n i c S i l i c a

... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... . . 48 T o t a l S u s p e n d e d

S o l i d s

... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... . . 52 T o t a l V o l a t i l e S o l i d s

... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 54 C hl or op hy ll a a n d

P h a e o p i g m e n t s

... ... ... ... ... ... ... ... ... ... ... ... ... ... ... . . 55

A P P E N D I X A

... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 59

A P P E N D I X B

... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 60

A C K N O W L E D G E M E N T S

... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... . . 65

### LIST O F TABLES

T abl e 1. Para m et er s rou ti nel y ana lyzed at the Nutr ie nt Ana lyti cal Servic es La bor at ory at CBL

. . . . . 1

Table 2. S am

pling and storag e for org anic a naly tes

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

T a b l e 3

. M i n i m

u m l i m i t s o f d e t e c t i o n o f

C B L

... ... ... ... ... ... ... ... ... ... ... ... ... ... . . 62



### LIST O F FIG URES
Fig ure 1

. Manifold assem bly for orthop hosp hate m ethod

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Fig ure 2

. Manifold assem bly for am m onium m ethod

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

F i g u r e 3

. M a n i f

o l d a s s e m b l y f o r n i t r i t e m e t h o d

... ... ... ... ... ... ... ... ... ... ... ... ... ... . 14
Fig ure 4

. Manifold assem bly for nitrite + nitrate m ethod

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

F i g u r e 5

. M a n i f

o l d a s s e m b l y f o r s i l i c a t e m e t h o d

... ... ... ... ... ... ... ... ... ... ... ... ... ... 21

F i g u r e 6

. F l o w d i a g r a m

o f n i t r o g e n a n a l y s i s

... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 27

F i g u r e 7

. F l o w d i a g r a m

o f p h o s p h o r u s a n a l y s i s

... ... ... ... ... ... ... ... ... ... ... ... ... ... 28
Fig ure 8

. Manifold assem bly for total dis solv ed p hosp horus m ethod

. . . . . . . . . . . . . . . . . . . . . . . . . . 29
Fig ure 9

. Manifold assem bly for total dis solv ed n itrog en m ethod

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Fig ure 1 0. Sc hem atic diag ram of the E xeter A naly tical, I nc. (EA I ) C E- 440 Elem ental A naly z er

. . . . 39
Fig ure 1 1. Manifold

assem bly for particulate phos phoru s m ethod

. . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Fig ure 1 2. Manifold

assem bly for particulate biog enic s ilica m ethod

. . . . . . . . . . . . . . . . . . . . . . . . . . 48
Fig ure 1 3. Pe rcentag e reco v ery of g lutam ic acid

as an in ternal sta ndard

for total dis solv ed

n i t r o g e n d i g e s t i o n / a n a l y s i s

... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... . . 64
Fig ure 1 4. Pe rcentag e reco v ery of g ly ceroph osph ate as an in ternal sta ndard for total dis solv ed p h o s p h o r u s d i g e s t i o n / a n a l y s i s

... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... . . 641


## I NTRO DUCTI ON

The follow ing pag es d ocum ent th e an aly tical m ethodo log ies pe rform ed b y the Nutrien t A naly tical S er v ic es L ab or at or

y a t t he U ni v er si ty

o f Ma ry la nd

C he sa pe ak e B io lo g ic al L ab or at or y ( C B L ). Th is

m an ua l i nc lu de s se ct io ns o n di ss ol v ed in or g an ic n ut ri en ts , d is so lv ed o rg an ic n ut ri en ts a nd

p ar ti cu la te
nu tr ie nt s. W it hi n ea ch o f th es e se ct io ns , s am pl e co ll ec ti on , s to ra g e, p re pa ra ti on a nd a na ly si s ar e di sc us se d. A f

in al s ec ti on

a dd re ss es d at a m an ag em en t a nd q ua li ty a ss ur an ce /q

ua li ty c on tr ol ( Q A /Q C ).
Many of the p rocedu res de scribed are u sed for the Mary land Mainstem po rtion o f the Che sapeak e B ay P r o gr a m.

I nstrum entation include s:

C T e c h n i c o n Au t o A n a l yze r I I,

C Tw o ch an ne l Te ch ni co n TrA A cs
- 80 0 N ut ri en t A na ly z er ,

C S him adz u TOC

- 5000 and 50 00 A Total Org anic C arbon A naly z ers,

C Tu rn er D es ig ns TD

- 70 0 F lu or om et er ,

C S hi m ad z u U V- 12 0- 02 S pe ct ro ph ot om et er ,

C E xeter A naly tical, I nc. CE

- 440 Elem ental A naly z er, and

C D ionex DX- 120 I on Chro m atog raph.

P C C om pu te rs w it h co m

pl et e sp re ad sh ee t p ac k ag es a re u se d fo r da ta r ed uc ti on a nd m an ag em en t.
Hig h q uality (18 .3 m eg ohm

- cm ) deio niz ed w ater is prov ided v ia 2 Barn stead NA NO pure I I sy stem s. The Barn stead sy stem

pro duces Ty

pe 1 R eag ent G rade water equal to or exceed ing stand ards e stablished by th e A m er ic an

S oc ie ty f or Te st in g a nd

Ma te ri al s. F ir st , w at er

is f il te re d th ro ug h a re v er se o sm os is

m embrane . Final prod uct wate r th en pa sse s th rou gh a ser ie s of f ive fi lt er s: one organi c col loi d, two

m ixed bed, on e org anic fre e, and one final 0 .2 : m filter. Throug hout this m anual, the term "deion iz ed wat er " re fe rs to 18.3 m egohm

- cm wat er and "f rozen" ref

er s to tempera tur e < - 20°C.

Tab le 1. P aram eters r outinely analyz ed a t the Nutrie nt A nalytical Services La bora tory a t C BL
D I S S O L VE D I

N O R G A N I C O R G A

N I C P A R TI C U L A TE

Am m onium

- Nitrog en Persulfa te N itrog en Carb on Nitrite- Nitrog en Persulfa te Ph osph orus Nitrog en Nitrite+N itrate- Nitrog en Disso lv ed C arbon Pho spho rus Pho sphate- Pho spho rus Total Susp ended So lids Silicate Total Volatile So lids Sulfate Biog enic S ilica C hl or id e C hl or op hy ll a N it ra te

- N it ro g en P ha eo pi g m en ts23



## DIS SOLVED I NOR GAN I C AN ALYTES

Instru mentation

All o f the dissolv ed in org anic n utrient p rocedu res, particulate phos phoru s an d b iog enic s ilica pro cedure s require the u se o f a seg m ented continu ous flow analy z er suc h as the Technicon Auto Ana ly z er I I or TrAAcs- 800. Sa m

ples a nd reag ents a re co ntinuo usly add ed in a specific seque nce along a path of g lass tubing and

m ixing coils. Within

the sy stem

of tubes, air b ubbles injected at precise interv als sw eep the walls of the tubin g and

prev ent d iffusion betw een success iv e sam ples. R eactions in the A utoA naly z er or TrAAcs- 800 do not d ev elop to co m pletion as in m anual m ethods , b ut reac h ide ntical sta g es o f de v el op m en t w it hi n ea ch s am pl e si nc e ev er

y s am pl e fo ll ow s th e sa m e pa th , ti m in g a nd e xp os ur e to specific reag ents.

The b as ic f

un ct io n of

e ac h co m

po ne nt o f th e se g m en te d co nt in uo us f lo w a na ly z er

is d is cu ss ed

b ri ef ly be lo w

. Th is e xp la na ti on

is s im il ar to th at o f S an bo rn a nd L ar ra nc e (1 97 2)

.
Au tomatic S ampler

At a tim ed in terv al, th e sam pler p robe alternately

draw s fluid from

a tray of discrete sam

ples a nd a w ash fluid rec eptacle. A fter each sam ple is draw n, the sa m

ple tray adv ances to the nex t sam ple p osition. A bubb le of air, w hich acts as a diffusion barrier, is a spirated into th e sam ple strea m

betw een sam

ple an d wash

. The ratio o f sam

ple- to- wash tim e an d the nu m ber o f sam

ples a naly z ed p er ho ur are controlled by t h e T r A A c s s o f

t w a r e , o r b y a c a m or t i me r

a s s e mb l y l o c a t e d

i n t h e t o p we l l o f

t h e Au t o A n a l yze r

I I

sam pler. C am s an d tim er asse m blies a re ad

j usted easily

to prov ide v arious sam

pling rates.

The w as h so lu ti on

s ep ar at es s uc ce ss iv e sa m pl es in th e sa m pl e st re am

a s in di ca te d on

th e g ra ph ic al r ec or d by alterna ting m inim a (w ash) a nd m axim a (sam ple). The sam

ple p robe is con nected to a stream div ider that d eliv ers ide ntical sa m ples s im ultaneo usly

to each m anifold v ia the pum p.
Proportion ing Pump

The p ro po rt io ni ng p um

p is a p er is ta lt ic

- ty

pe p um p th at c on ti nu ou sl y

d el iv er s ai r, r ea g en ts a nd s am pl es to the m anifold. P lastic pu m p tub es o f v arious diam eters are press ed b etwee n a series o f m ov ing rollers and a plate n. The m otion of the rollers along the tubes deliv ers a continu ous flow of sam ples a nd reag ents. The deliv ery

rate is dete rm ined by

the inside diam eter of the tu bes since the ro llers m ov e at a co nstant rate. These p um

p tub es are av ailable in

a larg e asso rtm ent o f deliv ery rates. The pum

p h olds a
m ax im um o f 28 tu be s an d ha s an

a ir b ar th at m ec ha ni ca ll y

m ea su re s an d in

ject s id en ti ca ll y

s iz ed a ir bu bb le s in to th e an al y ti ca l s tr ea m

. Th e pu m p tu be s de li v er in g r ea g en ts , a ir a nd s am pl es a re c on ne ct ed

to appro priate m anifolds.

Ma n ifol d

Each ana ly sis req

uires a m anifold specifically

des ig ned for the che m ical de term ination em ploy ed. The

m anifolds are co m

posed of a series of g lass co ils, in j ection fitting s an d h eating bath s arran g ed fo r the pr op er s eq ue nc e of r ea ct io ns le ad in g to c ol or d ev el op m en t. Th e g la ss c oi ls p er m it m ix in g o f th e sa m pl e and the rea g ents; a s tw o so lutions with different densities trav el arou nd each turn o f the m ixing coil, the de ns er s ol ut io n fa ll s th ro ug h th e le ss d en se o ne , c au si ng m ix in g a nd c re at io n of

a h om og en eo us m ix tu re4

of the tw o so lutions. The leng th of the co il determ ines th e am ount of tim e allow ed fo r che m ical reac tion betw een the ad dition of su ccessiv e reag ents. I njection fitting s for each of the reag ents a re plac ed betw een m ixing coils; thus , a sam ple en ters on e en d o f the m anifold, a reag ent is added , a nd m ixed; and then anothe r reag ent is added and m ixed. A fter the addition of all reag ents, and an a dequ ate reac tion ti m e, th e so lu ti on

f lo w s in to a c ol or im et er

.
Colo rimeter

The colorim eter m easures the ab sorption of m onoc hrom atic lig ht by the solution

in the flo w cell. Lig ht from a sing le sou rce p asses throug h tw o se parate but id entical in terference filters that pass lig ht w ithin a

narrow spe ctral ba nd. The lig ht the n p asses throug h the app ropriate flow cell an d is projected on to a photo tube. The photo tube g enerates an e lectrical sig nal in respo nse to the inten sity of the im ping ing lig ht. The ou tput fro m

each ph ototub e is a m easure of tran sm ittance a nd is con v erted e lectronically by the co lorim eter to a sig nal p roportion al to absorb ance. The relationsh ip be tween transm ittance a nd absorb ance is g iv en b y

the equatio n A = log 1/T; w here A = ab sorban ce an d T = transm ittance. The resulting sig nal is linear in abs orban ce an d d irectly

pro portion al to conce ntration. A s eac h sa m ple p asses th ro ug h th e fl ow c el l, th e si g na ls a re s en t t o a re co rd er

.
Reco rder

Resu lts of the an aly ses are con tinuou sly reco rded by

strip chart re corders and/or by com puter using an I BM c om patible DP 500 softwa re sy stem by La btronics I nc., or AA CE softw are sy stem by B ran a nd Lueb be. Eac h rec ord er can si m ult ane ous ly m oni tor two sep ar at e ana lyses . T he DP500 and AACE sy stem s can

each collec t and

ana ly z e da ta from

up to four d ifferent d etectors sim ultaneo usly

. The outp ut of the colo rim eter is propo rtional to abs orban ce an d stan dards of k now n co ncentratio n m

ust b e an aly z ed to relate absorb ance to co ncentratio n. The an alog sig nals c an b e co nv erted to abs orban ce v alues by

re fe rr in g to th e Tec hn ic on r ef er en ce c ur

v e an d th e st an da rd

c al ib ra ti on

c on tr ol

. Samplin g a nd Stora ge

C ollected water sam

ples a re filtered throug h Wha tm an G F/F filters (nom inal p ore siz e 0. 7 : m ), p laced in e it he r po ly

pr op y le ne b ot tl es o r di re ct ly in to 4 m l A ut oA na ly z er

c up s an d fr oz en

. S am

pl es f or s il ic at e are treated

in the sa m e m anner but a re refrig erated at 4° C. A ll sam ples a re rou tinely

ana ly z ed w ithin 2 8 day s.
Operatin g Proced ures

T h e f o l l o w i n g d e s c r i b e s s t e p -by -s t e p o p e r a t i n g p r o c e d u r e s f o r t h e Au t o A n a l yze r

I I s ys t e m.

1. Colo rimeter - T urn power on and allow 10 m inu tes f or w arm -up . Set sta

nd ard calibration setting f or desired det er m ina tion.

2. Recorder (

or Comp uter) - T urn pow er on a nd a llow 10 m inute s f

or r ec orde r w ar

m -up. Che ck

re cor der pa per s upply . I f usi ng c om

pute r f or da ta c olle ct ion, loa d s of

tw ar e and se le ct

a ppropr ia te s am

ple m et hod a nd s am

ple ta ble . R ef er to L abt ronic s Inc . D P500 and B ra n a nd L uebbe A A CE us er s’ m anua ls f or de sc ript ions of

s y st em ope ra tion.

3. Sa mpler Water Re

serv oirs - Chec k a

nd f ill th e deioni zed w ater re serv oirs.

4. Pump - Conne ct

pum p tube s and at ta ch pla te n to pum p. S ta rt pum p w ith dei oniz ed w at er

f low ing thr oug h the s y st em

. Che ck f or l ea k s in t ubes a t c onnec tions a nd f or a re g ula r bubbl e pat te rn i n the m ani f old.5

5. Recorder

- Start rec ord er. Pap er shoul d begin to m ove.

6. Colo rimeter

- Che ck ZE RO a nd FU L L SC A L E k nob. Z ER O si m

ula te s a ze ro out

put s o that Z ER O adj ustm ent (s cr ew driv er ) of

the re cor der

c an be m ade . S et

k nob to NO RMA L a nd e st abl is h a ba se line w ith dei oniz ed w at er us ing the BA SEL IN E C ON T RO L a djus tm ent k nob a nd a s ta ndar d c al ibra

tion (ST D CA L ) s et ting of 1.0; i. e. , w

her ei n the f

ull sc al e abs orba nce is 1.00.

7. A llow

re ag ent s to pum

p thr oug h the s y st em

a nd note a ny

ri se in bas el ine a nd re adj ust the ba se line to ze ro. Re f er to this ris e as the RE A G EN T BL A NK (a t a ST D CA L of

1.0)

.
8

. An e x t r e me l y wi d e r a n g e o f n u t r i e n t c o n c e n t r a t i o n s fo u n d i n C h e s a p e a k e B a y wa t e r s , b o t h t e mp o r a l l y a n d s p a t i a l l y, requi res use of a

standard curve cove ring a larg e range and th at cove rs a f ew STD CA L cont rol settin gs.

9

. Res et zero ba sel in e at t h e STD C AL co n tr o l se tt in g no rmall y us ed for t h at de te rmin at io n (e. g., pa rt ic u la te ph o sp h o ru s S TD CA L of 4.0) . N ex

t, sw itc h the ST D CA L s et ting to 1.0. T her e shoul d be no de f le ct ion of the pe n a t z er o ba se line . N ote pea k he ig hts of

s ta ndar ds at the v ar ious ST D CA L s et ting s al ong w ith the ST D CA L s et ting s. T his a llow s the ope ra tor to
use ST D CA L s et ting s in t he ra ng e of 1 t o 4 ( f or t his e x am ple ) i n a nal y zi ng s ta

ndar ds and sa

m ple s tha t ot

her w is e w ould have gone off

sca le. Intersperse s tand ards in th e run af ter appro xim ately

ev ery 20 sam ples, inclu din g a

standard analy zed at e ac h ST D CA L s et ting e m ploy ed during the pre ce ding 40 s am ple s. A v is ual c om

par is on w ith the da y ' s init ia l s ta ndar d

cur v e shoul d indi ca

te no g re at er

v ar ia nce tha n 5% of the pe ak he ig ht ( e. g ., i niti al s ta

ndar d pe ak

he ig ht 60. 0; s ubse quent standard s ac ceptable in th e range of 57 .0 to 63. 0). If the variance ex cee ds 5%, iden tify the source of the pro blem , correct and re -a nal y ze a f f ec te d s am ple s. A djus t ba se line a f

te r a pprox im at el y

e v er y 20 s am ple s. I f a n a djus tm ent

of m ore tha n 1

un it is required , iden tify

the source of the pro blem , correct and re-analy ze af f ected sa m ples.

10. A t c om

ple tion of the run, re m ov e line s f

rom re ag ent s and pla ce tube s in de ioniz ed w at er

.
11. Sh ut-d own - T urn off recorder. W ash sy stem

w ith 1 N hy dro chlo ric ac id for 15 m inu tes, f ollo w ed by

a 15 m inu te w ash

w ith dei oniz ed w at er . T urn of f pum p, r el ea se propor

tioni ng plate n a nd loos en pum p tube s. T urn of

f c olori m et er

.
Gla ssw are

G la ss w ar e ar e cl ea ne d ap pr op ri at el y f or th e de te rm in at io n. Mo st

a re a ci d- w as he d w it h 10 % h y dr oc hl or ic

ac id f ol lo w ed b y n um er ou s ri ns es w it h de io ni z ed w at er

. O th er

c le an in g p ro to co ls a re li st ed in th ei r specific sections.

Calib ration an d S tand ardiz ation

Please refer to each specific determ ination for the app ropriate STD C AL con trol setting and for the standard con centration s use d.

The STD CA L c ontrol setting locate d o n the colo rim eter allow s the operato r to adjust the ele ctrical outpu t to the co ncentratio n ran g e of the sa m ples. Ex trem ely

low v alues ( : g/L) re qui re high STD CAL setting s (hig h se nsitiv ity ) w hereas hig h v alues (m g /L) req uire lo wer STD C AL setting s (low er
se ns it iv it y ).

Con centration s of nutrients are ca lculated from the linear reg ression of the stand ard c oncen tration (indepe nden t v ariable) a g ainst th e co rrespon ding pea k heig ht (de pend ent v ariable). A ll stand ards

an al y z ed a t a p ar ti cu la r S TD C A L s et ti ng a re in cl ud ed in th e re g re ss io n fo r th at

s et o f ca lc ul at io ns

. O nl y
sam ples w hose peak

heig hts w ere m easured

at th at ind iv idual STD C AL setting are calculated from that reg ression. I f a

bro ad ra ng e of sam

ple co ncentratio ns req

uires th at m ore th an o ne S TD C AL setting be

use d thr oughout the co urs e of a r un, th en a se par at e re gress ion m ust be em plo yed for ea ch ST D CAL setting

. For exam

ple, peak heig hts o btained

from stand ards re ad a t STD CA L 9 .0 are u sed to ob tain the linear reg ression for calc ulating o nly the conce ntrations of sam ples w hose peak

heig hts w ere read

at6

S TD C A L 9

. 0. L ik ew is e, p ea k h ei g ht s ob ta in ed

f ro m s ta nd ar ds r ea d at

S TD C A L 2

. 0 ar e us ed

to o bt ai n the line ar reg ression for calc ulating on ly the conce ntrations of sam ples w hose peak

heig hts w ere read

at STD C AL 2. 0.

P ea k h ei g ht s ar e re ad

m an ua ll y f ro m th e st ri p ch ar t o r au to m at ic al ly b y th e D P 50 0 or

A A C E s of

tw ar e sy st em s. O pe ra to r v ig il an ce is n ec es sa ry th ro ug ho ut

th e ru n to e ns ur e th at

a ll p ea k s in di ca te s te ad

y s ta te
co nd it io ns f

or th e re ac ti on

f or e ac h sa m pl e. I f st ea dy

s ta te c on di ti on s ar e no t o bt ai ne d, th e sa m pl es a re
re- analy z ed.

Stock stand ards a re prep ared with prim ary stand ard g rade chem icals o f each nu trient in deioniz ed w ater. A s a g en er al r

ul e, s to ck s ol ut io ns a re m ad e ev er

y 6 m on th s an d th e pr ep ar at io n da te lo g g ed

. S ec on da ry st an da rd s, w he re a

pp ro pr ia te , a re p re pa re d w it h de io ni z ed w at er

. Wo rk in g s ta nd ar ds a re p re pa re d da il y with deioniz ed w ater or the ap propriate m atrix as describe d b y the specific determ ination m ethod and shou ld en com

pass the ran g e of the sa m ples.

All a naly sis do cum ents a re k ept ele ctronically and in notebo ok s. A copy

is a lso g iv en to the inv estig ator or g ranting ag ency

. I nform ation prov ided include s:

C n am e of the m ethod;

C c ollection date;

C s ource of sam

ples;

C a na ly st ;

C a naly sis da te;

C s am pl e nu m be r;

C s am ple co ncentratio n;

C r es ul ts o f du

pl ic at e an al y se s;

C re sults o f spik e an aly ses; an d.

C re sults o f ana ly ses o f com m ercially prep ared standard

referen ce m aterials.

Litera ture Cited

Sanb orn, H . a nd J . L arrance. 19 72. A n o peration s m anual of the A utoA naly z er for seaw ater nu trient analy sis. NO AA /NMFS . S eattle, Washing ton. 44pp

.7 

## O RTH O PH O SPH ATE Re v i s e d 2/ 2004

Am m onium m oly bdate and antim ony

po tassium

tartrate react in an acid m edium w ith dilu te solu tions of phos phoru s to form

an antim ony
- phos pho- m oly bdate com

plex whic h is reduce d to an in tensely blue

colored com plex by asco rbic ac id. C olor is propo rtional to ph osph orus conce ntration.

Method ology

Technicon I ndus trial Method N o. 155- 71W/Tentativ e. 19 73. Techn icon I ndus trial Sy stem s. Tarry town , New Y ork , 1 0591

.
US EPA

. 1979

. Method N o. 365. 1 in Meth ods for ch em ical an aly sis of water and waste s. U nited States E nv ir on m en ta l P ro te ct io n A g en cy , O ff ic e of R es ea rc h an d D ev el op m en t. C in ci nn at i, O hi o. R ep or t No. EP A- 600/4

- 79- 020 March 1 979. 4 60pp

.
F r o e l i c h , P. N

. a n d

M

. E

. Q

. Pi l s o n

. 19 7 8

. Sys t e ma t i c a b s o r b a n c e e r r o r

wi t h T e c h n i c o n Au t o A n a l yze r I I colorim eter. Water R es. 12:59 9- 603.

Instru mentation

T e c h n i c o n Au t o A n a l yze r I I

Ma n ifol d A ss em b ly

Figu re 1

. Manifold assem bly fo r orth opho spha te m ethod.8
Spe cif ication s

Stan dard C alibra tion Setting s: 9 .0, 6. 0, 2.0 , 1 .0 Damp : N orm al S am p li n g R at e: 40 /h ou r, 9 :1 s am pl e/ w as h ra ti o Filter: 8 80 nm Pho to tu b e: 19 9- B02 1- 04 Flo w ce ll : 50 mm R el at iv e A b so rb an ce ( 1 8 μM P): ~.85

In te rfer en ce s: Silicon at a lev el of 100 : M Si causes an in terference equiv alent to app roxim ately 0. 04 : M P. H ig h iron

m ay cau se p recipitation (i.e., loss) of p hosp horus. H ig h sa m ple tu rbidity

and colo r m ay interfere.

R ea ge n ts

Deio niz ed Water Dilue nt

3 2 1 1 3 Sod ium do decy l sulfate, (SD S)[C H (CH ) OS O Na;

m .w .=28 8.3 8; ph osph ate # 0.0 001 %] 0.0 5 g D e i o n i ze d

wa t e r u p t o 5 0 0 mL

A dd 0.05g of

s odium dode cy l s ulf

at e to a pprox im at el y

500 m L of de ioniz ed w at er . M ix w el l.

Sulfuric A cid S olution , 4 .9 N

2 4 S u l f u r i c a c i d ( H S O ) , c o n c e n t r a t e d

( s p

. gr

. 1

. 8 4 ) 1 3 6 mL D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

T o a 1000 m L v olum et

ric f la sk , a dd 136 m

L of c once ntra

te d s ulf uric a ci d to appr ox im at el y 800 m L of de ioniz ed w at er

w hile cool ing (c old w at er

ba th). A f

te r t he sol ution is c oole d, di lute to 1000 m L w ith dei oniz ed w at er

.
Ammoniu m Molybd ate S olution

4 6 7 2 4 2 A m m on iu m m ol y bd at e [( N H ) Mo O @ 4 H O] 40 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

In a 1000 m L v olum et

ric f la sk , di ss olv e 40 g of

a m m onium m oly bdat e in 800 m L of

de ioniz ed w at er . D ilut e to 1000 m L w ith dei oniz ed w at er . S tore in pla st ic bott le a w ay f rom dir ec t s unlig ht.

Asco rbic Acid So lution

686 Asco rbic ac id (C H O ) 18 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

In a 100 0 m L volume tric f lask, dissolve 18 g of a scorbic acid in 8 00 m L of deionized wa ter. Dilute to 10 00 m L w ith d eion ized

w ater a nd dispen se - 40 m L aliqu ots in to clean po ly bo ttles and fre eze . T haw overnight in th e ref

rigerator b ef

ore use.

An timony Potassium T artrate Solu tion

446 2 A nt im on y

p ot as si um

ta rt ra te [ K(S bO )C H O @ 1⁄2 H O] 3 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL9

In a 1000 m L v olum et

ric f la sk , di ss olv e 3 g of

a ntim ony

pota ss ium

ta rtr at e in 800 m L de ioniz ed w at er . D ilut e to 1000 m L w ith dei oniz ed w at er

.

Wo rk in g R ea ge n ts

R ea ge n t A S u l f u r i c Ac i d So l u t i o n , 4

. 9 N 5 0 mL A mmo n i u m M o l yb d a t e So l u t i o n 1 5 mL A n t i mo n y P o t a s s i u m T a r t r a t e So l u t i o n 5 mL

3 2 1 1 3 Sod ium do decy l sulfate, (SD S) [C H (CH ) OS O Na;

m .w . 2 88. 38; p hosp hate # 0.0 001%] 0.1 5 g

R ea ge n t B A s c o r b i c Ac i d So l u t i o n 3 0 mL SD S 0.0 5 g

Stan dard s

Stock Orthop hosp hate Stan dard , 1 2,0 00 : M P ot as si um

p ho sp ha te , m on ob as ic

2 4 (KH PO ), d

ried at 45° C 1.6 32 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

3 C h l o r o f o r m ( C H C l ) 1 mL

In a 1000 m L v olum et

ric f la sk , di ss olv e 1.632 g of

pota ss ium

phos phat e in
- 800 m L de ioniz ed w at er . D ilut e to 1000 m L w ith dei oniz ed w at er

the n a dd 1 m

L of c hlorof orm a s a pre se rv at iv e (1 m

L c onta ins 12 : m ole s P).

Seco nda ry Orth oph osph ate S tand ard S t o c k Or t h o p h o s p h a t e St a n d a r d 1 mL D e i o n i ze d

wa t e r u p t o 1 0 0 mL

In a 100 m L v olum et

ric f la sk , di lute 1 m

L of Stoc k O rthophos phat e Sta

ndar d to 100 m L w ith dei oniz ed w at er (1 m L c onta ins 0.12 : m ole s P).

Workin g Ortho pho sph ate S tand ards

Di lute 0.1, 0.25, 0. 5, 1.0, 1.5, 2.5, 5 and 10 m L of Se conda ry O rthophos phat e Sta

ndar d to 100 m L w ith dei oniz ed w at er to y ie ld

conc ent ra tions of 0.12 : M P (0.00372 m g /L ), 0.3 : M P (0.0093 m g /L ), 0.6 : M P (0.0186 m g /L ), 1.2 : M P (0.0372 m g /L ), 1.8 : M P (0.0558 m g /L ), 3.0 : M P (0.093 m g /L ), 6.0 : M P (0.186 m g /L ) a nd 12.0 : M P ( 0

. 3 7 2 mg / L) , r e s p e c t i v e l y.

Refractive I nde x C orrectio n

Wo rk in g R ea ge n ts

R ef ra ct iv e R ea ge n t A S u l f u r i c Ac i d So l u t i o n , 4

. 9 N 5 0 mL D e i o n i ze d

wa t e r 2 0 mL SD S 0.1 5 g10

R ef ra ct iv e R ea ge n t B A s c o r b i c Ac i d So l u t i o n 3 0 mL SD S 0.0 5 g

Procedur e

1. Obt ai n pe ak

he ig hts f or a ll sa m ple s and st anda

rds w ith Re f

ra ct iv e Re ag ent

A a nd Re ag ent

B in pl ac e of

R ea g ent A a nd Re ag ent B of the ort hophospha

te de te rm ina tion. Pea

k he ig hts f or t he re f ra ct iv e inde x c orr ec tion m ust be obta ine d a t t

he

sa m e ST D CA L s et ting a nd on t he sa m e col orim et er

a s the c orr es ponding s am ple s and st anda

rds (F roe lic h a nd Pilson 1978).

2. Subtr ac t t

he re f ra ct iv e inde x pe ak

he ig hts f rom the he ig hts obta ine d f

or t he orthophos phat e det er

m ina tion.

3. Calculate the reg ression equ ation using the corrected standard p eak heights.

4. Calculate the concentratio n of sa m ples f rom the regre ssion equ ation using the corrected sam ple peak heig hts.

When a larg e da ta set has been am assed in w hich each sam ple' s salinity

is k now n, a reg ression for the refractiv e ind ex c orrection on the co lorim eter can be calculated

. For each sam ple, the ap parent
or th op ho sp ha te c on ce nt ra ti on d ue to r ef ra ct iv e in de x is c al cu la te d fr om it s pe ak h ei g ht o bt ai ne d w it h R ef ra ct iv e R ea g en t A a nd

R ea g en t B , a nd

th e re g re ss io n of

o rt ho ph os ph at e st an da rd s ob ta in ed w it h

or th op ho sp ha te R ea g en t A a nd R ea g en t B

. Th e sa li ni ty is e nt er ed

a s th e in de pe nd en t v ar ia bl e an d it s appare nt orth opho sphate conce ntration due to its refractiv e ind ex in that colorim eter is entered as the de pe nd en t v ar ia

bl e. Th e re su lt in g r eg re ss io n al lo w s th e op er at or to s ub tr ac t a n ap pa re nt

o rt ho ph os ph at e
conce ntration whe n the salinity is k now n, as lon g as other m atrix effec ts are not p resent; th us, the operato r is n ot req

uired to ob tain the refractiv e ind ex p eak heig hts fo r all sa m

ples w hen a larg e da ta set ha s be e n f

ou nd to yie l d c on s i s t e nt or t ho ph os ph a t e co nc e nt r a t i on s du e to sa l i ni t y.11


## A MM ONIU M Re v i s e d 2/ 2004

D et er m in at io n of

a m m on iu m is b y th e B er th el ot

R ea ct io n in w hi ch a b lu e- co lo re d co m po un d si m il ar

to indop heno l form s w hen a so lution of am m onium salt is add ed to sod ium

ph enox ide, follow ed b y

the ad di ti on o f so di um

h y po ch lo ri te

. The a dd it io n of a p ot as si um

s od iu m ta rt ra te a nd

s od iu m c it ra te
s o l u t i o n p r e ve n t s p r e c i p i t a t i o n o f h yd r o x i d e s o f

c a l c i u m an d

mag n e s i u m.

Method ology

Technicon I ndus trial Method N o. 804- 86T. Aug ust 1 986. Techn icon I ndus trial Sy stem s. Tarry town , New Y ork , 1 0591

.
Ker ou el , R

. a nd

A

. A m in ot

. 1 98 7. P ro cé du re o pt im is ée h or s- co nt am in at io ns p ou r l’ an al y z e de s él ém en ts nutritifs d issous dans l’eau d e m er. Mar. E nv iron. Res. 22 :19- 32.

Instru mentation

Technicon TrAAcs- 800 Nutrien t A naly z er

Ma n ifol d A ss em b ly

Figu re 2

. Manifold assem bly fo r am mon ium meth od.12
Spe cif ication s

G ai n : 40 - 45 Bas el in e: 32 - 38 S am p li n g R at e: 60 /hour Filter: 6 30 nm Flo w ce ll : 5 0 mm Rela tive A bsor ban ce (1 2 : M N ): 0 .22 - 0. 24

C on ta min at io n : Am

m onium determ ination is hig hly

susc eptible to sk in an d atm osph eric co ntam ination (Kerouel an d A m inot 1 987). R ea ge n ts
Comp lex ing Rea gent

446 2 Sod ium po tassium

tartrate (KNaC H O @ 4 H O) 39 g

2 2 2 Sod ium citrate [HO C(C OO Na)(C H OO Na) @ 2 H O] 28 g

2 4 Sulfuric acid (H SO ), c oncen trated (sp . g r. 1 .84 ) as req uired D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL B r i j -35 , 3 0 % 1 mL

In a 1000 m L be ak er , di ss olv e 39 g of pota ss ium

s odium ta rtr at e and 28 g of

s odium c itr at e in 950 m L of

de ioniz ed w at er

. A djus t t he pH of the s oluti on to 5.0 us ing c once ntra te d s ulf uric a ci d. D ilut e to 1000 m L w ith dei oniz ed w at er in a v olum et

ric fl a s k

. Ad d 1 mL o f 3 0 % B r i j

- 3 5 a n d mi x t h o r o u g h l y. S t o r e i n a l i g h t r e s i s t a n t c o n t a i n e r

a n d r e fr i g e r a t e

. M a k e fr e s h we e k l y.

Alk aline Phenol Rea gent

6 5 P h e n o l ( C H O H ) , 8 8 % 2 3

. 6 mL Sod ium hy droxid e (N aOH ), 5 0% (w /w) 18 g D e i o n i ze d

wa t e r u p t o 2 5 0 mL

In a 250 m L v olum et

ric f la sk , s low ly

a dd 23.6 m

L of 88% phe nol t o - 150 m L of de ioniz ed w at er . W ei g h out ex ac tly 18 g of 50% (w /w ) s odium

hy drox ide , t hen w

hile in an ic e bat

h, s low ly a dd the s odium

hy drox ide to the phe nol/w at er s oluti on. D ilut e to 2 5 0 mL wi t h d e i o n i z e d wa t e r

. S t o r e i n a l i g h t r e s i s t a n t gl a s s c o n t a i n e r a n d r e fr i g e r a t e

. M a k e fr e s h we e k l y.

WAR N I N G: P h en o l i s e xt r eme l y da n ge r o u s a n d sh o u l d b e h an d l ed ac co r d i n gl y. P r ep ar e r ea ge n t fre sh wee kl y un d er a fu me ho od and w earing protective gloves.

Sod ium Hypo chlorite , 1 % S o d i u m hy p o c h l o r i t e ( C L O R O X Ul t r a - 6

. 0 % ) 4 4 mL D e i o n i ze d

wa t e r u p t o 2 0 0 mL

In a 250 m L v olum et

ric f la sk , di lute 44 m

L of a ny c om m er ci al ly a v ai la ble ble ac h c onta ining 6.0% s odium hy pochl orit e to 250 mL w i t h d e i o n i z e d wa t e r

. M a k e fr e s h e v e r y o t h e r d a y.

Sod ium N itropru sside (Sod ium N itroferricyanid e), 0.0 5%

2 5 2 Sodi um nit rop rus si de [Na Fe( CN) NO @ 2 H O] 0.5 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

In a 1000 m L v olum et

ric f la sk , di ss olv e 0.5 g of

s odium nit ropr uss ide in 900 m L of

de ioniz ed w at er . D ilut e to 1000 m L w ith dei oniz ed w at er . S tore in a lig ht r es is ta nt c onta ine r.13

Stan dard s

Stock A mmonium S tand ard, 1, 500 : M

4 2 4 Am m onium sulfate [ (N H ) SO ], dried at 45 ° C 0.1 00 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

3 C h l o r o f o r m ( C H C l ) 1 mL

In a 1000 m L v olum et

ric f la sk , di ss olv e 0.1 g of

a m m onium s ulf at e in
- 900 m L of

de ioniz ed w at er . D ilut e to 1000 m L w ith dei oniz ed w at er . A dd 1 m L c hlorof orm to ac t a s a pre se rv at iv e (1 m L c onta ins 1.5 : m ole s N)

.
Workin g A mmonium S tand ards

Di lute 0.1, 0.2, 0. 4, 0.6 and 0.8 m L of Stoc k A m m onium Sta ndar d to 100 m L w ith dei oniz ed w at er to y ie ld c once ntra

tions of 1.5 : M N (0. 021 m g /L ), 3 : M N (0. 042 m g /L ), 6 : M N (0. 084 m g /L ), 9 : M N (0. 126 m g /L ) a nd 12 : M N (0. 168 m g /L ),

r e s p e c t i v e l y.

Procedur e

1

. C h e c k

t h e l e v e l o f a l l r e a g e n t s a t s t a r t

- u p t o e n s u r e a d e q u a t e s u p p l y.

2

. P l ac e a l l l i n es i n t o t h ei r re sp ec t i ve co n t ai n er s a n d st ar t t h e p r o p o r t i o n i n g p u mp

. All o w - 10 m inu tes e qu ilib ration tim e.

3. P lace a sa line pri m er (artif icial se aw ater) bef

ore the actual sam

ples to avoid an abn orma l peak f or th e f irst sam ple. P lace a dei oniz ed w at er

c up be f ore a n int er sa

m ple c al ibra

nt.

A ty pical tray prot ocol appears a s f

ollo w s:
P, 5C, H @2, 2L @6, A @119, 20S> 7, W @120, 2I@ 3, S@ 119, 20S> 26,...

.

w her e: P = prim er (100% FS st anda

rd) , C = c al ibra

nt, H = 100% FS st anda

rd, L = 20% FS st anda

rd, A = a rti f ic ia l s ea w at er , S = sam ples, W = de ioniz ed w at er , a

nd I = i nte rs am

ple c al ibra

nt.

4. A t shut -do w

n, rem ove a ll reag ent lin es f rom their respective cont ainers ex clud ing the sam pler solu tion line and p lace them in de ioniz ed w at er

c onta ining 1 m

L /L of 30% Br ij- 35.

5. A f te r 5 m inute s, a llow

a ll line s to pum

p dry . S top t

he pum p a nd re le as e the plate n.

NO T ES:

1

. The gain set ti n g of 4 0 - 4 5 sh o u ld y ie ld pe aks o f
- 70 - 80 li nes w ith a 12 : M N s ta ndar d.

2. Re ag ent

bla nk s appe ar

to be quite v ar ia

ble . I f the re ag ent

bla nk is > 10 li nes w ith the a

ppropr ia te g ai n s et ting (40 - 45) a nd bas el ine s et ting (32 - 38) , t hen pre par e f re sh re ag ent

.
3. Cl ose a tte ntion shoul d be pa id t o pre v ent c onta m ina tion of

s am ple s. Sm ok ing a nd the us e of a m m onium -c onta ining cl ea ning s oluti ons shoul d be prohi bite d w ithi n the a ir- handl ing li m its s urr ounding the la bora tory . S am

ple bott le s shoul d be stored in a c lean environme nt and onl y open ed w

hen n ece ssary . Sam

ples shou ld n eve r be pip etted by m ou th. Sam

ples sh o u ld be an al y zed imm ed ia te ly up o n op en in g.14


## N ITRITE Re v i s e d 2/ 2004

Nit ri te rea ct s unde r ac idi c con dit ion s wit h sul fa nil amide to f orm a diazo com

poun d tha t cou ple s wit h N1- naph thy lethy lenediam ine d ihy droch loride to form a reddish

- purple az o d

y e m easured

at 5 20 nm

..
Method ology

Technicon I ndus trial Method N o. 818- 87T. Febru ary

19 87. Techn icon I ndus trial Sy stem s. Tarry town , New Y ork , 1 0591

.
Instru mentation

Technicon TrAAcs- 800 Nutrien t A naly z er

Ma n ifol d A ss em b ly

Figu re 3

. Manifold assem bly fo r nitrite meth od.
Spe cif ication s

G ai n : 53 Bas el in e: 36 - 37 S am p li n g R at e: 60 /hour Filt er : 52 0 n m Flo w ce ll : 50 mm Rela tive A bsor ban ce (3 : M N ):
- 0.1 6515

R ea ge n ts

Sulfanilamide Solu tion

2 6 4 2 2 Sulfan ilam ide (4 N H C H SO NH ) 2.5 g H yd r o c h l o r i c a c i d

( H C l ) , c o n c e n t r a t e d 2 5 mL D e i o n i ze d

wa t e r u p t o 5 0 0 mL Brij- 35, 30 % few drops

In a 500 m L v olum et

ric f la sk , a dd 2.5 g of

s ulf ani la m ide a nd 25 m

L of c once ntra

te d hy droc hlori c ac id t o - 30 0 m L of deionized

w at er . S w irl to dis sol v e and dilut e to 500 m L w ith dei oniz ed w at er . A dd a f ew drops of Br ij- 35, 30% a nd m ix thor oug hly

. Sto re in light resistant contain er. Re f rigerate w hen n ot in use.

N-1-N aph thyleth ylened iamine D ihyd roch loride Solu tion N- 1- naph thy lethy lenediam ine d ihy droch loride

12 14 2 ( CHN @ 2HC l) 0.2 5 g D e i o n i ze d

wa t e r u p t o 5 0 0 mL Brij- 35, 30 % few drops

In a 500 m L v olum et

ric f la sk , di ss olv e 0.25 g of

N -1- napht

hy le thy le nedi am ine dihy droc hlori de in
- 300 m L of de ioniz ed w at er

. Di lute to 500 m L w ith dei oniz ed w at er . A dd a f ew drops of Br ij- 35, 30% a nd m ix thor oug hly . S tore in a lig ht r es is ta nt cont ainer. Ref

rigerate w hen n ot in use.

Stan dard s

Stock N itrite Sta nda rd, 5, 000 : M

2 Sod ium nitrite (NaN O ), d

ried at 45° C 0.3 45 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

3 C h l o r o f o r m ( C H C l ) 1 mL

In a 1000 m L v olum et

ric f la sk , di ss olv e 0.345 g of

s odium nit rit e in
- 600 m L of

de ioniz ed w at er . D ilut e to 1000 m L w ith dei oniz ed w at er . A dd 1 m L of c hlorof orm a s a pre se rv at iv e (1 m

L c onta ins 5 : m ole s N)

.
Seco nda ry N itrite Sta nda rd S t o c k Ni t r i t e St a n d a r d 0

. 8 mL D e i o n i ze d

wa t e r u p t o 1 0 0 mL

In a 100 m L v olum et

ric f la sk , di lute 0.8 m

L of Stoc k N itr ite Sta

ndar d to 100 m L w ith dei oniz ed w at er to y ie ld a c once ntra

tion of 40 : M N.

Workin g N itrite Sta nda rds

Di lute 0.5, 1, 2.5, 5 a

nd 7.5 m L of Se conda ry N itr ite Sta

ndar d to 100 m L w ith dei oniz ed w at er to y ie ld c once ntra

tions of 0.2 : M N (0.0028 m g /L ), 0.4 : M N (0. 0056 m g /L ), 1 : M N (0. 014 m g /L ), 2 : M N (0. 028 m g /L ) a nd 3 : M N (0. 042 m g /L ),

r e s p e c t i v e l y.16

Procedur e

Run this analy sis concurrent ly w ith am m on ium, using a sy

nth etic se aw ater c up prio r to a sa m ple set and a deioni zed w ater cup prior to an inte rs am

ple c al ibra

nt ( Se e A m

m onium m et hodolog y ). T he 3-w ay

s tre am div ide r ( PT 2 conne ct or, P/N 116- B000- 01 ) used to spl it the sam

ple is the appro priate size f or th e pum

p tu bes used. Stream

dividers w ith sm aller ports create gas b u b b le s with th e rap id m o vement of sampl e th ro u gh th e fitt in g.

NO T ES:

1

. A gai n set ti n g of 5 3 sh o u ld y ie ld pe aks o f
- 80 li nes us ing a 3 : M N s ta

ndar d.

2

. Th i s i s a

ve r y s t e a d y a n a l ys i s wi t h a s mo o t h b a s e l i n e , we l l d e fi n e d p e a k s , mi n i ma l b a s e l i n e c o r r e c t i o n a n d mi n i ma l c a r r yov er c orr ec tion.17


## N IT RI TE + 

## N ITRATE Re v i s e d 2/ 2004

Filtered sam

ples a re pa ssed throug h a g ranulated cop per- cadm ium

colu m n to reduce nitrate to nitrite. The nitrite (o rig inally

prese nt plu s redu ced nitrate) the n is determ ined by diaz otiz ing w ith su lfanilam ide

an d co up li ng w it h N

- 1- na ph th y le th y le ne di am in e di hy dr oc hl or id e to f or m a c ol or ed a z o dy e. N it ra te
co nc en tr at io n is o bt ai ne d by s ub tr ac ti ng th e co rr es po nd in g n it ri te v al ue f ro m th e ni tr it e + ni tr at e
conce ntration.

Method ology

Technicon I ndus trial Method N o. 158- 71 W/A† Tentativ e. 19 77. Techn icon I ndus trial Sy stem s. Tarryto wn, N ew York , 1 0591

.
US EPA

. 1979

. Method No. 35 3.2 in Meth ods for ch em ical an aly sis of water and waste s. U nited States E nv ir on m en ta l P ro te ct io n A g en cy , O ff ic e of R es ea rc h an d D ev el op m en t. C in ci nn at i, O hi o. R ep or t No. EP A- 600/4

- 79- 020 March 1 979. 4 60pp

.
Instru mentation

T e c h n i c o n Au t o A n a l yze r I I

Ma n ifol d A ss em b ly

Figu re 4

. Manifold assem bly fo r nitrite + n itrate m ethod.18
Spe cif ication s

S ta n d ar d C al ib ra ti on S et ti n gs : OR N/G RN sam

ple tu be: 3 .0, 1. 0 OR N/Y EL sam

ple tu be: 2.0 , 1 .0 BL K/BLK sam

ple tu be: 9.0 , 6 .0, 2. 0, 1.0 Dilution loop : 3. 0, 1.0 D am p : Norm al S am p li n g R at e: 4 0/ ho ur , 9 :1 s am pl e/ w as h ra ti o Filt er : 550 nm Phototub e: 1 99- B02 1- 01 Flo w ce ll : 5 0 mm

3 R el at iv e A b so rb an ce ( 20 μ M N O -N ; B LK/BL K sam ple tu be): ~. 55 -

In te rfer en ce s: Me t a l io ns m a y in t e r f e r e if pr e s e nt in su f

f i c i e nt co nc e nt r a t i on s . T he pr e s e nc e of la r ge
conce ntrations of su lfide an d/or s ulfate g reatly redu ce the sen sitiv ity

of the co pper- cadm ium

colu m n.

R ea ge n ts

Alk aline Water Sod ium hy droxid e (N aOH , p ellets) 0.2 0 ± 0 .02 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

A dd 0.20 g of

s odium hy drox ide pe lle ts to 1000 m L of de ioniz ed w at er

. Cop per Sulfate R eagen t, 2 %

4 2 Cop per su lfate (C uSO @ 5 H O) 2 g D e i o n i ze d

wa t e r u p t o 1 0 0 mL

In a 100 m L v olum et

ric f la sk , di ss olv e 2 g of

c opper s ulf at e in
- 80 m L of deionized wa ter. Dilute to 10 0 m L w ith d eion ized

w at er

.
Ammoniu m Ch loride Rea gent

4 Am m onium chlo ride (N H Cl) 10 g A l ka l i n e W a t e r u p t o 1 0 0 0 mL Cop per S ulfate R eag ent, 2% 6 d rops

In a 1000 m L v olum et

ric f la sk , di ss olv e 10 g of

a m m onium c hlori de in
- 800 m L of

A lk al ine W at er

a nd dil ute to 1000 m L w ith A lk al ine W at er . A

tta in a pH ba la nce of 8.5. A dd 6 dr ops of

2% Coppe r S ulf

at e Re ag ent

.
Colo r R eagen t

6822 Sulfan ilam ide (C H N O S) 20 g

3 4 P h o s p h o r i c a c i d

( H P O ) , c o n c e n t r a t e d

( 8 0 % ) 2 0 0 mL N- 1- naph thy lethy lenediam ine d ihy droch loride

12 14 2 ( CHN @ 2HC l) 1 g D e i o n i ze d

wa t e r u p t o 2 0 0 0 mL B r i j -35 , 3 0 % 1 mL

In a 2000 m L v olum et

ric f la sk , a dd 200 m

L c once ntra

te d phos phoric a ci d a nd 20 g of s ulf ani la m ide to - 15 00 m L deio nized19

w at er . D is sol v e com

ple te ly . A dd 1 g of

N -1- napht

hy le thy le nedi am ine dihy droc hlori de and dis sol v e. D ilut e to 2000 m L w ith deio nized w ater a

nd add 1 m L of 30% Brij-35 . Store in refrig erator. P repare e very 6 w eek s.

Pre p ar at io n o f C op p er -C ad miu m C ol u mn

1. Us e g ood qua lit

y c adm ium f ili ng s (25 - 60 m es h s iz e)

.
2. Cl ea

n 10 g of c adm ium w ith 20 m L of

a ce tone . R ins e tw ic e w ith 20 m L of de ioniz ed w at er . C le an ca dm ium ne x t w ith 50

m L of 1 N hy droc hlori c ac id f

or 1 m inute . C adm ium

tur ns si lv er in col or. D ec ant

the hy droc hlori c ac id a

nd w as h the cadm ium w ith ano ther 50 m L of 1 N hy dro chlo ric ac id for 1 m inu te.

3. De ca

nt t he hy droc hlori c ac id a

nd w as h the c adm ium

s ev er al ti m es w ith dei oniz ed w at er

.

4 2 4. De ca

nt t he dei oniz ed w at er

a nd a dd 20 m

L of 2% (w /v ) c opper

s ulf at e (C uSO @ 5H O) . W as h the c adm ium unti l no blue col or r em ai ns in t

he sol ution.

5. A dd a nothe r 20 m L of 2% (w /v ) c opper

s ulf at e and w as h the c adm ium unti l no blue c olor re m ai ns in t he sol ution. T he

ca dm ium w ill be da rk

brow n in col or.

6

. D e c a n t a n d wa s h t h o r o u g h l y (
- 10 ti m es ) w ith dei oniz ed w at er

.
7. Fil l t

he re duct or c olum

n (22 cm le ng th of 0.110- inc h ID tubi ng ) w ith A m m onium Chl oride Re ag ent

a nd tr ans f

er the pre par ed ca dm ium

pa rti cl es to the c olum

n us ing a Pas te ur pi pet te or som e othe r m et hod that pr ev ent s cont ac t of the

ca dm ium pe lle ts w ith ai r. D o not al low a ny a ir bubble s to be tr appe d in the c olum n. Pac k the e ntir e col um

n unif orm ly

w ith filings such that, visually , the packed f ilin gs have separation gaps #- 1 mm

.
8. W hen the e ntir e col um n is w el l-pa ck ed w ith g ra nule s, ins er

t g la ss w ool pl ug s at

the outl et

e nd of the c olum

n. W ith

re ag ent s running thr oug h the s y st em , a tta ch the c olum n. R em em

ber to hav e no a ir bubble s in t he v al v e and to atta ch the col um n to the int ak e si de of

the v al v e f

irs t.

9. Che ck

f or g ood f low c har ac te ris tic s (g ood bubble pa tte rn) a f te r t he addi tion of

a ir bubble s bey ond the c olum n. I f the

co lu m n is p acked to o ti gh tl y , an in co n si st en t flo w p at te rn w il l r esu lt . All o w Am m o n iu m Ch lo ri d e Reage n t t o flo w th ro u gh the c olum n f or one hour.

1 0

. P r i o r

t o sa mp l e a n al ysi s, co n d i t i o n t h e c o l u mn wit h ap p r o xi mat el y 10 0 mg n i t r at e/ L fo r 5 min u t es fo l l o wed b y 10 0 mg nitri te/L f or 10 m inu tes.

3 2 11. A nal y ze W ork ing Sta ndar d A (40 : M N O -N ) a

nd W ork ing N itr ite Sta

ndar d (40 : M N O -N ). If the pe ak he ig ht of

- - W ork ing Sta

ndar d A is < 90% of

the pe ak he ig ht of W ork ing N itr ite Sta

ndar d, pr epa

re a ne w c adm ium re duct ion c olum

n.

Stan dard s

Stock N itrate S tand ard, 5, 000 : M

3 Potass ium

nitrate (KNO ), d

ried at 45° C 0.5 055 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

In a 1000 m L v olum et

ric f la sk , di ss olv e 0.5055 g of pota ss ium

nit ra te in
- 800 m L of

de ioniz ed w at er . D ilut e to 1000 m L w ith dei oniz ed w at er

(1 m L c onta ins 5 : m ole s N)

.
Workin g S tand ard A S t o c k Ni t r a t e St a n d a r d 0

. 8 mL D e i o n i ze d

wa t e r u p t o 1 0 0 mL

In a 100 m L v olum et

ric f la sk , di lute 0.8 m

L of Stoc k N itr at e Sta

ndar d to 100 m L w ith dei oniz ed w at er to y ie ld a c once ntra

tion of 40 : M N (0. 56 m g N /L ).20

Workin g N itrate S tand ards

Di lute 2.5, 5, 10, 15 a nd 25 m L of W ork ing Sta

ndar d A to 100 m L w ith dei oniz ed w at er to y ie ld c once ntra

tions of 1 : M N (0.014 m g N /L ), 2 : M N (0. 028 m g N /L ), 4 : M N (0. 056 m g N /L ), 6 : M N (0. 084 m g N /L ) a nd 10 : M N (0. 140 m g N /L ),

r e s p e c t i v e l y.

Di lute 0.8 m

L of Stoc k N itr at e Sta

ndar d to 200 m L w ith dei oniz ed w at er to y ie ld c once ntra

tions of 20 : M N (0. 28 m g N /L ).

Di lute 1.0 m

L a nd 1.5 m

L of Stoc k N itr at e Sta

ndar d to 100 m L w ith dei oniz ed w at er to y ie ld c once ntra

tions of 50 a nd 75 : M N

4 (0.70 and 1.05 m g N /L ), re spe ct iv el y f or us e w ith the O RN /Y EL s am ple tube , Y EL /B L U NH Cl tube a nd s am ple c once ntra

tions of 0.50 - 1. 00 m g N /L (ni tri te + nit ra te ).

Di lute 2.0 m

L of Stoc k N itr at e Sta

ndar d to 100 m L w ith dei oniz ed w at er to y ie ld c once ntra

tions of 100 : M N (1. 40 m g N /L ) f or

4 use w ith the O RN /G RN s am ple tube , Y EL /B L U NH Cl tube a nd s am ple c once ntra

tions of 1.00 - 1.40 m g N /L (ni tri te + nit ra te ).

Di lute 3.0, 5.0, 6. 0 a nd 8.0 m L of Stoc k N itr at e Sta

ndar d to 100 m L w ith dei oniz ed w at er to y ie ld c once ntra

tions of 150, 250, 300 a nd 400 : M N ( 2

. 1 , 3

. 5 , 4

. 2 an d 5

. 6 mg N/ L) fo r u se wit h t

h e d i l u t i o n l o o p an d sa mp l e c o n ce n t r at i o n s o f 1

. 4 0 - 5

. 6 0 mg N/L (nitr ite + nitrate). T he nitrate sam ple is sam pled originally

w ith an ORN/WHT sam

ple tub e a nd YEL /YEL deio nized w ater

4 dilu ent tub e w

hich is then "res am

pled " to an ORN/YEL resam ple tub e a nd YEL /BLU

NH Cl tub e.

Stock N itrite Sta nda rd, 5, 000 : M

2 Sod ium nitrite (NaN O ), d

ried at 45° C 0.3 45 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

3 C h l o r o f o r m ( C H C l ) 1 mL

In a 1000 m L v olum et

ric f la sk , di ss olv e 0.345 g of

s odium nit rit e in
- 600 m L of

de ioniz ed w at er . D ilut e to 1000 m L w ith dei oniz ed w at er . A dd 1 m L of c hlorof orm a s a pre se rv at iv e (1 m

L c onta ins 5 : m ole s N)

.
Workin g N itrite Sta nda rd

Di lute 0.8 m

L of Stoc k N itr ite Sta

ndar d to 100 m L w ith dei oniz ed w at er to y ie ld a c once ntra

tion of 40 : M N (0. 56 m g N /L ).21 

## S ILICATE Re v i s e d 2/ 2004

This reaction is bas ed o n the redu ction of silico m oly bdate in acid ic solu tion to "m oly bden um blue " by ascorbic acid. O xalic a cid is added to m inim iz e interfere nce from ph osph ates.

Method ology

Technicon I ndus trial Method N o. 811- 86T. Nov em

ber 1 986. Techn icon I ndus trial Sy stem s. Tarry town , New Y ork , 1 0591

.
Instru mentation

Technicon TrAAcs- 800 Nutrien t A naly z er

Ma n ifol d A ss em b ly

Figu re 5

. Manifold assem bly fo r silicate meth od.
Spe cif ication s

Ga in: 8 Baseline: 3 9 - 42 S am p li n g R at e: 60 /hour Filt er : 800 nm Flo w ce ll : 5 0 mm Rela tive A bsor ban ce (7 5 : M S i) : - 1.1 0 Interferences: Sa linity

m ay interfere.22

R ea ge n ts

Ox alic A cid S olution

2 2 4 2 O xa li c ac id

( H C O @ 2H O) 100 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

In a 1000 m L v olum et

ric f la sk , di ss olv e 100 g of

ox al ic a ci d in
- 90 0 m L deio nized w ater a nd dilu te to 10 00 m L w ith d eion ized

w at er

.
Asco rbic Acid So lution

2 2 4 2 O xa li c ac id

( H C O @ 2 H O) 5 g

686 Asco rbic ac id (C H O ), U .S. P. quality 100 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

In a 1000 m L v olum et

ric f la sk , di ss olv e 5 g of

ox al ic a ci d in
- 800 m

L of de ioniz ed w at er . A dd 100 g of a sc orbic a ci d a nd m ix until dis sol v ed. D ilut e to 1000 m L w ith dei oniz ed w at er

.
Ammoniu m Molybd ate S olution

4 6 7 2 4 2 A m m on iu m m ol y bd at e [ (N H ) Mo O @ 4 H O] 7.5 g D e i o n i ze d

wa t e r u p t o 2 5 0 mL

In a 250 m L v olum et

ric f la sk , di ss olv e 7.5 g of

a m m onium m oly bdat e in
- 200 m L of

de ioniz ed w at er . D ilut e to 250 m L w ith dei oniz ed w at er

.
Stock Phosp hate Solu tion (Baseline S pike)

2 4 Potass ium

ph osph ate (KH PO ), d

ried at 45° C 0.4 394 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

In a 1000 m L v olum et

ric f la sk , di ss olv e 0.4394 g of pota ss ium

phos phat e in
- 6 0 0 mL of d ei o n i ze d wat er

. Di l u t e t o 1 0 0 0 mL

w ith dei oniz ed w at er . Pre par e f re sh w hen m ak ing 0.7 N Sulf

uric A ci d Solut ion.

Sulfuric A cid S olution , 0 .7 N

2 4 S u l f u r i c a c i d ( H S O ) , c o n c e n t r a t e d

( s p

. gr

. 1

. 8 4 ) 4

. 0 6 mL S t o c k P h o s p h a t e So l u t i o n 2 1

. 4 mL

3 2 1 1 3 Sod ium do decy l sulfate, (SD S) [C H (CH ) OS O Na;

m .w . = 28 8.3 8; ph osph ate # 0. 00 01 %]
- 0.2 5 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

In a 1000 m L v olum et

ric f la sk , a dd 4.06 m

L of c once ntra

te d s ulf uric a ci d a nd 21.4 m

L of Stoc k Phospha te Solut ion t o - 6 0 0 mL of de ioniz ed w at er . D ilut e to 1000 m L w ith dei oniz ed w at er . A dd
- 0.25 g of SD S.

Sod ium Hydr ox ide Wa sh Solu tion Sod ium hy droxid e (N aOH ) 4 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

In a 1000 m L v olum et

ric f la sk , di ss olv e 4 g of

s odium hy drox ide in
- 800 m L of de ioniz ed w at er . D ilut e to 1000 m L w ith dei oniz ed w at er

.23

Sampler (Baseline) Solu tion

3 2 Is o p r o p a n o l [ ( C H ) C H O H ] 1 0 0 mL D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

In a 1000 m L v olum et

ric f la sk , di lute 100 m

L of is opropa nol t o 1000 m

L w ith dei oniz ed w at er

.
Stan dard s

Stock Silica te S tand ard, 10 ,00 0 : M

2 6 Sod ium silicofluo ride (N a SiF ), d ried at 45° C 1.8 8 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

In a 1000 m L v olum et

ric f la sk , di ss olv e 1.88 g of

s odium s ili cof luori de in
- 900 m L of

de ioniz ed w at er . D ilut e to 1000 m L w ith dei oniz ed w at er

(1 m L c onta ins 10 : m ole s Si) . S tore in a pla st ic c onta ine r.

Workin g S ilicate S tand ards

Di lute 0.05, 0.1, 0. 2, 0.3, 0.4, 0.5 a nd 0.75 m

L of Stoc k Sil ic at e Sta

ndar d to 100 m L w ith dei oniz ed w at er to y ie ld

conc ent ra tions of 5 : M Si (0. 14 m g /L ), 10 : M Si (0. 281 m g /L ), 20 : M Si (0. 562 m g /L ), 30 : M Si (0. 843 m g /L ), 40 : M Si (1.124 m g /L ), 50 : M Si (1. 41 m g /L ) a nd 75 : M S i ( 2

. 1 0 mg / L) , r e s p e c t i v e l y.

NO T E:

1. T he use of

g la ss w ar e w ith this m et

hod s hould be a v oide d be ca use it m ay be a s ourc e of s ili ca c onta m ina tion. A ll chem icals used f

or reage nts and the deion ized w ater s ho uld be of good qualit y and lo w in silica.

2. Sa m

ple r ( Ba se line ) S oluti on of is opropa nol i s use d ins te ad of

de ioniz ed w at er

a s bas el ine s oluti on to count er the e f f ec t of

sa lt w at er on t he sy st em

due to its hig h re f

ra ct iv e inde x re la tiv e to de ioniz ed w at er

.
Procedur e

1

. At s t a r t

- u p , c h e c k

t h e l e v e l o f a l l r e a g e n t s t o e n s u r e a n a d e q u a t e s u p p l y.

2. W ith the m oly

bdat e line pum

ping a ir, pla ce a ll othe r l ine s into the ir re spe ct iv e cont ai ner s and st ar t t he propor tioni ng pum

p.

3

. Aft er 2 - 3 min u t es , p l ac e t h e mo l yb d at e l i n e i n t o i t s c o n t ai n er

. All o w - 10 m inute s equi libr at ion.

4. A ty pical tray prot ocol appears a s f

ollo w s:
P, 5C, H , 2L , n S, I ,... .

w her e: P = pr im er (100% FS st anda

rd) , C = c al ibra

nt, H = 100% FS st anda

rd, L = 20% FS st anda

rd, S = s am

ple s, a nd I = i nte rs am

ple c al ibra

nt.

5. A t s hut-dow

n, r em ov e al l r ea g ent

li nes a nd pla ce int o the Sodium H y drox ide W as h Solut ion c onta ine r. A llow to pum p 5

m inu tes.

6. Rem ove a ll lin es f

rom the w ash contain er and place into d eion ized w ater w ith S DS. A llow to pump 10 m inu tes.24


## S U L F A T E , 

## C HLORI DE AND 

## N I TRATE Re v is e d 2/ 2004

A sm all v olum e of filtered sa m ple is introdu ced into a n ion chro m atog raph (I C). The sam ple is pum

ped th ro ug h a pr e- co lu m

n, s ep ar at or

c ol um n, s up pr es so r co lu m

n an d co nd

uc ti v it y d et ec to r. A ni on s ar e separated in the p re- colum

n an d se parator colum

n b ased on their affin ity

for resin e xchan g e sites in the

co lu m ns

. Th e su

pp re ss or c ol um n co nv er ts th e sa m

pl e an io ns to th ei r ac id

f or m

. Th e se pa ra te d an io ns a re
m easured by the cond uctiv ity detec tor. The conc entration of an ions is dete rm ined by com paring pea k

areas o f un k

now ns to a calibration curv e g enerated from k now n stan dards.

Method ology
Pfaff, J .D ., C. A. B rock

hoff a nd J .W. O’D ell. A ug ust 1 991. U SEP A Test Method No. 30 0.0 . The determ ination of ino rg anic a nions in w ater by ion chrom atog raphy . E nv ironm ental Mo nitoring and Sy stem s L aborato ry , C incinna ti, O H 45 268.

US EP A. 19 87 . S e c t i on 11 .0 . De t e r min a t i on of ch l or i de , n i t r a t e an d s ul f a t e by io n c hr oma t ogr a ph y in Ha nd bo ok o f

m e t ho ds fo r

ac i d d e po s i t i on st ud i e s : La bo r a t or y an a l yse s fo r su r f a c e wa t e r ch e mis t r y. United State s E nv ironm ental P rotection Ag ency , O ffice of Rese arch and Dev elopm ent. Washing ton, D. C. . R eport # E PA 60 0/4- 87/02 6 S eptem ber 1 987. 33 6pp.

Instru mentation

D io ne x- 12 0 I on

C hr om at og ra

ph w it h P ea k

N et s of tw ar e (v er si on

5

. 21 ) w as b ro ug ht

o n- li ne in

Jan ua ry 2003 . A D ionex AS

- 40 Auto Sam pler was added

to the sy stem

in J anuary

20 04.

R ea ge n ts

2 3 3 Elu ent, 3. 5M N a CO /1.0M NaHC O So lution

2 3 Sod ium carb onate (Na CO ) 0.7 42 g

3 Sod ium bicarb onate (NaH CO ) 0. 164 g D e i o n i ze d

wa t e r u p t o 2 0 0 0 mL

I n a 2 0 0 0 mL vo l u met r i c fl as k, d i ss o l ve 0

. 7 4 2 g of so d i u m ca r b o n at e an d 0

. 1 6 4 g o f so d i u m b i ca r b o n at e i n ap p r o xi mat el y 15 0 0 mL of de ioniz ed w at er . D ilut e to 2000 m L w ith dei oniz ed w at er

. Reg enera nt

2 4 S u l f u r i c a c i d ( H S O ) , c o n c e n t r a t e d

( s p

. gr

. 1

. 8 4 ) 2. 8 mL D e i o n i ze d

wa t e r up t o 2 0 0 0 mL

2 4 I n a 2 0 0 0 mL vo l u met r i c fla sk , ad d 2

. 8 mL o f co n ce n t r at ed H S O t o ap p r o xi mat el y 15 0 0 mL of d ei o n i ze d wat er

. Di l u t e t o 2 0 0 0 mL

w ith dei oniz ed w at er . The reg ene ra nt re se rv oir m

ust be fi lle d to the absol ute top of the c onta ine r. CB L ’s s y st em holds s ix li te rs of

ac id.

T his e lue nt/r eg ene ra

nt s y st em is c al le d dis pla ce m ent che m ic al re g ene ra tion. A s the re g ene ra nt is use d up, t he use d e lue nt t ak es it s pla ce in the re se rv oir. It

is c ruc ia l t

hat e v er y ti m e tha t t

he el uent is c hang ed, that t

he re g ene ra

nt i s al so cha

ng ed.25

Stan dard s

Stock So dium C hlorid e S tand ard Sod ium C hloride (NaC l), d

ried at 45° C 1.6 485 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

In a 10 00 m L volume tric f lask, dissol ve 1.6 48 5 g of sodi um chlorid e in ~ 80 0 m L deio nized w ater. Dilute to 10 00 m L w ith d eion ized

w at er (1m L c onta ins 1 m g Cl ).

-

Stock So dium S ulfate Stan dard

2 4 Sod ium sulfate (Na SO ), d

ried at 45° C 1.8 141 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

In a 100 0 m L volume tric f lask, dissol ve 1.8 14 1 g of sodi um sulfa te in ~ 80 0 m L deio nized w ater. Dilute to 10 00 m L w ith d eion ized

4 w at er (1m L c onta ins 1 m g SO ). -2

Stock So dium N itrate S tand ard

3 Sod ium nitrate (NaN O ), d

ried at 45° C 6.0 679 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

In a 100 0 m L volume tric flask , dissol ve 6.0 67 9 g of sodiu m nitrate in ~ 8 00 m L deio nized w ater. Dilute to 10 00 m L w ith d eion ized

3 w at er (1m L c onta ins 1 m g N O -N ).

-

Wor king 5:5: 0.5 Chl ori de/ Sulf ate /Ni tr ate Standa rd (m g/L) S t o c k S o d i u m Ch l o r i d e St a n d a r d 0

. 5 mL S t o c k S o d i u m Su l f

a t e St a n d a r d 0

. 5 mL S t o c k S o d i u m Ni t r a t e St a n d a r d 0

. 0 5 mL D e i o n i ze d

wa t e r u p t o 1 0 0 mL

In a 100 m L v olum et

ric f la sk , di lute 0.5 m

L of s toc k s odium c hlori de st anda

rd, 0.5 m L s toc k s odium s ulf at e st anda rd a nd 0.05 mL st o ck so d i u m n i t r at e s t an d ar d t o 1 0 0 mL wit h d ei o n i ze d wat er

. Th e fi n al co n ce n t r at i o n o f t h i s s t an d ar d i s 5 mg Cl / L, 5 mg -

4 3 SO /L a nd 0.5 m g N O -N /L

. -2

-

A compar able proc edu re may be us ed to mak e d if f erent w orkin g sta nda rds base d o n th e
conc entra tions of the samp les to be ana lyz ed.

Procedur e

E ac h sa mp l e t o b e a n al yze d mu st b e p r e- t r ea t ed t o re mo ve o r ga n i c p ar t i cl es t h at may b i n d t o t h e c o l u mn b ea d s, re d u ci n g c o l u mn ef f ic ie ncy , r el ia

bili ty a nd a ls o c olum

n li f e. O rg ani c par

tic le s ar e re m ov ed by

a Ba k er Bond S PE C-18 col um n a nd f ilt ra tion t h r o u gh a Gel man o r

Wh at man 0

. 2 μm Acr o d i sk d es i gn ed fo r

u se wit h an IC sys t em. 1. To prepare the C-18 columns, pass one colum n volu m e of

HP LC gra de m ethano l thro ugh each colum n. Then pass three col um n v olum es of

de ioniz ed w at er

thr oug h e ac h c olum

n.

2. T o pre par e a sa

m ple , f irs t r em ov e al l t

he dei oniz ed w at er

f rom the c olum

n a nd the n im

m edi at el y pla ce 1 m

L of s am ple int o the col um n.

3. P lace an acrodisk at the e nd of the colu m n con tainin g the sa

m ple.

4. P ass the sam

ple thro ugh th e c olu m

n usin g positive pressure (pushin g a ir thro ugh a sy ringe). Disca

rd th e f irst f ive dro ps.26

Ca pture the ne x t 11 drops in a 0.5 Ml D ione x s am ple v ia l, pla ce a

D ione x s am

ple c ap on top of

the v ia l a nd re f rig er at e until analy sis.27 

## ORGA NIC ANA LYTES

R at io n al e

Methods for m easuring disso lv ed o rg anic c arbon , n itrog en a nd phos phoru s are describe d in the fo llowin g sections. D issolv ed o rg anic c arbon con centration s are determ ined by hig h tem perature com

bustion . F or total dis solv ed n itrog en a nd phos phoru s, potassiu m persu lfate is added

to a sam ple, whic h, whe n u nder he at a nd p re ss ur e, b re ak s do w n th e or g an ic c on st it ue nt s to in or g an ic f or m s. I no rg an ic f

ra ct io ns th en

a re subtracted

from the total dis solv ed s am

ple to y ield the disso lv ed o rg anic c oncen tration. Se e F ig ures 6 and 7.
Samplin g a nd Stora ge

C ollected water sam

ples a re filtered throug h Wha tm an G F/F filters (nom inal p ore siz e 0. 7 : m ) and placed in ap propriate contain ers an d p reserv ed. Table 2 prese nts sam pling and storag e pra ctices fo r org anic a naly tes. C ontaine rs for dissolv ed o rg anic c arbon are acid w ashed in 10% H Cl and rinsed thorou g hly

w ith de ioniz ed w ater. C ontaine rs for nitrog en a nd phos phoru s are autoclav ed w ith po tassium pe r s ul f a t e so l ut i on be f

or e us e , t he n r i ns e d t ho r ou ghl y wi t h d e i on i zed wa t e r on l y.

Tab le 2. S

amp ling a nd storag e for orga nic a nalytes
AN AL YTE VOLU ME (m L) STOR AG E CO NTAI NE R
Disso lv ed o rg anic c arbon
- 30

- 20° C Teflon or Boro silicate G lass Vial 1 Disso lv ed n itrog en/pho spho rus 10

- 20° C Boro silicate G lass S crew- cap Tube

Te fl on v ia ls c an b e us ed f or a ll s am pl es

. Wh ea to n- 33 lo w e xt ra ct ab le b or os il ic at e g la ss v ia ls 1 ® (recom m ended by EP A) are u sed for saltw ater sam ples.

Fig ure 6. Flow diag ram of nit rog en ana ly si s (TN = tot al ni tr oge n, PN = part ic ula te nit rog en, TDN = tot al di sso lv ed ni tr oge n, DIN = dis sol ved inor gani c nit rog en, DON = dis sol ved organ ic nit rog en).28

Figu re 7

. Flow

diag ram of phos phor us a nalysis (

TP = tota l pho spho rus, PP = p articulate ph osph orus, T D P = to ta l d is so lv ed p ho sp ho ru s, D

IP = d is so lv ed

in or ga ni c ph os

ph or us , D O

P = d is so lv ed

o rg an ic phos phor us).29


## T O TAL 

## D IS SOLV ED 

## N ITRO G EN Re v i s e d 2/ 2004 

### AN D 

## P HO SP HO RU S

Thi s m et ho d is a p er su lf

at e ox id at io n te ch ni qu e fo r ni tr og en a nd p ho sp ho ru s w he re , u nd er in it ia ll y alk aline c ondition s, nitrate is the so le nitrog en p roduc t. Pho sphate is the sole p hosp horus produ ct after acidic cond itions a re ach iev ed fo llowin g furthe r auto decom position of the p ersulfate in the hea ted oxidatio n tub es.. Dig ested sam

ples a re pa ssed throug h a g ranulated cop per- cadm ium

colu m n to reduce nitrate to nitrite. The nitrite the n is determ ined by diaz otiz ing w ith su lfanilam ide an d co upling w ith N

- 1- naph thy lethy lenediam ine d ihy droch loride to form a colored az o d y e. C olor is propo rtional to nitrog en
conce ntration.

Am m onium m oly bdate and antim ony

po tassium

tartrate react in an acid m edium w ith dilu te solu tions of phos phoru s to form

an antim ony
- phos pho- m oly bdate com

plex whic h is reduce d to an in tensely blue

colored com plex by asco rbic ac id. C olor is propo rtional to ph osph orus conce ntration.

Method ology

D' Elia, C. F., P. A. Steu dler an d N . C orwin

. 19 77. D eterm ination of tota l nitrog en in aqu eous sam

ples using persu lfate dig estion. L im nol. Ocea nog r. 2 2:760

- 764.

Valderram a, J .C

. 1981

. The sim ultaneo us a naly sis of total nitro g en a nd total ph osph orus in na tural waters. Mar. C hem . 1 0:109

- 122.

Instru mentation

T e c h n i c o n Au t o A n a l yze r I I

Ma n ifol d A ss em b ly

Figu re 8

. Manifold assem bly fo r tota l disso lved p hosp horu s m ethod.30

Figu re 9

. Manifold assem bly fo r tota l disso lved n itrogen m ethod.
Spe cif ication s

D am p : N orm al Samplin g R ate: 4 0/ ho ur , 9 :1 s am pl e/ w as h ra ti o Filt er s: 55 0 nm

f or n it ra te a nd

8 80 n m f or p ho sp ha te Pho to tu b es : 1 99

- B 02 1- 01 f or n it ra te a nd

1 99

- B 02 1- 04 f

or p ho sp ha te Flo w ce ll : 5 0 mm

3 R el at iv e A b so rb an ce ( 40 μ M N O -N ; OR N /YE L s am p le t u b e) : ~.59 - R el at iv e A b so rb an ce ( 18 μ M P) : ~.85

Interferences: Me t a l io ns m a y in t e r f e r e if pr e s e nt in su f

f i c i e nt co nc e nt r a t i on s . T he pr e s e nc e of hi gh

co nc en tr at io ns o f su lf

at e w il l r ed

uc e th e se ns it iv it y

o f th e co pp er
- ca dm iu m

c ol um n. S il ic on

a t a
conce ntration of 1 00 : M Si causes interference s eq uiv alent to
- 0.0 4 : M P.

R ea ge n ts

Ox idiz ing Rea gent - L ow , Mo dera te an d High N an d P C once ntratio ns Sod ium hy droxid e (N aOH ) 3 g

2 2 8 Potass ium

persu lfate (K S O ), lo w N (<0.0 01 %) 20. 1 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

In a 1000 m L v olum et

ric f la sk , di ss olv e 3 g of

s odium hy drox ide a nd 20.1 g of

pota ss ium

pe rs ulf at e in
- 80 0 m L of deionized

wa t e r

. D i l u t e t o 1 0 0 0 mL wi t h d e i o n i z e d wa t e r

. M a k e fr e s h d a i l y.31

Ox idiz ing Rea gent - V ery High N an d P C once ntratio ns Sod ium hy droxid e (N aOH ) 3 g

2 2 8 Potass ium

persu lfate (K S O ), lo w N (<0.0 01 %) 20. 1 g D e i o n i ze d

wa t e r u p t o 3 0 0 0 mL

In a 3000 m L v olum et

ric f la sk , di ss olv e 3 g of

s odium hy drox ide a nd 20.1 g of

pota ss ium

pe rs ulf at e in
- 80 0 m L of deionized

wa t e r

. D i l u t e t o 3 0 0 0 mL wi t h d e i o n i z e d wa t e r

. M a k e fr e s h d a i l y.

Buf f er S olution

3 3 Boric acid (H BO ) 61. 8 g Sod ium hy droxid e (N aOH ) 8 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

In a 1000 m L v olum et

ric f la sk , di ss olv e 61.8 g of

bori c ac id i n - 300 m L de ioniz ed w at er . A dd 8 g of s odium hy drox ide a nd dilu te to 10 00 m L w ith d eion ized w ater. T he solu tion is sta ble f or seve ral w eek s.

Intern al S tand ards

Stock G lutamic A cid S tand ard, 5, 040 : M

3 2 2 2 Glutam ic acid

[C H NH (CO OH ) ], d

ried at 45° C 0.3 705 g D e i o n i ze d

wa t e r u p t o 5 0 0 mL

3 C h l o r o f o r m ( C H C l ) 0

. 5 mL

In a 500 m L v olum et

ric f la sk , di ss olv e 0.3705 g of g luta m ic a ci d in
- 400 m L of

de ioniz ed w at er

a nd dil ute to 500 m L w ith dei oniz ed w at er . A dd 0.5 m l of c hlorof orm a s a pre se rv at iv e (1 m

L c onta ins 5.04 : m ole s N)

.
Workin g G lutamic A cid S tand ard - L ow , Mo dera te an d High N an d P C once ntratio ns S t o c k Gl u t a mi c Ac i d

St a n d a r d 1 mL D e i o n i ze d

wa t e r u p t o 1 0 0 mL

In a 100 m L v olum et

ric f la sk , di lute 1 m

L of Stoc k G luta m ic A ci d Sta

ndar d to 100 m l w ith dei oniz ed w at er to y ie ld a

conc ent ra tion of 50.4 : M N (0. 7056 m g N /L ).

Workin g G lutamic A cid S tand ards - V ery High N an d P C once ntratio ns S t o c k Gl u t a mi c Ac i d

St a n d a r d 6 mL D e i o n i ze d

wa t e r u p t o 1 0 0 mL

In a 100 m L v olum et

ric f la sk , di lute 6 m

L of Stoc k G luta m ic A ci d Sta

ndar d to 100 m L w ith dei oniz ed w at er to y ie ld a

conc ent ra tion of 302.4 : M N (4. 23 m g N /L ).

Stock G lycerop hosp hate Stan dard , 3 09 : M $

- G ly ce ro ph os ph or ic a ci d, d is od iu m s al t, 5

- hy dr at e

2 2 3 2 2 [( HOCH ) CHOPO Na @ 5 H O] 0.0 473 g D e i o n i ze d

wa t e r u p t o 5 0 0 mL

3 C h l o r o f o r m ( C H C l ) 0

. 5 mL

In a 50 0 m L vol u m et ri c flask, di sso lve 0. 0 4 7 3 g of $ -G ly ce rophos phoric a ci d, di sodi um s al t, 5-hy dra te in
- 40 0 m L of deionized

w at er . D ilut e to 500 m L w ith dei oniz ed w at er . A dd 0.5 m L of c hlorof orm a s a pre se rv at iv e ( 1 m L c onta ins 0.309 : m ole s P).32

Workin g G lycerop hosp hate Stan dard - L ow , Mo dera te an d High N an d P C once ntratio ns S t o c k Gl yc e r o p h o s p h a t e St a n d a r d 1 mL D e i o n i ze d

wa t e r u p t o 1 0 0 mL

In a 100 m L v olum et

ric f la sk , di lute 1 m

L of Stoc k G ly ce rophos phat e Sta

ndar d to 100 m L w ith dei oniz ed w at er to y ie ld a

conc ent ra tion of 3.09 : M P (0. 096 m g P/L ).

Workin g G lycerop hosp hate Stan dard - V ery High N an d P C once ntratio ns S t o c k Gl yc e r o p h o s p h a t e St a n d a r d 2 mL D e i o n i ze d

wa t e r u p t o 1 0 0 mL

In a 100 m L v olum et

ric f la sk , di lute 2 m

L of Stoc k G ly ce rophos phat e Sta

ndar d to 100 m L w ith dei oniz ed w at er to y ie ld a

conc ent ra tion of 6.18 : M P (0.192 m g P/L ).

Workin g In orga nic S tand ards

Stock N itrate S tand ard, 5, 000 : M

3 Potass ium

nitrate (KNO ), d

ried at 45° C 0.5 055 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

3 C h l o r o f o r m ( C H C l ) 1 mL

In a 1000 m L v olum et

ric f la sk , di ss olv e 0.5055 g of pota ss ium

nit ra te in 1000 m L of

de ioniz ed w at er . A dd 1 m L of c hlorof orm
as a pre se rv at iv e (1 m

L c onta ins 5 : m ole s N)

.
Workin g N itrate S tand ards - L ow N an d P C once ntratio ns

Di lute 0.5, 1.0 a

nd 1.5 m L of Stoc k N itr at e Sta

ndar d to 100 m L w ith dei oniz ed w at er to y ie ld c once ntra

tions of 25 : M N (0. 35

m g N /L ), 50 : M N (0. 70 m g N /L ) a nd 75 : M N ( 1

. 0 5 mg N / L) , r e s p e c t i v e l y.

Workin g N itrate S tand ards - Mo dera te N an d P C once ntratio ns

Di lute 0.5, 1.0 a

nd 2.0 m L of Stoc k N itr at e Sta

ndar d to 100 m L w ith dei oniz ed w at er to y ie ld c once ntra

tions of 25 : M N (0. 35

m g N /L ), 50 : M N (0. 70 m g N /L ) a nd 100 : M N ( 1

. 4 0 mg N / L) , r e s p e c t i v e l y.

Workin g N itrate S tand ards - High N an d P C once ntratio ns

Di lute 1.0, 2.0 a

nd 4.0 m L of Stoc k N itr at e Sta

ndar d to 100 m L w ith dei oniz ed w at er to y ie ld c once ntra

tions of 50 : M N (0. 70

m g N /L ), 100 : M N (1. 40 m g N /L ) a nd 200 : M N ( 2

. 8 0 mg N / L) , r e s p e c t i v e l y.

Workin g N itrate S tand ards - V ery High N an d P C once ntratio ns

Di lute 3.0, 6.0 a

nd 8.0 m L of Stoc k N itr at e Sta

ndar d to 100 m L w ith dei oniz ed w at er to y ie ld c once ntra

tions of 150 : M N (2. 1

m g N /L ), 300 : M N (4. 2 m g N /L ) a nd 400 : M N ( 5

. 6 mg N / L) , r e s p e c t i v e l y.

Stock Orthop hosp hate Stan dard , 1 2,0 00 : M P ot as si um

p ho sp ha te , m on ob as ic

2 4 (KH PO ), d

ried at 45° C 1.6 32 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

3 C h l o r o f o r m ( C H C l ) 1 mL33

In a 1000 m L v olum et

ric f la sk , di ss olv e 1.632 g of

pota ss ium

phos phat e in
- 800 m

L of de ioniz ed w at er . D ilut e to 1000 m L w ith dei oniz ed w at er . A dd 1 m L of c hlorof orm a s a pre se rv at iv e (1 m

L c onta ins 12 : m ole s P).

Seco nda ry Orth oph osph ate S tand ard S t o c k Or t h o p h o s p h a t e St a n d a r d 1 mL D e i o n i ze d

wa t e r u p t o 1 0 0 ml

In a 100 m L v olum et

ric f la sk , di lute 1 m

L of Stoc k O rthophos phat e Sta

ndar d to 100 m L w ith dei oniz ed w at er (1 m L c onta ins 0.12 : m ole s P).

Workin g Ortho pho sph ate S tand ards - L ow , Mo dera te an d High N an d P C once ntratio ns

Di lute 0.5, 2.0 a

nd 4.0 m L of Se conda ry O rthophos phat e Sta

ndar d to 100 m L w ith dei oniz ed w at er to y ie ld c once ntra

tions of 0.6 : M P (0.0186 m g /L ), 2.4 : M P (0.0744 m g /L ) a nd 4.8 : M P ( 0

. 1 4 8 8 mg / L) , r e s p e c t i v e l y.

Workin g Ortho pho sph ate S tand ards - V ery High N an d P C once ntratio ns

Di lute 2.0, 5.0 a

nd 10.0 m

L of Se conda ry O rthophos phat e Sta

ndar d to 100 m L w ith dei oniz ed w at er to y ie ld c once ntra

tions of 2.4 : M P (0.0744 m g /L ), 6.0 : M P (0.186 m g /L ) a nd 12.0 : M P ( 0

. 3 7 2 mg / L) , r e s p e c t i v e l y.

Procedur e

1. Se le ct

a ppropr ia te s ta

ndar ds and ox idiz ing re ag ent f or s am ple s to be a nal y ze d. F or 0 - 200 : M N a nd 0 - 5.0 : M P , use 10 m L s am

ple s and 5 m L of

O x idiz ing Re ag ent

f or L ow , Mode ra te a nd H ig h N a nd P Conc ent ra tions , f ollow ing s te ps 2 - 9. F or 150 - 400 : M N a nd 2.4 - 12.0 : M P, use 5 m L s am ple s and 15 m L of

O x idiz ing Re ag ent

f or Ve ry H ig h N a nd P Con centration s, f

ollo w ing steps 2V H - 9VH.

2. Plac e 10 m L of

f ilt er ed w at er

(W hat m an G F/F f

ilt er , 0.7 : m ) in a 30 m L screw ca p test tub e a

nd f reez e.

2VH. Plac e 5 m L of

f ilt er ed w at er

(W hat m an G F/F f

ilt er , 0.7 : m ) in a 30 m L screw ca p test tub e a

nd f reez e.

3. P lace 10 m L of e ach standard (3 repl icates of e ach) in 30 m L screw ca p test tub es a

nd treat e xac tly

as sam ples.

3V H. P lace 5 m L of e ach standard (3 repl icates of e ach) in 30 m L screw ca p test tub es a

nd treat e xac tly

as sam ples.

4. When rea dy

to analy ze, thaw

sam ples at room tem

perature.

5. A dd 5 m

L of O x idiz ing Re ag ent . A

pre ci pita te w ill f orm w ith se aw at er

s am ple s. C ap te st

tube s f ai rly ti g htly . I nv er

t tw ice.

5VH. A dd 15 m

L of O x idiz ing Re ag ent . A

pre ci pita te w ill f orm w ith se aw at er

s am ple s. C ap te st

tube s f ai rly ti g htly . I nv er

t tw ice.

6. P lace test tubes in a pressure cooker a t 10 0 - 11 0°C a

nd 3 - 4 psi for 60 m inu tes. Bring back to atm osph eric pressure ove r 1 hour.

7. Re m ov e te st

tube s, ti g hte n c aps a nd c ool t o room te m per at ure . S am ple s ca n be s tore d a t t

his point

.
8. A dd 1 m

L of Buf f er Solut ion t o e ac h tube a nd s hak e. T he pH of the s am ple s hould be 4 - 4.5 a f

te r t he addi tion of

the bu f f er solutio n. Tra

nsfe r an aliquo t of e ach sam ple to AutoAnaly zer c up s using a Pasteur pi pette.

9

. An al yze fo r

n i t r i t e + n i t r at e a n d o r t h o p h o sp h at e a s d es cr i b ed i n t h e D i ss o l ve d In o r ga n i c An al yte s s ec t i o n

. Us e t h e p u mp

3 4 tube s f or l ow s am ple c once ntra

tions of 0.00 - 1.00 m g N /L ; O RN /Y EL N O s am ple tube a nd Y EL /B L U NH Cl tube , f or -

3 4 m o d era te sampl e co n cen tr at io n s o f 0. 3 5 - 1 .4 m g N/L; ORN /GRN N O sampl e tu b e an d YEL/ BLU NH Cl tu b e, for h igh

-

3 sa m ple c once ntra

tions of 0.70 - 2.80 m g N /L ; O RN /W HT N O s am ple tube a nd Y EL /Y EL de ionz ed w at er dil uent on t he

-34

4 dilu tion loo p "resa

m pled " to a BL K/BL K res am ple tub e a nd YEL /YEL NH Cl tub e.

9V H. A naly ze f

or ni trite + nitrate and orth op ho sphate as desc ribed in the Dissolved Ino rganic A naly tes se ction . Use a

3 BLK/ BLK NO sampl e tu b e an d YEL/ YE L dei o n zed w at er d il u en t o n th e d il u ti o n lo o p "resa m p le d " to a BLK/ BLK

-

4 resam ple tub e a nd YEL /YEL NH Cl tub e.

NO T ES:

1. T he use of

int er nal org ani c st anda

rds (g luta m ic a ci d a nd g ly ce rophos phat e) a llow s the c hec k f or pe rc ent ag e re cov er

y a nd is inc orpor at ed into ea ch dig es tion bat ch.

2

. Th e p r o ce d u r e i n cl u d es an i n t er n al d i l u t i o n fac t o r o f sa mp l es an d st an d ar d s d u e t o ad d i t i o n o f re ag en t s o f 1 : 1

. 6 fo r l o w N and P concentratio n sam

ples and 1 :4. 2 for v ery

high N and P concentratio n sam ples.

3. Reag ent Blanks: Ox idizin g re age nt o nly (3 replicates) is digested in 30 m L test tubes, and b uff ered. T he analy zed peak hei g hts of the nit ra te a nd phos phat e re ag ent bla nk s ar e norm al iz ed to t

he sa m ple + re ag ent

v olum e by

a dding 10 m

L of dei oniz ed w at er

im m edi at el y be f ore a nal y zi ng f

or ni tri te + nit

ra te a nd phos phat e. T he re sul ta nt r ea g ent bla nk pe ak he ig ht then is subtracted f

rom the sam

ple peak heig hts before c alculating the concentratio ns based on the reg ression from the peak heights of the sta

nd ards. Reag ent blan k f

or N shou ld b e # 8% of

the pe ak he ig ht of the 75 : M N s ta

ndar d. R ea g ent

bla nk f or P shou ld b e # 6% of

the 4.8 : M P st anda

rd.

4. De ioniz ed W at er

Bl ank s: 10 m

L of de ioniz ed w at er

(3 re plic at es ) a

re dig es te d, a

nd a nal y ze d w ith the s am ple s and st anda rds . T hei r pe ak

he ig hts a re inc lude d w ith the pe ak he ig ht of the dig es te d inor g ani c w ork ing s ta ndar ds in t

he

re g re ss ion t o obta in t

he sl ope use d to ca lc ula te c once ntra tions of the s am ple s. T he v al ue of

the re ag ent

bla nk is g ene ra lly about 0 - 5 c har

t l ine s le ss tha

n the v al ue of

the y int er ce pt obt ai ned in t

he re g re ss ion of

inor g ani c w ork ing s ta

ndar ds and deio nized w ater blanks.35


## T OTAL AN D 

## D IS SOLV ED 

## O RG ANIC Re v i s e d 02 / 2004



## C A R B ON

The Shim adz u TOC 50 00/50 00A utiliz es h ig h tem perature com

bustion in determ ining org anic c arbon , and is use d to analy z e all rang es o f salinity

.

S HIM A D Z U T OC -5 00 0 T OT A L OR G A N IC C A R BON A N A L YZ E R ME T HOD Method ology
Sug im ura, Y. and

Y . S uz uk i. 1988

. A hig h tem perature cataly tic ox idation m ethod for the determ ination of n on
- v ol at il e di ss ol v ed

o rg an ic c ar

bo n in s ea w at er

b y d ir ec t i njec ti on

o f a li qu id

s am pl e. M ar

. Che m . 2 4:105

- 131.

Instru mentation

The Shim adz u TOC

- 5000 /5000 A uses a hig h tem perature com

bustion m ethod to an aly z e aq

ueou s sam

ples fo r TI C , TO C a nd

n on
- pu rg ea

bl e or g an ic c ar

bo n (N P O C ). S am

pl es a re tr ea te d w it h hy dr oc hl or ic a ci d and sparg ed w ith ultra pure carrier g rade air to driv e off inorg anic c arbon

. Hig h tem perature com

bustion (680° C) on a cataly st bed of platinum

- coated alum ina b alls brea k s do wn org anic c arbon into carbon

2 2 di ox id e (C O ). Th e C O is c ar ri ed b y u lt ra p ur e ai r to a n on
- di sp er si v e in fr ar ed d et ec to r (N D I R ) w he re

2 CO is d etected.
Spe cif ication s

A n al yt ic al R an ge : 100 ppb

- 40 00 ppm usin g 25 0 : L s y ring e an d 4 - 10 0 : L inj ection v olum e, using

re g ul ar s en si ti v it y

c at al y st

. C al ib ra ti on : U p to 4 co ncentratio ns m ay be used to calib rate the instrum ent

R ea ge n ts
Hydroch loric A cid, 2 N H yd r o c h l o r i c a c i d

( H C l ) , c o n c e n t r a t e d 1 7 2 mL D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

In a 1000 m L v olum et

ric f la sk , a dd 172 m

L of c once ntra

te d hy droc hlori c ac id t o - 6 0 0 mL of d ei o n i ze d wat er

. Di l u t e t o 1 0 0 0 mL

w ith dei oniz ed w at er

.
Stan dard

S to ck Po ta ss iu m Hy d ro ge n Ph th al at e (KH P) S ta n d ar d , 1 00 0 mg /L

6 4 P ot as si um

h y dr og en

p ht ha la te ( H O C O C H C O O K), dried at 45 ° C 2.1 25 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

In a 1000 m L v olum et

ric f la sk , di ss olv e 2.125 g of

pota ss ium

hy drog en phtha la te in
- 800 m L of de ioniz ed w at er . D ilut e to 100036

m L w ith d eion ized w ater. Mak e f resh ev ery

4 - 6 m on ths. Sto re at 4°C.

Procedur e

1. Sa m

ple s ar e st ore d f

roz en at -20° C. When re ady

to ana ly ze , t haw

s am ple s at room te m per at ure a nd tr ans f

er to auto sam pler vials.

2. Ma k e 3 w ork ing s ta ndar ds (g ene ra lly

< 20 m g /L ) f

rom the Stoc k

K HP Sta ndar d f or t he st anda rd c urv e, a

nd loa d int o auto sam pler. Use deionized wa

ter as the zero concentration . Num erous stand ard concentratio ns m ay be analy zed as

sam ples.

3. A ci dif

y e ac h s am ple w ith 100 : L of

2 N H y droc hlori c A ci d a nd a dd 250 : L of 2 N H y droc hlori c A ci d to ea ch st anda rd

v i a l

. I t i s p o s s i b l e t o p r o g r a m t h e i n s t r u me n t t o d o t h i s a

u t o ma t i c a l l y.

4. T he st anda

rds a nd s am

ple s ar e spa

rg ed w ith ultr a pure c ar

rie r ( UPC) g ra de A ir f

or 6 m inute s at

60 m L /m inute , dr iv ing off any ino rganic c arbon in the sam ple.

5. Sta

ndar ds and sa

m ple s ar e inje ct ed onto a

c at al y st be d of platinum -c oat ed al um ina ba lls a nd c om

bust ed at 680° C. T he

2 re sul ting CO g as is c ar

rie d to the N DI R de te ct or by the U PC g ra de A ir.

6. For the s ta

ndar d c urv e, us e the a re a of the de ioniz ed w at er

a s the w at er

bla nk in ca lc ula ting the 4 s ta

ndar d concentratio ns.

7. Ca lc ula te a

li nea r r eg re ss ion of

the 4 s ta ndar d c once ntra

tions , f irs t s ubtra ct ing the w at er

bla nk f rom e ac h s ta ndar d.

8. T he abs olute v al ue of the y -int er ce pt i s an indic at or of the ins trum ent

bla nk a nd is s ubtra ct ed f

rom the a re a of

the

sam ples. T he w ater blank includ es the instrume nt b lank, so on ly one value is s ub tracted f

rom the standard s.

C al cu la ti on o f T OC

TOC conce ntration is calcu lated u sing the follow ing equ ation:

s whe re: A = area of the sam ple, b = y
- intercept, and

m = slop e of the reg ression line.

D is so lv ed in or ga n ic c ar b on , c ar b on at e al k al in it y, a n d h ar d n es s:

Disso lv ed in org anic c arbon also can be d eterm ined with direct rea ction with 25% p hosp horic acid. O nce the in org anic c oncen tration is determ ined, carb onate alk alinity can be calculated

usin g the follow ing equatio n:37

NO T E: T hi s m e t ho d c a n a l s o b e us e d o n s a l i ne wa t e r s of < 1 0 p pt sa l i ni t y. Titr im et ri c ha rd ne ss d et er m in at io n is a pp ro pr ia te f

or f re sh w at er s am pl es ( A P H A , 1 97 5)

.
Method ology

AP HA

. 1975

. Method 309B

. ED TA Titrim etric Metho d in Stand ard Meth ods for the Ex am ination of Water and

Wastew ater, 14 Ed ition. A m erican Pub lic H ealth A ssociation

. Washing ton, D. C. th 1193 pp.38


## PARTI CU LATE ANA LYTES

R at io n al e

The direct m easurem ent o f particu late carb on, particu late nitro g en a nd particulate phos phoru s is the preferred m ethod used by the Nutrien t A naly tical Se rv ices L aborato ry

. A larg e v olum e can be filtered onto the p ad, y ielding a represen tativ e sam ple. The altern ativ e, subtractio n o f the dissolv ed c oncen tration from the total sam

ple co ncentratio n to determ ine th e pa rticulate c arbon , n itrog en o r ph osph orus
conce ntration, is im

precise, som etim es y ielding neg ativ e co ncentratio ns. D irect m easurem ent is rapid, sensitiv e an d m ore p recise.

Instru mentation

Particulate ph osph orus and biog enic s ilica pro cedure s requ ire the use of a seg m ented continu ous flow analy z er suc h as the Auto Ana ly z er I I , p rev iously des cribed in the sectio n D issolv ed I norg anic A naly tes. P ar ti cu la te c ar

bo n an d pa rt ic ul at e ni tr og en p ro ce du re s re qu ir e th e us e of a n el em en ta l a na ly z er

. Samplin g a nd Stora ge

A k no w n v ol um e of

th e co ll ec te d w at er

is f il te re d th ro ug h Wh at m an G F /F f il te rs ( 25 m

m f or p ar ti cu la te carbon and nitrog en, and 47m

m for particulate phos phoru s, nom inal p ore siz e 0. 7 : m ). Th e fi lt er is folded, place d in an a lum inum foil pouc h an d froz en u ntil ana ly sis. Fo r biog enic s ilica, w ater is filtered throug h a 0.4 μm

N uclepo re po ly carbon ate filter. The filter is placed in a 50 m L p lastic cen trifug e tub e and stored in a refrig erator.

Sedim ent sa m

ples a re co llected, dried and g round w ith a m ortar an d p estle to thorou g hly blen d the

sam ple.39



## P ARTICULATE 

## C AR BON A ND 

## N IT ROGEN Re v i s e d 2/ 2004

2 Sam ples a re co m

busted in pure oxy g en (O ) un der sta tic con ditions. P roduc ts of com bustion are passed

ov er suitab le reag ents in

the com bustion tube w here com

plete o xidation

occ urs. I n the redu ction tube,

2 ox id es o f ni tr og en

( N ) ar e co nv er te d to m ol ec ul ar N

. Th e ca rb on

d io xi de ( C O ), w at er

v ap or a nd N a re
m ixed and released into th e therm al con ductiv ity detec tor w here the co ncentratio ns o f the sam ple g ases
are m easured

.
Instru mentation

Exete r A naly tical, I nc. (EA I ) C E- 440 Elem ental A naly z er
Operatin g Princip les

2 2 2 C Carb on (as C O ), h y drog en (a s H O) and nitrog en (N ) con tent in org anic a nd inorg anic c om poun ds

can be d eterm ined.

2 C C om bu st io n of

th e w ei g he d or

f il te re d sa m

pl e oc cu rs in

p ur e O u nd er

s ta ti c co nd it io ns ( F ig ur e 13 ).

Figu re 1 0. Sch ema tic diag ram of the E

xeter A nalytical, Inc. (

EAI) C E-44 0 E lemen tal A nalyzer

.40

C Helium

(H e, ultra- pure carrier g rade) c arries the com bustion pro ducts throug h the ana ly tical sy stem to th e at m os ph er e. H el iu m a ls o pu rg es th e in st ru m en t.

C Solen oids A

- G con trol the g as flow throu g h the sy stem

. Valv es H and I are used for au tom atic leak t e s t i ng.

C Prod ucts o f com

bustion pas s ov er suitab le reag ents in the com bustion tube w here com

plete o xidation

occurs.

2 C I n th e re du ct io n tu

be , o xi de s of

N a re c on v er te d to m ol ec ul ar N

. Th e C O , w at er

v ap or a nd N a re then flushed

into a m ixing v olum e w here they are thorou g hly

ho m og eniz ed a t a p recise v olum e, tem perature and pressure

. The m ixture is release d thro ug h the sam ple v olum e into the therm al

co nd uc ti v it y d et ec to r.

C Betw een the first of thre e pa irs of therm al con ductiv ity cells, an absorp tion trap rem ov es w ater

2 (H O) from

the sam ple g as. The differe ntial sig nal rea d b efore a nd after the trap reflec ts the am ount

2 of h y dr og en

( as H O ) in th e or ig in al s am pl e. A s im il ar

m ea su re is m ad e of

th e si g na l o ut

pu t o f a

2 secon d p air of therm al con ductiv ity cells betw een whic h a trap rem ov es C O

. The rem aining g as

2 consists only

of N and

H e. This g as p asses throug h a therm al con ductiv ity cell and the o utput

2 sig nal is com

pared to a reference cell thro ug h w hich pure He flows. This g iv es the N con centration

.
Stan dard

3 6 5 Stand ard is acetanilide (C H CO NH C H ) stored at 4 5° C. C om

position of acetanilide is 7 1.0 9% C , 6 .71 % H and 10.3 6% N.

Procedur e

1. Fil te r a

k now n v olum e of

s am ple w at er thr oug h a

W hat m an G F/F f

ilt er pa d (25 m

m ; 0. 7 : m

pore s iz e; pre -c om

bust ed in oven set a t 50 0°C f or 90 m inu tes a

nd cooled to room te m

perature in a dessicator. T his setting on CBL ’s oven has been determ ined to be 55 0°C.) Filter sam ples in du plicate f

or back-up and 10% QA

/QC.

2. Fol d filter pads in half inward, and place in an alum inu m f oil p ou ch. L abel and fre eze f or later analy sis.

3. W hen re ady

f or a nal y si s, re m ov e al um inum f oil pouche s f

rom f re ez er a nd pla ce in a dry ing ov en at 45° C ov er

nig ht.

4. Load nickel sleev es (pre-com bu sted at 875 ° C f

or 90 m inu tes a

nd cooled to room te m

perature) int o th e f

ilter ram . L oad f ilt er pa ds into the nic k le s le ev es . Pla ce loa ded nic k el s le ev es int o the s am ple w hee l.

5. Dr y s edi m ent s am ple s at 45° C ov er

nig ht a nd w ei g h int o a lum inum

c aps ule s (pr e- com

bust ed at 550° C f or 90 m inute s and cool ed to r oom

te m per at ure ). L oad nic k el s le ev es int o the s am ple w hee l a nd pla ce a lum inum

c aps ule s cont ai ning the sedim ent sam

ples into the nickel s leev es.

6. Load standards (
- 1500 : g a cetanilide) and blanks (em pty ca psul e f or stand ards and w eighed sam ples; f ilter pad for f

iltered

sa m ple s) int o the s am

ple w hee l. A pprox im at el y

50 s am ple s ca n be run per

da y

.
7. Plac e 3 s ta

ndar ds at the be g inning of

e ac h run to de te rm ine the K -Fa ct or.

8. Ins er

t s ta ndar d c hec k s bet

w ee n e v er

y 15 - 20 s am

ple s and at

the e nd of

e ac h run.41

T h e R u n C yc le

1. A t t he st ar t of e ac h run, the e ntir e sy st em is f lus hed w ith hel ium

(H e) a t a hig h f low ra te w hile the s am

ple is in the c ool

zone.

2. The injectio n bo x is auto m atically purged usin g the P valve

.

2 3. T he com

bust ion t ra in i s f ill ed w ith O a nd the s am ple is inj ec te d.

4. Short ly

a f te r s am ple inj ec tion, D v al v e cl ose s to s ea l of f the c om bust ion t ra in f rom the re st of the a nal y tic al s y st em

that i s still bein g f

lush ed w ith He.

2 5. Com

bust ion oc cur s under

s ta tic c onditi ons in a

n e x ce ss of

O at a bout 975° C. D uring thi s tim e, the m ix ing v olum e is be ing purg ed w ith E a

nd F v al v es ope n.

6. V alve F closes to allow the pressure in the m ixing v olu m e to reac h atm osph eric pressure.

7. Cl ose to the e nd of the c om bust ion pe riod, a

hig h-te m per at ure he at ing c oil ar ound the c om bust ion t ube v apor iz es a ny cond ensates at the entrance of the com bu stion tub e that m ay

have been pro du ced by

diff usio n of the sa

m ple du ring init ial st ag es of c om bust ion.

2 8. T o a ss ure c om

ple te c om bust ion, the la dle is re tra ct ed
- 1 inc h, a s m al l a m ount of O is a dded and the la dle is f ully inj ec te d.

9. Dur ing hig h he at , v al v e E c los es , A a nd D re -ope n a nd the c om bust ion pr oduct s ar e com ple te ly f lus hed f rom the

com bu stion train into the m ixing v olu m e.

1 0

. Wh en a p r es su r e o f 1 5 0 0 mm Hg i s r ea ch ed , va l ve D c l o se s, t r ap p i n g t h e s amp l e g as i n t h e mi xi n g vo l u me. Th e t i me requi red to reach this pressure is ca lled "f ill tim e" and is usually 20 - 50 second s.

11

. The com

bu stion train rem ains un der po sitive press ure un til th e e nd of the com plete cy cle.

12

. While the sa

m plin g g ases are m ixing, pure He f lows f rom valv e C throu gh the sam ple volume and th rou gh the detectors.

13. T he si g nal f

rom e ac h de te ct or br idg e is re ad and st ore d in m em ory to prov ide a

ba se line re adi ng w ith no s am ple g as in the det ec tor.

1 4

. Aft er mix i n g i s c o mp l et e a n d b as el i n e r ea d i n g i s s et , F an d G o p en al l o wi n g t h e s amp l e g as ca p t u r ed i n t h e mi xi n g vo l u me to exp an d th ro u gh th e sampl e vo lu m e to th e atmo sp h ere . Du ri n g th is ti m e, valve C is cl o sed an d th ere is l o w flo w th ro u gh the de te ct or.

2 2 2 15. W hen sa

m ple g as es a re ne ar

a tm osphe ric pre ss ure , v al v es F a nd G c los e and C ope ns. T he H O, CO a nd N c once ntra tions of the sa m ple are m easured by

displ acing the sa

m ple gas thro ugh th e detectors to the atm osph ere.

16

. The volume of sam ple gas in the sy stem

is larg e e no ugh so th at the He f low a llows m easurem ent of the contents of ea ch detector in sequ ence, und er ste ady -state c on diti on s.

17. T he sa

m ple g as pa ss es thr oug h the de te ct ors a t a c onst ant f low , pr es sur e and te m

per at ure . T his e lim ina te s any

v ar ia tion in wat er va p o r co n ce n t r at i o n d u e t o ch an ge s i n wat er ad so r p t i o n o n t h e wa l l s o f t h e p n eu mat i c s yst em.

18. W hile the s am

ple g as is dis pla ce d thr oug h the de te ct ors , t he output si g nal s ar e re cor ded.

19

. The diff

erence in m icrovolts ( : V) be tw ee n e ac h " re ad" s ig nal a nd the ba se line (" ze ro" ) l ev el f or t he sa m e det ec tor is dire ct ly propor tiona l t o the c once ntra

tion of the s am ple g as m ea sur ed.

20

. A t the end o f

a c y cle, the ex haust valve s open to allow the sam ple gas es to esc ape to the atm osph ere.

21. T he com

pute r t hen print s out t

he ca lc ula te d re sul ts , pl ac es the ins trum ent in st andby

w ith C v al v e open and w ai ts f or t he

nex t c om m and.42

22

. With the HA

automa tic injector, the results are prin ted af ter ea ch run . T he run cy cle c on tinu es until th e pre-sele cted

num ber of runs ha v e bee n c om

ple te d.

D efin it io n o f T er ms

Bla n k : B la nk

v al ue = b la nk

r ea d (s ee R ea d S ig na l b el ow ) m in us b la nk z er o (s ee Z er o Val ue b el ow ).

C ap su le : P re
- co m

bu st ed a lu m in um

o r ni ck el c on ta in er

. U se d fo r se al in g s am pl es w it h an

a cc ur at e weig ht an d m aintains integ rity prior to co m

bustion

.

2 Comb ustion T ime: Ti m e fo r sa m pl e to f ul ly c om bu st in O e nv ir on m en t.

C om b u st io n T u b e: Qua rtz tube use d fo r pac k ing reag ents a nd for sam ple co m bustion

.
C on d it io n er : C oa ts th e w al ls o f th e sy st em s ur fa ce s, p ar ti cu la rl y

th e m ix in g a nd

s am pl e v ol um es , w it h

2 2 2 H O v ap or , C O a nd

N th at s im ul at e ac tu al s am

pl e ru nn in g c on di ti on s. Th e co nd it io ne r w e us e is acetanilide as recom

m ended by the m anufac turer. A sam ple is consid ered con ditioner whe n p laced be fo re a b la nk

o r a st an da rd

in a r un

. B la nk s sh ou ld

b e ru n im

m ed ia te ly a ft er a c on di ti on er

.
Detec tor: The heart o f the analy z er co nsisting of three b ridg es. D eterm ines th e pe rcentag es o f C , H and N i n t he sa mpl e via th e r mal co nd uc t i vit y.

D et ec to r Ov en : Ke ep s th e te m pe ra tu re o f th e de te ct or , p re ss ur e tr an sd uc er , m ix in g v ol um e an d sa m pl e
v ol um e co ns ta nt

.
D ou b le D ro p : O n H A autom ation, tw o sa m ples a re dro pped

for one run used for >2 5 m m filters. Sam ple I D requires a "+" p refix.

Fill Time: T i me r e qu i r e d t o b ui l d u p t he pr e s s ur e in th e m i xi ng vol ume to 15 00 m m Hg.

Fur n ac e: H eats th e red

uction and com

bustion tube s to operatin g tem

perature.
Injectio n: Mo v ing the ladle in to the com bustion furna ce.

I nj e c t i on B ox: F or

th e H A a ut om at io n, th e bo x as se m bl y th at h ou se s th e sa m

pl e w he el

.
K-Fa ct or : I nstrum ent se nsitiv ity factor in : V/ : g , c alibrated using a chem ical stan dard.

L ad le : Transports th e bo at or capsu le con taining the sam ple in to the com

bustion furna ce.

Mix ing Volu me: S pherical bottle in w hich sam

ple g ases b ecom e ho m og enou s.

Mother Board: The m ain circ uit bo ard. Where all C E- 440 pow er sup plies a re loca ted.

R ea d S ig n al : S te ad

y
- st at e si g na l p ro du ce d by th e de te ct or

w he n sa m

pl e g as es a re p re se nt

in s ta bl e
conce ntration.43

2 Red uction T ube : Q uartz tube w ith red uced copp er that rem ov es ex cess O from the sam ple g as an d

2 re duc es ox ide s of N to fr ee N.

Ru n: O ne s am

pl e an al y si s fr om

s ta rt to f in is h, in cl ud in g p ri nt

- ou t.

R u n C yc le : The en tire ana ly tical seq uence of runs from

the first run to the last run in a day , in cluding t he tr a ns f e r of th e ru n c ycl e da t a to th e di s k.
Sample Volu me: Tube whe re sam ple g as is exhau sted fro m the m ixing v olum e prio r to entering the de te ct or

.

2 S cr u b b er s: Rem ov es w ater an d C O from g as su pplies.

2 Tra ps: U sed for rem ov ing w ater an d C O from the sam ple g as.

Z er o V al u e: B ri dg e si g na l w it h on ly p ur e H e fl ow in g th ro ug h th e de te ct or

.

Calib ration

T he fo ll owin g for m

ula is us ed to cal cul at e K

- Fac tor s, as well as C, N and H conce ntr at ion s in unknown

sam ples:

w he re : K = c al ib ra ti on f ac to r of th e in st ru m en t, W = s am pl e w ei g ht , R = read sig nal o f sam

ple g as, Z = z ero rea ding or instrum ent b aseline, and B = b la nk

s ig na l ( in st ru m en t, la dl e an d ca ps ul es ).

Al wa ys r un a c on di t i on e r be f or e a s t a nd a r d a nd be f

or e an d a f

t e r a b l a nk.

K- Factors v ary

g reatly am ong instrum ents b ut sh ould be w ithin th e follo wing rang es:

C K = 18 - 25

H K = 55 - 76

N K = 7 - 1044


## P ARTI CULATE 

## P HOS P HORUS Revi sed 2/ 2004



### AND 

## P ARTI CULATE 

## I NORGANI C 

## P HOS P HORUS

Am m onium m oly bdate and antim ony

po tassium

tartrate react in an acid m edium w ith dilu te solu tions of phos phoru s to form

an antim ony
- phos pho- m oly bdate com

plex whic h is reduce d to an in tensely blue

colored com plex by asco rbic ac id. C olor is propo rtional to ph osph orus conce ntration.

Method ology and R elated Reference
Asp ila, I ., H. A g em ian an d A .S. Y. C hau. 1 976. A sem i- autom ated m ethod for the determ ination of inorg anic, org anic a nd total ph osph ate in sedim ents. A naly st 10 1:187

- 197.
Keefe, C .W. 1994

. The Co ntribution of I norg anic C om

poun ds to the Particulate C arbon , N itrog en, and Pho spho rus in

Su spend ed Matter and Surface Se dim ents o f C hesap eak e B ay

. Estuarie s 17 :122- 130.

Instru mentation

Tec hn ic on

A ut oA na ly z er

I I w it h I B M c om

pa ti bl e B ra n an d L ue bb e A A C E v er

. 5

. 22 S of tw ar e da ta
c o l l e c t i o n s ys t e m.

Ma n ifol d A ss em b ly

Figu re 1 1. Manifold

asse mbly for particu late p hosp horu s m ethod.45
Spe cif ication s

S ta n d ar d C al ib ra ti on S et ti n g: 4. 0 D am p : N orm al S am p le R at e: 40 /h ou r, 9 :1 s am pl e/ w as h ra ti o Filt er : 88 0 n m Pho to tu b e: 199- B02 1- 04 Flo w ce ll : 5 0 mm R el at iv e A b so rb an ce ( 48 μ M P) : ~.27 In te rfer en ce s: Silico n (S i) at a naly sis tem perature >40° C and/or <2.2 N sulfu ric acid in the m ixed reag ent so lution causes interference in the co ncentratio n ran g e of >0.0 5 m g /m L S i in the ex tract. These cond itions a re av oided by m aintaining an acid c oncen tration o f 2. 45 N sulfuric acid in the reag ents a nd

ana lysis at 37°C.

R ea ge n ts
Hydroch loric A cid, 1 N H yd r o c h l o r i c a c i d

( H C l ) , c o n c e n t r a t e d

( s p

. gr

. 1

. 9 ) 8 6 mL D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

In a 1000 m L v olum et

ric f la sk , a dd 86 m

L of c once ntra

te d hy droc hlori c ac id t o - 800 m L of de ioniz ed w at er . A f

te r c oole d, di lute to 1000 m L w ith dei oniz ed w at er

.
Deio niz ed Water Dilue nt

3 2 1 1 3 Sod ium do decy l sulfate, (SD S) [C H (CH ) OS O Na]

m .w . = 28 8.3 8; ph osph ate # 0.0 001 %] 0.0 5 g D e i o n i ze d

wa t e r u p t o 5 0 0 mL

A dd 0.05 g of

s odium dode cy l s ulf

at e to 500 m L de ioniz ed w at er . M ix w el l.

Sulfuric A cid S olution , 4.9 N

2 4 S u l f u r i c a c i d ( H S O ) , c o n c e n t r a t e d

( s p

. gr

. 1

. 8 4 ) 1 3 6 mL D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

T o a 1000 m L v olum et

ric f la sk , a dd 136 m

L of c once ntra

te d s ulf uric a ci d to appr ox im at el y 800 m L of de ioniz ed w at er

w hile cool ing (c old w at er

ba th). A f

te r t he sol ution is c oole d, di lute to 1000 m L w ith dei oniz ed w at er

.
Ammoniu m Molybd ate S olution

4 6 7 2 4 2 A m m on iu m m ol y bd at e [( N H ) Mo O @ 4 H O] 40 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

In a 1000 m L v olum et

ric f la sk , di ss olv e 40 g of

a m m onium m oly bdat e in 800 m L of

de ioniz ed w at er . D ilut e to 1000 m L w ith dei oniz ed w at er . S tore in pla st ic bott le a w ay f rom dir ec t s unlig ht.

Asco rbic Acid So lution

686 Asco rbic ac id (C H O ) 18 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

3 2 1 1 3 Sod ium do decy l sulfate, (SD S) [C H (CH ) OS O Na] 1. 66 g46

In a 1000 m L v olum et

ric f la sk , di ss olv e 18 g of

a sc orbic a ci d a nd 1.66 g of SD S in 800 m L of

de ioniz ed w at er . D ilut e to 1000

m L w ith d eion ized w ater a nd dispen se - 40 m L into clea n po ly bo ttles and fre eze . T haw overnight in refrig erator bef ore use.

An timony Potassium T artrate Solu tion

446 2 A nt im on y

p ot as si um

ta rt ra te [ K(S bO )C H O @ 1⁄2 H O] 3 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

In a 1000 m L v olum et

ric f la sk , di ss olv e 3 g of

a ntim ony

pota ss ium

ta rtr at e in 800 m L de ioniz ed w at er . D ilut e to 1000 m L w ith dei oniz ed w at er

.
Wo rk in g R ea ge n ts

R ea ge n t A S u l f u r i c Ac i d So l u t i o n , 4

. 9 N 5 0 mL A mmo n i u m M o l yb d a t e So l u t i o n 1 5 mL A n t i mo n y P o t a s s i u m T a r t r a t e So l u t i o n 5 mL SD S 0.1 5 g

R ea ge n t B A s c o r b i c Ac i d So l u t i o n 3 0 mL SD S 0.0 5 g

Stan dard s

Stock Phosp horu s S tand ard, 12 ,00 0 : M P ot as si um

p ho sp ha te , m on ob as ic

2 4 (KH PO ), d

ried at 45° C 1.6 32 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

3 C h l o r o f o r m ( C H C l ) 1 mL

In a 1000 m L v olum et

ric f la sk , di ss olv e 1.632 g of

pota ss ium

phos phat e in
- 800 m L of de ioniz ed w at er . D ilut e to 1000 m L w ith dei oniz ed w at er . A dd 1 m L of c hlorof orm to ac t a s a pre se rv at iv e (1 m L c onta ins 12 : m ole s P).

Workin g Phosp horu s S tand ards

Di lute 0.05, 0.1, 0. 2, 0.3 and 0.4 m L of Stoc k Phosphorus Sta

ndar d to 100 m L w ith 1 N H y droc hlori c A ci d to y ie ld

conc ent ra tions of 6 : M P (0.185 m g P/L ), 12 : M P (0.37 m g P/L ), 24 : M P (0.74 m g P/L ), 36 : M P (1.11 m g P/L ) a nd 48 : M P ( 1

. 4 8 mg P / L) , r e s p e c t i v e l y.

Particulate Phosph orus Procedu re

1. Fil te r a

k now n v olum e of

w at er thr oug h a

W hat m an G F/F f

ilt er (47 m m ; 0. 7 : m

pore s iz e, pre -dr ie d a t 103 - 105° C f or 24 ho urs, if a lso usin g pad f

or to tal suspend ed solid s). Using f orceps, fold filter pad in half , sam ple insid e, and place in an al um inum

f oil pouch. C olle ct

s edi m ent s am ple s in a c le an cont ai ner

.
2. Freeze alum inu m

f oil p ou ches and/or sedim ents.

3. If f ilt er s ar e use d a ls o f

or T ota l Sus pende d Soli ds, dry ov er nig ht at 103- 105 C. D ry s edi m ent s at

50° C ov er

nig ht or unti l o d r y. Gr i n d s e d i me n t s a mp l e s t o b l e n d t h o r o u g h l y.

4. Fold f

ilt er s in qua

rte rs us ing f

orc eps a nd pla ce in num

ber ed cr uci ble s w ith lids . W ei g h s edi m ent int o c ruc ible s. I n our la bora tory , g ene ra lly

a bout 25 m g of

s edi m ent is us ed. C ov er

c ruc ible s. R ec ord s am

ple num ber s and cor

re spondi ng47

crucible nu m

bers. P repare 5% of

sam ples in du plicate.

5. Com

bu st sam ples in crucib les in an oven set at 500 ° C f or 90 m inu tes. T his setting on CBL ’s oven has been determ ined to b e 550 ° C.

6. When crucibles are cool, rem ove f rom m uff le f urn ace . P lace f

ilter or sedim ent in a labelled 50 m L plastic screw ca p centrifuge tub e.

7. A dd 10 m L of 1 N Hy dro chlo ric A cid to each tub e c on tainin g a f ilter. For sedime nts, add 20 m L to each tub e.

8. Ca p tube s and le t s ta

nd f or a m inim um of 24 hour s. Sha

k e tube s se v er al ti m es duri ng the 24-hour pe riod.

9. Tra nsfe r supernatan t to an Auto A naly zer c up w ith a P asteur pipette.

10. A nal y ze f

or phos phat e usi ng a n A

utoA nal y ze r I I s y st em

e quippe d w ith a T ef

lon s am ple probe . S pik e 5% of

s am ple s 1:1

w ith 1 .11 m g P/L standard solu tion and analy ze.

11. Bl ank

f ilt er pa ds shoul d be c ar

rie d thr oug h the proc edur e abov e; a pprox im at el y

one bla nk pe r - 20 sam

ples.

Calc ulation of Phosph orus C once ntratio n

Pho spho rus co ncentratio n is calculated usin g B ran a nd Lueb be A AC E v er.5. 22 softwa re. The calc ulation is bas ed o n the follow ing equ ation:

whe re: B = m ean reading of blank s, F = inv erse o f the reg ression slope of the stand ards,

E V = v olum e of hy droch loric ac id us ed fo r extrac tion (L )

( i.e. , 0 .01 for filters and

0. 02 for sed im ents), and

F V = v ol um e of

s am pl e fi lt er ed

( L ).

Technicon Auto Ana ly z er I I w ith 50 m m flow cell an d S TD C AL setting of 4.0 ty pically g iv es the follow ing results:
slope 43 .63

r 0 .99 99 2

Particulate Inor gan ic Phosp horu s Proced ure

1. Fol low procedure for Particu late Ph osph oru s Steps 1 thro ugh 3. Do no t com bu st the sam

ples. P lace f

ilter or w eighed

se dim ent s am ple dir ec tly

in la bel ed 50 m L s cr ew

c ap ce ntri f

ug e tube . Fol low

proc edur e f

or S te ps 7 thr oug h 11. 2. Ca lc ula te Part ic ula te Inor g ani c Phosphorus c once ntra tion.48


## P ARTICULATE 

## B IO G ENIC 

## S IL IC A Re v i s e d 2/ 2004

Silicom oly

bdate is redu ced in acid

solu tion to "m oly

bden um blue " by asco rbic ac id. O xalic a cid is added t o e l i min a t e in t e r f

e r e nc e fr om ph os ph a t e s . De t e c t i on of th e si l i c omol ybd a t e co mpl e x i s by co l or i met r y.

Method ology

T e c hn i c on I nd us t r i a l Me t ho d No . 1 86

- 72 W / B . T e c hn i c on I nd us t r i a l Sys t e ms. T a r r yto wn , Ne w Yo r k, † 1059 1.

Paasc he, E. 19 73. S ilicon a nd the ec olog y of m arine p lank

ton d iatom s. I

. Tha lassiosira pse udon ana ( Cyclo tella na na) g rown

in a ch em ostat w ith silicate as lim iting nu trient. Mar. Biol. 19 :117- 126.

Instru mentation

Tec hn ic on

A ut oA na ly z er

I I w it h I B M c om

pa ti bl e B ra n an d L ue bb e A A C E v er s. 5

. 22 S of tw ar e da ta collection sy stem

Ma n ifol d A ss em b ly

Figu re 1 2. Manifold

asse mbly for particu late b iogenic silica meth od.49
Spe cif ication s

Stan dard C alibra tion Setting : 5. 0 D am p : N orm al Samplin g R ate: 4 0/ ho ur , 9 :1 s am pl e/ w as h ra ti o Filt er : 660 nm Pho to tu b e: 19 9- B02 1- 04 Flo w ce ll : 5 0 mm R el at iv e A b so rb an ce ( 42 μ M S i) : ~.27

Interferences: Tannin , larg e qu antities o f iron and sulfide, stron g colo ration and turbidity m ay interfere.
Dige stion Eq uipmen t

2 di g it al ti m er s 10 0° C w at er

b at h ic e ba th

c e nt r i f uge tu be ra c ks 2 p oly propy lene re

- pipettes 2 1 00 0 mL po l ypr op yle ne vol ume t r i c fl a s ks

D ig es ti on R ea ge n ts

Sod ium Hydr ox ide , 0. 2 N Sod ium hy droxid e (N aOH ) 8 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

In a 1000 m L poly propy le ne v olum et

ric f la sk , di ss olv e 8 g of

s odium hy drox ide pe lle ts in
- 800 m L of de ioniz ed w at er . D ilut e to 1000 m L w ith dei oniz ed w at er

.
Sulfuric A cid S olution , 1 N

2 4 S u l f u r i c a c i d ( H S O ) , c o n c e n t r a t e d

( s p

. gr

. 1

. 8 4 ) 2 8 mL D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

In a 1000 m L v olum et

ric f la sk , c ar ef

ully a dd 28 m

L of c once ntra

te d s ulf uric a ci d to
- 800 m L of

de ioniz ed w at er . W

hen cool , dilut e to 1000 m L w ith dei oniz ed w at er

. Sample Collec tion

1. Fil te r a

k now n v olum e (v olum e depe ndent on w at er s ourc e) of

w at er thr oug h a

0.4 : m N ucl epor e poly ca

rbona te f ilt er

. ®

2. Fol d filter in half

, sam ple insid e, and place in a labelled 50 mL pol y pro py lene centrif

uge tube, cap and ref

rigerate.

Procedur e

1. Fill wa

ter bath to depth that w ill cover liquid in tub es a nd heat to 10 0°C.

2. Prepa

re ic e bat h.

3

. To t h e sampl e p ad in th e cen tr ifu ge tu b e, ad d 10 m L of 0. 2 N So d iu m Hy d ro xid e fro m

a po lypr o p y le n e re- p ip et te . M ake50

sur e tha t t he f ilt er pa d is c ov er ed by

the Sodium

H y drox ide . C ap the tube le av ing it loos ene d 1⁄4 tur

n.

4. P lace the centrifuge tub e in the 10 0°C w ater bath f or exac tly 20 m inu tes.

5. A f ter ex actly

20 m inu tes, rem ove the tube from the ho t w ater bath and p lace in ice bath for ex actly 4 m inu tes.

6. A dd 2.5 m

L of 1 N Sulf uric A ci d Solut ion t o tube to neut ra liz e. C ap and sha

k e. T he sa

m ple s ca n be s tore d now , unt il

ana ly ze d.

7. Tra nsfe r ex tract to an A

uto A naly zer c up using a poly ethy lene P asteur pipette. A void p articulate pieces. Sam ples m ay need to b e c entrifuged at 30 00 rpm f or 10 m inu tes.

8. A nal y ze f

or s ili con on a n A

utoA nal y ze r I I s y st em

.
9. Di g es t bl ank s and st anda rds int er spe rs ed w ith sa

m ple s. D ig es t 5% of

s am ple s in dupl ic at e and spi k e 5% of s am ple duplic at es w ith 0.4 m

L of Se conda ry Sil ic a Sta

ndar d (2. 5 : M Si /m L ). A nal y ze bla

nk s and st anda

rds be f ore s am ple s to establish stand ard curve.

A n al ys is R ea ge n ts

Ox alic A cid S olution

2 2 4 2 O xa li c ac id

( H C O @ 2H O) 50 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

In a 1000 m L poly propy le ne v olum et

ric f la sk , di ss olv e 50 g of

ox al ic a ci d in
- 9 0 0 mL de i o n i ze d wat er an d d i l u t e t o 1 0 0 0 mL

w ith dei oniz ed w at er

.
Asco rbic Acid So lution

686 Asco rbic ac id (C H O ), U .S. P. quality 17. 6 g

3 2 A c e t o n e [ ( C H ) C O ] 5 0 mL

3 2 1 1 3 S od iu m d od ec y l s ul fa te , ( S D S ) [C H (C H ) O S O N a] , m .w . = 28 8.3 8; ph osph ate # 0.0 001 %] 0.3 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

In a 1000 m L poly propy le ne v olum et

ric f la sk , di ss olv e 17.6 g of

a sc orbic a ci d in 500 m L of de ioniz ed w at er

a nd 50 m

L of

ac et one. Mi x a nd dil ute to 1000 m L w ith dei oniz ed w at er . A dd 0.3 g of s odium dode cy l s ulf

at e. D is pens e 200 m L a liquot s into po ly bot tles and f

reez e.

Sulfuric A cid S olution , 0 .07 N

2 4 S u l f u r i c a c i d ( H S O ) , c o n c e n t r a t e d

( s p

. gr

. 1

. 8 4 ) 1

. 9 6 mL D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

In a 1000 m L poly propy le ne v olum et

ric f la sk , a dd 1.96 m

L of c once ntra

te d s ulf uric a ci d to
- 800 m L of

de ioniz ed w at er . D ilut e to 1000 m L w ith dei oniz ed w at er

.
Ammoniu m Molybd ate S olution

4 6 7 2 4 2 A m m on iu m m ol y bd at e [ (N H ) Mo O @ 4 H O] 7 g S u l f u r i c Ac i d So l u t i o n , 0

. 0 7 N u p t o 1 0 0 0 mL

Di ss olv e 7 g of

a m m onium m oly bdat e in 1000 m L of

0.07 N Sulf

uric A ci d Solut ion. Stor e in a n a m ber pla st ic c onta ine r i n the dark. Prep are f

resh ev ery

f ew day s.51

Stan dard s

Stock Silica Sta nda rd, 10 ,00 0 : M

2 6 Sod ium silicofluo ride (N a SiF ), d ried at 45° C 1.8 8 g D e i o n i ze d

wa t e r u p t o 1 0 0 0 mL

In a 1000 m L poly propy le ne v olum et

ric f la sk , di ss olv e 1.88 g of

s odium s ili cof luori de in
- 800 m L of

de ioniz ed w at er . D ilut e to 1000 m L w ith dei oniz ed w at er (1 m L c onta ins 10 : m ole s Si)

.
Seco nda ry S ilica S tand ard S t o c k S i l i c a St a n d a r d 2 5 mL D e i o n i ze d

wa t e r u p t o 1 0 0 mL

I n a 1 0 0 mL po l yp r o p yle n e v o l u met r i c fl as k, d i l u t e 2 5 mL of S t o ck S i l i ca S t an d ar d t o 1 0 0 mL wit h d ei o n i ze d wat er (1 mL cont ai ns 2.5 : m ole s Si)

.
Workin g S ilica S tand ards

To labelled 50 m L po ly

pro py lene scre w

ca p centrifuge tub es, add 0, 0 .1, 0.2 , 0.3 , 0.4 , 0.5 and 0. 6 m L of Sec on dary Silica Sta ndar d. D ig es t s ta

ndar ds as s am

ple s. A f te r di g es tion, s ta

ndar d tube s w ill c onta in 0, 7.0, 14.0, 21. 1. 28.1, 35.1 a nd 42.2 : g Si,

r e s p e c t i v e l y.

NO T E: T he use of

g la ss w ar e w ith this m et

hod s hould be a v oide d be ca use it m ay be a s ourc e of Si cont am ina tion. A ll che m ic al s
use d f or r ea g ent s and the de ioniz ed w at er s hould be of

g ood qua lit

y a nd low in Si c once ntra

tion.

Calc ulation of Silica Con centra tion

S il ic a co nc en tr at io n is c al cu la te d us in g B ra n an d L ue bb e A A C E v er

. 5

. 22 s of tw ar e. Th e ca lc ul at io n is based on the follow ing equ ation:

whe re: B = m ean reading of the b lank s, F = inv erse o f the reg ression slope of the stand ards, and V = v olum e filtered (L)

Technicon Auto Ana ly z er I I w ith 50 m m flow cell an d S TD C AL setting of 5.0 ty pically g iv es the follow ing results:
slope 1 .60

r 0 .99 98 252


## T O TAL 

## S US PE ND ED 

## S O LIDS Re v i s e d 2/ 2004

Tot al s us pe nd ed

s ol id s (TS S ) is th e re ta in ed m at er ia l o n a st an da rd

g la ss f il te r pa d af

te r fi lt ra ti on

o f a well- m ixed sam

ple o f w ater. R esults a re ex pressed in m g /L.

Method ology

AP HA

. 1975

. Method 208 D. Total N onfilterable Resid ue D ried at 103

- 10 5 C (Total Su spend ed Ma tt er ) in Stan dard Methods for the Ex am ination of Water and Wastewa ter, 1 4 Ed ition. A m erican th Pub lic H ealth A ssociation

. Washing ton, D. C. 1 193p p.

U S E P A

. 1 97 9. M et ho d N o. 1 60

. 2 (w it h sl ig ht m od if ic at io n) in Method s for chem ical an aly sis of water and waste s. U nited States Env ironm ental P rotection Ag ency , O ffice of Rese arch and Dev elopm ent. Cinc innati, Ohio

. Rep ort N o. EPA

- 600/4

- 79- 020 March 1 979. 4 60pp

. Spe cif ication s

In te rfer en ce s: E xc es si v e re si du e m ay f or m a w at er
- tr ap pi ng c ru st

. S am pl e si z e sh ou ld

b e li m it ed

to y ield <2 00 m g of residue.
Sample Collec tion and Hand ling

1. Se quent ia lly

num ber W hat m an 47 m m G F/F f ilt er s (0.7 : m

pore s iz e) a long the outs ide e dg e, w her e the s am

ple w ill not pas s throug h, us ing a

f ine -ti pped per

m ane nt m ar

k er

.
2. Dr y f ilt er s at 103 - 105° C ov er

nig ht.

3. W ei g h f

ilt er s (in g ra

m s) to 4 de ci m al pla ce s to obt ai n pre -w ei g ht. Pre- w ei g hed f ilt er s ar e st ore d in se al ed cont ai ner s until ready f or use.

4

. Fi lt er a kn o wn vo lu m e o f w at er t h ro u gh th e filt er p ad (co n d u ct ed in th e fiel d ). Rin se fil te r wit h de io n iz ed w at er t o remove
sa lts . F old pa d in hal f

, s am ple ins ide , a

nd pla ce in al um inum

f oil pouch.

5. Freeze f

ilter pads for storage

.
Procedur e

1. Dr y f ilt er s at 103 - 105° C ov er

nig ht. A llow

s am ple s to c ool t o room

te m per at ure in des si ca

tor.

2. W ei g h f

ilt er s and re cor d w ei g hts (i n g ra m s)

.
3. Subs am

ple a port ion of

the f ilt er s and re pla ce the m

in the dry

ing ov en f

or a m inim um of 1 hour

.
4. Re-w eigh subsam

pled f ilters. If there is > 0.5 m g w eight loss betw een the f irst and second w eight of the sub sam pled f ilter pads , t hen al l f

ilt er pa ds shoul d be re -dr ie d a nd re -w ei g hed.

5

. R e p e a t s t e p s 3 a n d 4 a s n e c e s s a r y.53

Calc ulation of TS S

TSS co ncentratio n is calculated usin g the follow ing equ ation:

p o st w he re : W = d

ry w ei g ht o f fi lt er p ad a ft er f il te ri ng ( g ),

p re W = d ry w eig ht of filter pad

befo re filtering (g ), a nd

L V = v ol um e of

w at er f il te re d (L ).54



## T O TAL 

## V O LATILE 

## S US PE ND ED 

## S OLID S Re v i s e d 2/ 2004

Total v olatile so lids (TVS) is th e v olatiliz ed m aterial tha t is lo st on ig nition from

Total Su spend ed S olids (TSS). I t is c alculated from the m easurem ent o f a total su spend ed s olid (TSS ) sam ple m inus the

m easurem ent o f the quan tity rem aining after com

bustion . R esults a re ex pressed in m g /L.

Meth odo logy

A P H A

. 1 97 5. Me th od

2 08 E ( w it h m od if

ic at io n)

. To ta l v ol at il e an d fi xe d re si du e at 5 50 ° C

. in

S ta nd ar d Me th od s fo r th e E xa m in at io n of Wa te r an d Wa st ew at er , 1 4 th E di ti on

. A m er ic an P ub li c H ea lt h Asso ciation. Washin g ton, D. C. 11 93pp

.

Sample Collec tion and Hand ling

1. Com

bust What m an 47 m m G F/F f ilt er s (0.7 : m

pore s iz e) in an ov en se t at 500° C f or 90 m inute s. T his s et ting on CB L ’s ov en has be en det er m ine d to be 550° C.

2. W ei g h f

ilt er s (g ) t o 4 de ci m al pla ce s to obt ai n pre -w ei g ht a nd s tore indi v idua l pa ds in a num ber ed pe tri dis h.

3. Foll ow

s am ple f ilt ra tion proc edur e f

or t ota l s uspe nded sol id s am ple . Ma k e note of pa d num ber on f oil pouch.

Procedur e

1. Dr

y f ilt er s ov er

nig ht at 103° -105° C. A llow to cool to room

te m per at ure in des si ca

tor.

2. W ei g h f

ilt er s and re cor d w ei g hts (i n g ). Ca lc ula te T SS.

3. T ra

nsf er f ilt er to a porc el ai n c ruc ible a nd c om bust a t 500° C f

or 90 m inute s. A llow

to cool to room

te m per at ure in des si ca tor.

4. W ei g h f

ilt er a nd re cor d w ei g hts (i n g ). Ca lc ula te T VS.

Calc ula ti on of

TVS

com b ust w he re : W = d ry

w ei g ht o f fi lt er p ad a nd s am pl e af te r co m bu st io n (i n g ra m s)

.

p o st W = d ry

w ei g ht o f fi lt er p ad a ft er f il te ri ng ( in g ra m s)

.

L V = v olum e of water filtered (L )55 

## C H LO RO PH YLL a AN D Re v i s e d 2/ 2004 

## P H AEO PIG M ENTS

Tw o fl uo ro m et ri c m et ho ds a re a v ai la bl e fo r an al y si s of

C hl or op hy ll a

. Chlo rophy ll, in a m easured

v ol um e of

w at er , is c on ce nt ra te d by f il te ri ng th ro ug h a g la ss f

ib er f il te r, a nd th e pi g m en ts o n th e fi lt er a re extracted in 90% ac etone. F luoresce nce is pro portion al to chlorop hy ll con centration

. I n o ne m ethod, fluoresce nce of the extrac t is m easured befo re an d after acidification usin g a fluorom eter. I n the seco nd

m ethod, the fluorom eter is equipp ed w ith sp ecific w av eleng th filters whic h rea d o nly em issions from ch lo ro ph y ll a , w it ho ut

in te rf er en ce s fr om

c hl or op hy ll b and

ph aeopig m ents. Therefore, the sam ple is not acidified.

Method ology

S tr ic k la nd , J. D

. H

. a nd

T. R

. P ar so ns

. 1 97 2. A p ra ct ic al h an db oo k o f se aw at er

a na ly si s. B ul le ti n 16 7 (2 nd ed.). F isheries Rese arch Boa rd o f C anada , O ttawa, C anada

.
P ar so ns , T. R

. , Y

. Ma it a an d C

. M. L al li

. 1 98 4. D et er m in at io n of c hl or op hy ll s an d to ta l c ar ot en oi ds : Spec tropho tom etric m ethod. p p. 101

- 11 2 in Pa rsons, T.R. , Y . Maita and C. M. L alli. A m anual of chem ical an d b iolog ical m ethods for seaw ater an aly sis. Pe rg am on Press, O xford.

We ls ch m ey er , N

. A

. 1 99 4. F lu or om et ri c an al y si s of c hl or op hy ll a in

th e pr es en ce o f ch lo ro ph y ll b and phaeo pig m ents. Lim

nol. Ocea nog r., 39: 1 985- 1992

.

Instru mentation Acid if ication Metho d: Flu or om et er : Tur

ne r D es ig ns Mo de l TD 70 0 ca li br at ed a g ai ns t a s pe ct ro ph ot om et er

u si ng p ur e ch lo ro ph y ll a from

spin ach (Sig m a C hem ical C om pany , C 57 53), or liquid standard s from Turner Desig ns, #10- 850. Ex citation f

ilter: Turner D esig ns 1 0- 050R , 3 40- 500 nm Emission filter: Turner D esig ns 1 0- 051R , > 66 5 n m Lamp : D ay lig ht White Lam p, Turner D esig ns 1 0- 045
Welschmeye r T echn ique: Fluorometer: Tu rn er

D es ig ns Mo de l TD 70 0 ca li br at ed a g ai ns t a s pe ct ro ph ot om et er

u si ng p ur e ch lo ro ph y ll a from

spin ach (Sig m a C hem ical C om pany , C 57 53), or liquid standard s from Turner Desig ns, #10- 850. Ex citation Filter: 43 6 n m Emission Filter: 68 0 n m Lamp : blue , F 4T4.5B 2 eq uiv alent

R ea ge n ts

Aceto ne, 90 %

3 2 A c e t o n e [ ( C H ) C O ] 9 0 0 mL D e i o n i ze d

wa t e r 1 0 0 mL

In a 1000 m L v olum et

ric f la sk , a dd 900 m

L of a ce tone a nd a dd 100 m

L de ioniz ed w at er

.56

Hydroch loric A cid, 1N H yd r o c h l o r i c a c i d

( H C l ) , c o n c e n t r a t e d 8

. 6 mL D e i o n i ze d

wa t e r up t o 1 0 0 mL

In a 100 m L v olum et

ric f la sk , a dd 8.6 m

L of c once ntra

te d hy droc hlori c ac id t o - 7 0 mL of d ei o n i ze d wat er

. Di l u t e t o 1 0 0 mL

w ith dei oniz ed w at er

.
Procedur e

1. Fil te r a

k now n v olum e of

w at er thr oug h a

W hat m an G F/F f

ilt er pa d (nom ina l por e si ze 0.7 : m ). O nly a f ai nt c olor is

nee ded on the pa d.

2. Fol d pad in half, sa m ple insid e, w

rap in aluminum f

oil, label and fre eze f

or analy sis w ithi n 4 we eks

.
3. Bef ore analy sis, briefly

thaw pads, th en place in a 15 m l centrif uge tube. A dd 10 m L of 90% A cetone.

4. Using a Te f

lon pestle, grind th e f

ilter ag ainst the side of the tube un til th e f ilter is w ell ground . A llow the sa m ple to extract f or 2 - 24 hours in the da rk unde r r ef rig er at ion. O v er

nig ht i s re com

m ende d.

5. Rem ove tubes f

rom ref rigerator and allow

to w arm to roo m tem perature.

6. Sha k e tube s, c ent

rif ug e at - 2300 rpm

f or 5- 10 m inute s and pipe tte li quid i nto a 5 m L round cuv et te f

or r ea ding on t he f luorom et er . I f

a re f rig er at ed ce ntri f

ug e is us ed, a

long er s pin t im e m ay

be us ed, but is not nec es sa ry

.
7. If pha eopi g m ent s ar e to be m ea sur ed, a dd 2 dr ops of 1N H y droc hlori c A ci d a nd re ad on the f luorom et er

a g ai n. I f us ing the W el sc hm ey er

te chni que, do not ac idify the sam ple.

C al cu la ti on o f C h lo ro p h yl l a an d Phae opigmen t C once ntratio n

C hl or op hy ll a , p ha eo pi g m en t, a nd a ct iv e ch lo ro ph y ll a con centration s are calculated usin g the follow ing equatio ns:

and57

b whe re: R = fluorom etric read ing befo re ad ding acid,

a R = fluorom etric read ing after adding acid,

s b F = c al ib ra ti ng s td

c on c. /r ea di ng o f st d, ( C /R ),

b a r = R /R determ ined on a calib rating extrac t, an d V = v ol um e fi lt er ed

( m L ). 10 = ex tract v olum e.

C al ib ra ti on o f th e T D 70 0: D et er m in e th e co nc en tr at io n of th e ch lo ro ph y ll a stock

solu tion o n the sp ec tr op ho to m et er , o r us e li qu id s ta nd ar ds f

ro m Tu rn er D es ig ns

. F ol lo w in st ru ct io ns in th e m an ua l t o calibrate for raw fluore scence or conce ntration.

Calc ulation for the Welschmeye r T echn ique :

T he in s t r ume nt ca n b e ca l i br a t e d t o r e a d i n c on c e nt r a t i on . T o c or r e c t fo r vol ume fi l t e r e d, th e ab ove calculation

is u sed.

C h lo ro p h yl l a C alibra tion of the Fluorometer :

1. E m pt y 1 m g o f dr ie d ch lo ro ph y ll a into a 10 0 m L v olum etric flask and dilute to 10 0 m L w ith 90 % A ce to ne ( 10 m g /L ).

2. Mak e serial dilutions of 1.0 m g /L, 0.1 m g /L, and 0.0 1 m g /L fro m the 10 m g /L so lution (10 m L o f

ea ch s ol ut io n di lu te d to 1 00 m L w it h 90 % A ce to ne ).

3. Read

the 10 m g /L a nd 1 m g /L so lutions on a sp ectroph otom eter w ith a 10 cm cell path leng th at 750

nm , 6 65 nm , 6 64 nm , 6 47 nm and 63 0 n m

. Add

2 drops of 1 N Hy droch loric A cid an d rea d ag ain. The reading at 7 50 nm

is a turbid ity blan k

. I f its v alue is g reater tha n . 005, cen trifug e the sam ple at 2300 rpm for 5 m inutes and read it ag ain. The read ing at 6 65 nm is u sed in the determ ination of phaeo pig m ents.

4. Read

the 10, 1, 0. 1 an d 0 .01 m g /L so lutions on the flu orom eter, using m any dilutio ns.

5. The calculatio ns fo r the Spec . n um bers a re as follow s:

664 647 630 m g C hl a /L = 1 1. 85 (R )

- 1

. 54 (R )

- 0

. 08 (R )58

n w he re : R = R ea di ng a t n nm

Deter mination of the calib ration factors:

Mu lt ip ly th e 1 m g /L v al ue b y

1 0, a nd a v er ag e th is v al ue a nd

th e 10 m g /L v al ue

. B y

u si ng th is

av er ag e an d th e fl uo re sc en ce r ea di ng s fr om e ac h st an da rd , in cl ud in g d il ut io ns , c al ib ra ti on f ac to rs
can be c alculated.

C = con centration of standard

.

b R = fluore scence read ing befo re ad ding acid.

Deter mination of r:

b a Determ ine th e ratio of R /R for all of the rea ding s. A v erag e. U se a r ol li ng a v er ag e of

th e la st th re e ca li br at io ns a s th e w or k in g f ac to r an d r.59



## A PPEND IX A

SAMP LE CUSTODY
Upo n arriv al at the lab oratory , s am ples a re co unted, ob serv ed fo r po tential p roblem s (m elting , b rok en
co nt ai ne rs , e tc

. ) an d pl ac ed in a f re ez er u nt il a na ly si s. S am

pl e in fo rm at io n an d da te o f ar ri v al a re
r e c or de d o n a lo g sh e e t , a nd en t e r e d i n a n AC CE SS fi l e fo r in t e r na l tr a c kin g.

INSTRUM ENT MAINTENANCE

Ana ly tical instru m ents a re m aintained on a reg ular b asis an d rec ords are k ept o f ho urs o f op eration, sched uled m aintenan ce, pum

p tub e ch ang es, etc.

A critical spa re pa rts inv entory is m aintained

for each instrum ent. I nstrum ent d own
- tim e is m inim iz ed b y tr ou bl es ho ot in g in st ru m en t p ro bl em s by te le ph on e w it h m an uf

ac tu re rs a nd

s er v ic e re pr es en ta ti v es

. S pa re parts c an b e rece iv ed w ithin 2 4 h ours v ia nex t- day air s erv ice.

TEMP ERATURE LOGS

The tem peratures in freez ers an d refrig erators used for sam ple sto rag e are m onitored w ith N I ST certified and calibrated therm om eters. Tem peratures in dry ing ov ens are m onitored w ith N I ST certified and calibrated therm om eters.

DATA HANDLING

D at a pr od uc ed b y c om pu te r dr iv en

in st ru m en ts a re tr an sf

er re d el ec tr on ic al ly

v ia L I M s y st em

to th e m ai n la bo ra to ry d at a ba se

. O th er

d at a ar e m an ua ll y

e nt er ed in L ot us 1 23 © o r Mic ro so ft E xc el © s pr ea ds he et s by data m anag em ent p ersonn el. A hard cop y of m anually

entere d d ata is produ ced and unde rg oes a
m an ua l p oi nt

- by

- po in t v er if ic at io n pr oc es s. A ny

n ec es sa ry

c or re ct io ns a re m ad e. S ub se qu en tl y , a ll d at a
a r e fu r t he r

ch e c ked by a s e r i e s of

pr ogr a ms t o e ns ur e th e fo l l ow i ng:

324 C NO + NO + NH < Total Disso lv ed N itrog en,

--+

4 C PO < Total Disso lv ed P hosp horus,

-3

C DO C < TOC , a nd

223 C NO < NO + NO

.

---
Any data indicating errors are g iv en s pecific error co des. F inal p rinted data files are created and sent al on g w it h an

e le ct ro ni c co py

to th e cl ie nt

.60


## A PPEND IX B

STATEMENT O N INSTRUMENT COM P ARABI LITY

The Nu trient A naly tical Se rv ices L aborato ry dev elops a da ta qu ality

m aintenan ce p rog ram

for each analy te w henev er ne w instrum entation is acq uired. I t is th e po licy

of the N utrient A naly tical Se rv ices Labo ratory

to report any

data from

new instrum entation only after thorou g h an d sa tisfactory side- by

- side

com parison s w ith ex isting instrum entation are p reform ed.

No predeterm ined num

ber o f data pairs a re use d to m ak e the assessm ent o n d ata co m parability betw een
new and existin g m ethodo log y

. Ev en in the case of ins trum entation with sim ilar m ethods of detection (i.e., auto m ated c olorim etric), a lthoug h n o sp ecific n um ber o f data pairs is used, there are at least 1 00 data p airs. C om

parability at lo w and hig h co ncentratio ns, salinity and othe r po ssible m atrix inte rferences,

se ns it iv it y

a nd p re ci si on a re a ll f ac to rs in d et er m in in g th e nu m

be r of p ai rs th at m us t b e ad dr es se d be fo re br i ngi ng a n i ns t r ume nt on
- l i ne an d i n d e t e r min i ng i ns t r ume nt co mpa r a bi l i t y.

The a na ly st

w ho p er fo rm s th es e co m pa ri so ns s ho ul d be e xp er ie nc ed , o pe n- m in de d an d im pa rt ia l. Th is person can g iv e an ev aluation of ea se o f instrum ent o peration and a v ery im portant g eneral statem ent o f

com parability

. This statem ent o n co m parability m ust th en b e su

bstantiated

v ia statistical analy sis of the da ta

. A s pr ev io us ly

m en ti on ed , th es e da ta m us t e nc om

pa ss th e en ti re c on ce nt ra ti on r an g e, m at ri x in te rf er en ce s, p er ce nt

r ec ov er y , r es ul ts o f st an da rd

r ef er en ce m at er ia l a na ly se s, e tc

. Th e da ta interpretation

m ust su pport com

parability

. The analy st and labo ratory

Q A/Q C officer m

ust co ncur and finally , s om e so rt of presenta tion (w ritten or v erbal) m ust b e g iv en to the contracto r exp laining w hat proced ure w as follo wed

and the results th at w ere o btained to bring this instrum ent o n- line.

STATEMENT O F QUALITY ASSURANCE/Q UALITY CONTRO L PRO CEDURES

A constan t con sideration of Nutrien t A naly tical Se rv ices is assuring the quality of data g enerated

by the proced ures p resented

in this m anual. F urther, indic ation of d ata qu ality is a ccom plished by ana ly z ing duplica tes, spik es, standard s- as- sam ples, standard

referen ce m aterials; an d p articipating in crosscalibration exe rcises.

Lab orato ry D uplica tes
App roxim ately

5% o f the total nu m

ber o f sam

ples a naly z ed c onsist of lab oratory du plicates. F or dissolv ed a naly tes, after a sam ple is analy z ed, the sa m e sam ple co ntainer is plac ed fa rther alo ng in the autom atic sam

pler tray and re- analy z ed. The m ean of the tw o v alues is repo rted as the conce ntration for that sa m ple. I f a difference of >10% is observ ed b etwee n rep licates, then all of the rep licates fo r that pa rt ic ul ar a na ly ti ca l r

un a re c ar ef ul ly r ev ie w ed

. I f on ly

o ne o f th e du

pl ic at e pa ir s is in

q ue st io n, th en

o nl y th at s am pl e is r e- an al y z ed

. I f al l s ho w a s im il ar tr en d, th en

in st ru m en ta ti on /r ea g en t p ro bl em s ar e
su sp ec te d an d th e an al y ti ca l r un is h al te d un ti l s uc h ti m e as th e pr ob le m is r es ol v ed

. Th is p ro ce du re is practiced for all disso lv ed a naly tes tha t are not c onsu m ed c om pletely in the an aly tical pro cedure

. For th os e th at a re c om pl et el y c on su m ed

a nd f or p ar ti cu la te a na ly te s, d

up li ca te a na ly se s ar e ac tu al ly d up li ca te
sam ples c ollected in the field and analy z ed in the sam e an aly tical run

.
Val ue s fo r ea ch d

up li ca te a na ly z ed

a re r ec or de d in a s ep ar at e Q A /Q C d at a fi le a lo ng w it h th e sa m pl e
num ber, sam ple co llection date a nd analy sis da te. The m ean conce ntration and standard dev iation o f the61

replicates are ca lculated in this data file.

I n th e ca se o f pa rt ic ul at e ca rb on a nd n it ro g en , to ta l s us pe nd ed s ol id s an d ch lo ro ph y ll a , 1 0% of the to tal

nu m be r of s am pl es a re a na ly z ed

a s du pl ic at es

. Th is g en er at es s uf fi ci en t q ua li ty a ss ur an ce d at a to
com pensa te for the o m ission of lab oratory spik es for these non- aqueo us sa m ples.

L ab or at or y d up li ca te s se rv e as a n in di ca to r of in st ru m en t s ta

bi li ty , c on si st en cy

in la bo ra to ry s am pl e pr e pa r a t i on an d a na l ysi s , a s we l l as an es t i mat e of fi e l d p r of

i c i e nc y.

Lab orato ry S pikes

A pp ro xi m at el y

5 % o f th e to ta l n

um be r of s am pl es a na ly z ed

c on si st o f la bo ra to ry s pi k es

. A s pi k e is pr ep ar ed b y a dd in g a k

no w n v ol um e of

s ta nd ar d to a k

no w n v ol um e of

p re
- an al y z ed

s am pl e. W e
ro ut in el y a dd e no ug h co nc en tr at ed

s ta nd ar d to p ro v id e a si g ni fi ca nt r es po ns e on

o ur in st ru m en ts th at

is di st in g ui sh ab le f

ro m th e or ig in al c on ce nt ra ti on o f th e sa m

pl e. Th is c on ce nt ra te d st an da rd is u se d to

m inim iz e an y

po ssible c hang e in sam

ple m atrix b y

the addition

of spik e. The spik ed s am

ple is analy z ed a nd its exp ected conce ntration calculated as the su m

of the o rig inal

co nc en tr at io n an d th e sp ik e co nc en tr at io n, n or m al iz ed f or th e co ns ti tu en t v ol um es

. A c om pa ri so n is

m ade betw een the ac tual v alue a nd the ex pected v alue. These c oncen trations (orig inal, expec ted an d actual) are reco rded in a separate QA /QC data file alon g w ith sam ple n um ber, sam ple co llection date,

an al y si s da te a nd

th e am ou nt

o f sp ik e ad de d. I n th e ca se o f pa rt ic ul at e ph os ph or us , th e v ol um e fi lt er ed

is

no t us e d i n t he ca l c ul a t i on to de t e r min e pe r c e nt a ge r e c ove r y.

I f a v alue o f >11 5% or <85% is observ ed fo r perce ntag e reco v ery of the sp ik e, then all of the sp ik es for th at p ar ti cu la r an al y ti ca l r

un a re c ar ef ul ly r ev ie w ed

. I f on ly

o ne o f th e sp ik es is in

q ue st io n, th en

o nl y th at s am pl e is r e- an al y z ed

. I f al l s ho w p oo r re co v er y , th en in st ru m en ta ti on /r ea g en t p ro bl em s ar e
su sp ec te d an d th e an al y ti ca l r un is h al te d un ti l s uc h ti m e th at th e pr ob le m

is r es ol v ed

. Th is p ro ce du re is adhere d to for all dissolv ed a naly tes an d fo r the extracts of p articulate phos phoru s an d b iog enic s ilica.

Doc umentatio n o f

Slo pes, etc.

A r un ni ng r ec or d of

th e sl op es o f th e st an da rd c ur v es ( th e so
- ca ll ed

"F , " " S " a nd

"K" f ac to rs ) is

m aintained

for each analy sis. R ando m up and do wn m ov em ent w ithin a predeterm ined rang e as a function of tim e ind icates th e an aly sis is unde r con trol. C onsisten t upw ard o r do wnw ard tren d o f these factors indicates the an aly sis is out o f con trol an d req uires im m ediate attention.

Limits of De tection

L im it s of d et ec ti on , th e lo w es t c on ce nt ra ti on o f an a na ly te th at

th e an al y ti ca l p ro ce du re c an r el ia bl y detect, hav e be en e stablished for all para m eters ro utinely m easured

by N utrient A naly tical Se rv ices. The lim it of detection

is 3 tim es the stand ard d ev iation o f a m inim um of 7 rep licates o f a sing le low

conce ntration sam

ple.

Table 3 presents the current m inim um lim its of detection

. These v alues are rev iewed and rev ised pe r i od i c a l l y.62

Tab le 3. Minim um limits o f detec tion o

f C BL, O ctober/N

ovem ber 1987

, Marc h 1 994, Febr uary/D ecem ber 1998 , F ebrua ry 1 999, Jan uary 2004 a

M E A N CO N C

. S T A

N D A R D D E T E C T ION LIM IT N U TR I E N T (m g /L ) D E VI A TI O N (m g /L ) ( : M)

Am m onium 0.0 007 0.0 01 0.0 030 0.2 1 Nitrite 0.0 002 0.0 001 0.0 003 0.0 2 Nitrite + N itrate 0.0 011 0.0 0023 0.0 007 0.0 5 Pho sphate 0.0 027 0.0 0025 0.0 007 0.0 2 Disso lv ed O rg anic C arbon 3.5 800 0.0 50 0.1 500 12 .50 Total Susp ended

So lids 13. 4000 0.8 00 2.4 000 Total Volatile So lids 3.9 5 0.6 7 1.9 8 Particulate Ph osph orus 0.0 187 0.0 008 0.0 024 0.0 8 Particulate I norg anic P hosp horus 0.0 027 0.0 002 0.0 006 0.0 2 Total Disso lv ed N itrog en 0.3 9 0.0 096 0.0 300 2.0 6 Total Disso lv ed P hosp horus 0.0 057 0.0 005 0.0 015 0.0 5 Silicate 0.2 500 0.0 03 0.0 100 0.3 2 Particulate N itrog en 0.3 170 0.0 041 0.0 123 0.8 8 Particulate C arbon 2.2 600 0.0 253 0.0 759 6.3 2 Particulate B iog enic S ilica 0.1 630 0.0 03 0.0 090 0.3 2 Sedim ent C (10 m g ) 2.1 830% 0.0 44 0.1 300% Sedim ent N (10 m g ) 0.1 950% 0.0 03 0.0 084% Sedim ent P (34 .8 m g ) 0.0 304% 0.0 03 0.0 087% C hl or op hy ll a 24. 26 0.2 2 0.6 5 ( : g /L)

F R E S H W A TE R

D E TE C TI O N L I M I TS

Chlo ride 5.8 4 0.0 8 0.2 3 6.5 7 Sulfate 4.9 0 0.0 3 0.0 9 2.8 0

R es ul ts b as ed

o n a m in im

um o f se v en r ep li ca te s co ll ec te d fr om o ne c ub it ai ne r an d an al y z ed r an do m ly a

on a ty pical d ay of analy ses.

S ta n d ar d a s S am p le

Stand ards a re an aly z ed a s sam ples th roug hout the an aly tical run

. This is an exc ellent m eans of ev aluating in st ru m en t p er fo rm an ce d ur in g th e co ur se o f an

a na ly ti ca l r

un

. S ta nd ar ds a s sa m

pl es a re a na ly z ed

e v er y 12

- 20 sam

ples, depen ding on

the instrum ent an d an aly te.

S ta n d ar d R efer en ce Ma te ri al s

Particu late C

arbo n, Nitrog en a nd Pho spho rus : B CS S- 1 is a m arine s edim ent refe rence m aterial pre pared by the Nation al R esearch Cou ncil o f C anada

. I t is c ertified b y the Cou ncil fo r carbo n co ntent, g iv es a
no n- ce rt if ie d ra ng e of

r es ul ts f or p ho sp ho ru s, b ut

n o in fo rm at io n fo r ni tr og en

. W e ha v e an al y z ed th is63

sedim ent fo r m any

y ears an d m aintain a su

bstantial databas e for nitrog en a nd phos phoru s, as w ell as carbon v alues. We analy z e this sedim ent at least q uarterly and com pare these results to the certified

v alue, non- certified ra ng e of v alues and our historical v alues.

Disso lved A nalytes and Har dness : S tandard

referen ce m aterials fo r am m onium , n itrite + n itrate, n itrite, orthop hosp hate, dissolv ed n itrog en, dissolv ed p hosp horus, disso lv ed o rg anic c arbon , s ulfate a nd chloride and hard nes s ar e sup pli ed by SPEX, a US EPA cer ti fi ed co m pan y. Th e samples arr ive in ampule s and we prepare final co ncentratio ns to app roxim ate ty pical e stuarine conce ntrations. The sam ples a re then

place d in pre

- cleaned

Teflon o r po ly

bo ttles, fro z en a nd analy z ed o n at least a quarterly bas is.
The analy sis of these froz en s tandard referen ce m aterials as a function of tim e also pro v ides d ata on the ef fe ct of our pres er vatio n tec hni que (fr eezin g) on the i nte grit y of t he co nce ntr at ion of samples . The US EPA reco m

m ends a ho lding tim e of 28 day s for m any of the p aram eters w e rou tinely ana ly z e.

D is so lv ed

o rg an ic c ar

bo n re su lt s pr ov id e a g oo d ex am pl e. S P E X s ta nd ar ds ( 2 an d 5 m g C /L ) w er e prepared in deioniz ed w ater on

31 May 19 95. S ev eral of each conce ntration were placed in pre

- cleaned Teflon bottles and

froz en fo r sub seque nt an aly sis. R esults fo r the Shim adz u TOC

- 5000 C arbon A naly z er
ar e pre sen te d bel ow.

SH I MAD Z U TOC- 5000 TOTAL OR GA NI C CA RB ON A

NA LY Z ER (Con centration s in m g C /L)

A N A L Y S I S D A TE

Stand ard 6/14/9 5 7/5/95 8/16/9 5 11/6/9 5

(Froz en) (Froz en) (Froz en) (Froz en)
5.0 0 5.3 0 5.2 3 5.3 2 5.4 2 2.0 0 2.1 1 2.1 3 2.1 5 2.1 3

Org anic c om

poun ds a re inclu ded with each dissolv ed n itrog en a nd dissolv ed p hosp horus dig estion to determ ine th e co m

pletenes s of the d ig estion proced ure. G lutam ic acid

and g ly ceroph osph ate are used as the N and P sources , re spectiv ely

. Fig ures 1 4 an d 1 5 illustrate the percen tag e reco v eries o f these intern al standard s for a sp ecific p roj ect in whic h w e pa rticipated in 19 94

- 19 95.

Cro ss C alibra tion Ex ercises

Nutrien t A naly tical Se rv ices h as p articipated in m any cross calibra tion e xercises. P articipation in su ch prog ram s is an e xcellent m eans of d eterm ining accu racy of results. Ex am

ples o f such

cross calibra tion exercises inclu de th e C hesap eak e B ay Pro g ram Q uarterly

Sp lit Sam

ples, Che sapeak e B ay

Pro g ram B lind Aud its, U SG S S tandard

R eference Sam

ple P roj ect, US EP A Method Validation S tudies and I nternation al C ou nc il f or th e E xp lo ra ti on

o f th e S ea

I nt er co m pa ri so n E xe rc is e fo r N ut ri en ts in S ea Wa te r.64

Figu re 1 3. Pe rcentag e rec overy of glu tamic acid as a n inte rnal standa rd fo r tota l disso lved n itrogen digestion /analys is. (

1995 )

Figu re 1 4. Pe rcentag e rec overy of gly cerop hosp hate as a n inte rnal standa rd fo r tota l disso lved ph os ph or us d ig es ti on /a na ly

si s

. ( 1995 )65



## AC KNOW LEDGEMEN TS

We app reciate D r. C hristoph er D ’Elia’s h elp in assem bling the earlier v ersions of this m anual. I n 1 978 he started a cquiring equ ipm ent an d h iring perso nnel to esta blish NA SL to su

pport research at C BL and , la te r, “ ou ts id e” p ro g ra m s. Th is m an ua l r ef le ct s hi s in si st en ce o n ap pr op ri at e m et ho ds f or th e sa m pl e mat r i x t o e ns ur e da t a ac c ur a c y.

Erin Con nor authore d p art of the 1 997 m anual, and assem bled all the sections and diag ram s in a consisten t form at. A driene Cap ers he lped assem ble th is m anual and, also, the prev ious v ersions.

We than k D r. Theod ore L oder and Dr. Le onard H aas fo r their editorial sug g estions and rev iew of the 1997 v ersion of this docu m ent.Final report of the land

- based testing of the 

### Hyde

- GuardianTM

- system 1 Texel , January 200 9 

# Final r eport of the land

- based testing of the Hyde

- GuardianTM

- System, for Type Approval according to the Regulation D

- 2 and the relevant IMO Guideline (April

– July 2008)

Marcel J.W. Veldhuis Ph

- D Royal Netherl ands Institute for Sea Research P.O.Box 59 1790 AB, Den Burg, TEXEL the Netherlands phone: +31

- 222

- 369512/300 email: veldhuis@nioz.nlFinal report of the land

- based testing of the 

### Hyde

- GuardianTM

- system 2 

# Final r eport of the land

- based testing of the Hyde

- GuardianTM

- System, for Type Approval according to the Regulation D

- 2 and the relevant IMO Guideline (April

– July 2008)

Marcel J.W. Veldhuis Ph

- D Frank Fuhr Dipl.

- Biol. Peter

- Paul Stehouwer MSc. Royal Netherlands Institute for Sea Research P.O.Box 59 1790 AB, Den Burg, TE XEL the Netherlands phone: +31

- 222

- 369512/300 email: veldhuis@nioz.nl , ffuhr@nioz.nl , pstehouwer@nioz.nlFinal report of the land

- based testing of the 

### Hyde

- GuardianTM

- system 3 

# Final r eport of the land

- based testing of the Hyde

- GuardianTM

- System, for Type Approval according to the Regulation D

- 2 and the relevant IMO Guideline (April

– July 2008)

Royal Netherlands Institute for Sea Research P.O.Box 59 1790 AB, Den Burg, TEXEL the Netherlands phone: +31

- 222

- 369512/300 email: veldhuis@nioz.nl or ffuhr@nioz.nlFinal report of the land

- based testing of the 

### Hyde

- GuardianTM

- system 4 

## Content page

1 Executive summary 5

2 Zusammenfassung 6

3 Summary table 7

4 Acknowledgements 8

5 Introduction 8

6 Description of the test facility 9

6.1 NIOZ Royal Netherlands Institute for Sea Research 9

6.2 Portrait of Hyde Marine ; producer of the Hyde

- Guardian TM

- system 11

6.3 The test facility 11

6.4 Technical description of the Hyde

- GuardianTM

- system 1 3

7 Requirements to meet the D2 Standard 1 5

7.1 Requirements to meet: guideline G8 1 5

7.2 Experimental design 18

8 Results 2 4

8.1 P hysical and chemical parameters 24

8.2 Biology 28

9 Environmental accepta bility 35

10 Discussion and evaluation of results 3 7

11 References 3 9
Appendix 1: species list of phytoplankton and zooplankton 41 present during the various test runs.Final report of the land

- based testing of the 

### Hyde

- GuardianTM

- system 5 

## 1 Executive summary

The Hyde

- GuardianTM

- system, a ballast water manageme nt system was tested according to the s tandard as set out in regulation D

- 2 of the “International Convention for the Control and Management of Ships’ Ballast Water and Sediments,” hereafter referred to as the “D2

- Standard” and consistent with the IMO Guide lines for Type Approval testing (G8) . The tests were conducted in the spring and early summer of 2008 in the harbour of the Netherlands Institute for Sea Research. In general most of the requirements for testing which concern water characteristics and abu ndance of organisms were met and in most test runs environmental conditions were far

more challenging than strictly needed. The Hyde

- GuardianTM

- system is designed as a UV

- light disinfection system (UV

- reactor) with a mechanical filter as a pre

- treatment ste p. The water is treated at intake and at discharge, i.e a two

- fold disinfection of the ballast water. In total 10 test runs in a row were successful for all relevant parameters and the BWT system performed on average much better than stated in Regulation

- D 2 by achieving values for organisms well below the requirements of the D2 Standard. For many size classes of organisms the residual number of organisms was often one order of magnitude lower than the number in D2

- Standard. This system should therefore be r egarded as an effective way of treating ballast water in ships, thereby minimizing the risk of new invasions originating from the ships ballast water. The sediment load was reduced in a pre

- treatment step (self

- cleaning disk filters) and the organisms wer e deactivated by ultraviolet light disinfection

. Although numbers of phytoplankton and bacteria were also largely reduced by the ultraviolet light disinfection step not all cells were fully disintegrated. Complementary tests indicated that the remaining o rganisms possessed a reduced viability and were (partly) deactivated. Treated water, which was transferred to favourable conditions, showed no regrowth within

a period of 10 days. Environmental acceptability tests however showed that the growth of organism s, mainly plankton, was not limited by the discharge water indicating that the discharged water was still vital.Final report of the land

- based testing of the 

### Hyde

- GuardianTM

- system 6 

## 2 Zusammenfassung

Das Hyde

- GuardianTM

- system zur Behandlung von Ballastwasser wurde im Frühjahr und Sommer 2008 gemäß der Regularien

- D2 (D2

- Standard), sowie der IMO Richtlinien über Tests für eine Typzulassung (G8) im Hafen des Königlich Niederländischen Meeresforschungsinst itut (NIOZ) getestet. Generell wurden die allermeisten Anforderungen bezüglich der abiotischen Parameter des Testwassers und zur Organismendichte erfüllt. In den meisten Tests waren die Bedingungen sogar schwieriger, als in den Richtlinien verlangt. Das Hy de

- GuardianTM

- System arbeitet mit UV

- Licht als Desinfektionseinheit (UV

- Reaktor). Zusätzlich verfügt es über einen mechanischen Filter als Vorabreinigungsstufe. Das Waser wird bei Aufnahme und Abgabe behandelt, d.h. es findet eine Doppelbehandlung des Balla stwassers statt. Es wurden 10 voneinander unabhängige Tests nacheinander, erfolgreich durchgeführt. Das System erfüllte alle Anforderungen der Regularien

- D2 und der Richtlinie G8. Die Organismenanzahlen nach der Behandlung lagen deutlich unter den Anforder ungen des D2

- Standards, für einige Gruppen im Durchschnitt sogar um eine Größenordnung. Das System sollte demzufolge als effektive und sichere Möglichkeit zur Behandlung von Ballastwasser betrachtet werden, die dazu beitragen kann, weitere biologische Inva sionen zu verhindern. Die Sedimentfracht wurde in einem Vorbehandlungsschritt reduziert (selbstreinigende Scheibenfilter) und die Organismen durch UV

- Bestrahlung inaktiviert. Obwohl die Anzahl von Phytoplanktern und Bakterien durch die UV

- Behandlung stark reduziert wurden, wurden nicht alle Zellen vollständig zerstört. Ergänzende Versuche ergaben, dass ein Teil dieser Zellen noch eine eingeschränkte Vitalität zeigte und nur teilweise inaktiviert war. Dennoch wurde in Inkubationsexperimenten über Zeiträume v on mehr als 10 Tagen unter vorteilhaften Umweltbedingungen kein Planktonwachstum im behandelten Wasser festgestellt. Gleichzeitig durchgeführte Umweltverträglichkeitstests (Zugabe von Zeigeorganismen und Planktonkulturen, Verdünnungsreihen) mit dem behande lten Wasser ergaben jedoch keine negativen Auswirkungen auf das Planktonwachstum und zeigten somit, dass das behandelte Wasser selbst das Wachstum gesunder Organismen nicht beeinflusst.Final report of the land

- based testing of the 

### Hyde

- GuardianTM

- system 7 

## 3 Summary table with results for the Type Approval Certificate of th e Hyde

- GuardianTM

- BWT

- System

Land

- based tests NIOZ Reference & Treated Reference Treated salinity 22.1 PSU Intake Discharge Discharge Discharge* natural plankton Average min. max. Average min. max. Average min. max. Average
t otal bact eria [counts/mL] 3.3+E6 1.3+E6 6.6+E6 2.1+E6 0.73+E6 3.6+E6 2.4+E6 0.38+E6 5.2+E6 0.31+E6
E. coli [cfu/mL] < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 Enterococci [cfu/mL] <1 < 1 <1 < 1 < 0.1 1 < 0.1 < 0.1 < 0.1 < 1 plankton <10 μm [c ounts/mL] 5446 235 13646 589 62 1939 <10 <10 <10 12 plankton 10

- 50 μm [counts/mL] 1028 304 1784 147 140 156 <10 <10 <10 2.3 plankton >50 μm [counts/m 3 ] 25.5+E5 10.1+E5 48.3+E5 3.95+E3 617 10.8+E3 2.9 n.d 7.3

-



### Land

- based tests NIOZ Reference & T reated Reference Treated salinity 31.9 PSU Intake Discharge Discharge Discharge* natural plankton Average min. max. Average min. max. Average min. max. Average total bacteria [counts/mL] 4.2+E6 0.72+E6 6.3+E6 2.1+E6 0.33+E6 5.4+E6 0.88 +E6 0.24+E6 1.2+E6 0.64+E6 E. coli [cfu/mL] <0.1 < 0.1 <0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 Enterococci [cfu/mL] 8.3 < 1 17 < 0.1 < 0.1 1 < 1 < 1 < 1 < 1 plankton <10 μm [counts/mL] 2366 1194 3754 290 173 632 <10 <10 <10 1.9 plankton 10

- 50 μm [counts/mL] 1378 514 2090 140 121 151 <10 <10 <10 1.0 plankton >50 μm [counts/m 3 ] 1.58+E5 0.52+E5 4.11+E5 1.54+E4 0.25+E4 0.84+E4 n.d. n.d. n.d.

-
Summary table (average and minimum and maximum) of collected data covering the major groups of o rganisms at two series of 5 test

- runs at a low and high salinity range, respectively. n.d.: not detectable in sample;

- : no data; * numbers as determined in bottle incubations of discharge water.Final report of the land

- based testing of the Hyde

- GuardianTM

- System 8 

## 4 Acknowledgements

The authors thank the technical staff o f the NIOZ and in particular Anna Noordeloos, Eveline Garritsen, Swier Oosterhuis, Siem Groot, Robert Lakeman, Santiago Gonzalez and Pieter van Kralingen. Their cooperation was essential for the successful completion of this certification project. Special thanks also to Yolanda van Iperen for providing the species list of plankton in the Wadden Sea. We also thank Mr. Rolf von Ostrowski, Mrs. Karin Sigler and Dr. Kai Trümpler of the Bundesambt für Seeshifffahrt und Hydrographie, acting on behalf of the Germ an Administration, for their excellent collaboration during this whole certification process.



## 5 Introduction

Ships transport 5

- 10 billion tons of ballast water annually all over the globe (Endrese n et al. 2004) . The ballast water is loaded with particulate sediment and an enormous variety of (living) organisms, which ranges from juvenile stages, larvae and eggs of fish and larger zooplankton (Williams et al. 1988 ; Carlton & Geller 1993) to macroalgae, phytoplankton (Hamer et al. 2000 ; Hallegraeff et al. 1997) , bacteria and viruses (Gollash et al. 1998) . In general these organisms belong to the natural ecosystem in and around the port of origin but they might not be occurring naturally in the coastal waters and port of destination at the

en d of a ship’s voyage . In hundreds of cases around the world, this has resulted in severe damage to the receiving ecosystem and to human health, because these non

- native organisms developed into a plague. This often has a high impact on the ecosystem and ca n cause economical damage (Hoagland et al. 2002) , as it results in a decrease of stocks of commercially valuable fish and shellfish species and occasionally outbreaks of diseases such as cholera (Ruiz et al. 2000 ; Drake et al. 2001) . If action is not taken , the problem of invasive species will increase in an exponential manner for several reasons. Ships are getting larger, faster and the amount of traffic across the oceans is expected to increase rapidly during the coming decades, and therefore also the chan c e of non

- indigenous organisms to have large enough numbers for settling and expanding. The problem of invasive species is considered as one of the 4 m ajor threats of the world’s oceans next to land

- based marine pollution, overexploitation of living marine resources, and physical alteration/destruction of habitats
To minimize these risks for the future, the International Maritime Organization (IMO) of t he United Nations has adopted the Ballast Water Convention in 2004 (Anonymous 2005) . The Convention states that finally ALL ships (>50,000 in number ) should install proper ballast water treatment (BWT) equipment on board between 2009 and 2016. As a temporary and intermediate solution for the time being ship may reduce the risk of invasive species by performing ballast water exchange during their voyag e when passing deep water (>200 m depth and 200 NM from the coast. Ballast water exchange faces many problems as to feasibility, safety and efficacy For a large part of ships’ voyages the required depth and/or distance to shore requirements are never met; BW exchange can affect the ships construction stability and in rough seas exchange is not possible because of the risk to ship and crew. Treatment of ballast water is therefore considered to be the best solution of reducing the risk of invasive species.

D uring the recent years numerous solutions for treatment of ballast water have been mentioned and tested with the ultimate goal to reduce the amount of organisms in ballast water (Rigby & Taylor 2001) . However, next to a high efficacy there is more needed for a BWT syst em to be a good system. Next to biologically effective the system should be practicable, environmentally acceptable and also cost effective.Final report of the land

- based testing of the Hyde

- GuardianTM

- System 9 Despite the fact that the treatment technology for drinking

- , waste

- and process water is well

- developed none of t hese techniques is directly applicable to ballast water (Rigby & Taylor 2001 ; MEPC 49/2/13, 2003 ). Besides reducing the load of organisms the sediment load should be reduced as well. There are also considerable differences in ships operation, types of ships, the amount of space available for a ballast water treatment system on board and the way ships are operated. Ballast water treatment will develop into a new field of technology of its own with a commercial market estimated for the next 10 years in the order of 8 bill ion Euro (Haskoning 2001)

. As a primarily scientific research institute NIOZ is defining its role in the certification process as to study 1) the numerical abundance and biodiversity of organisms prior, during and after a treatment with the Hyde

- GuardianTM

- Ballast Water Treatment s ystem (efficacy of the BWT system), 2) to determine the viability status of the remaining organisms during discharge, 3) to assess the environmental acceptability of discharging ultraviolet light disinfected balla st water by measuring residual effluent toxicity in order to determine latent effects, since ballast water treated with this specific UV

- system does not need a complete G9 procedure (environmental impact).

This research strategy allows for more in depth t esting, while it includes ALL organisms and not only the size classes as specified in the Convention D2

- standard.


## 6 Description of the treatment facility

6.1 NIOZ Royal Netherlands Institute for Sea Research

NIOZ Royal Netherlands Institute for Sea Re search is the National Oceanographic Institute of the Netherlands. NIOZ is part of the Netherlands Organization for Scientific Research (NWO). The institute employs around 200 people and the annual budget is approximately €20 million. The mission of NIOZ i s to gain and communicate scientific knowledge on seas and oceans for the understanding and sustainability of our planet. The institute also facilitates and supports marine research and education in the Netherlands and in Europe.

In order to fulfil its mi ssion, the institute performs tasks in three specific fields. Research: The emphasis is on innovative and independent fundamental research in continental seas and open oceans. The institute also carries out research based on societal questions when this me rges well with its fundamental work. The senior scientists at NIOZ all participate in international research projects. Education: The institute educates PhD and other students of universities and schools for professional education. Together with universiti es NIOZ also organises courses for PhD students and master students in the marine sciences. A number of our senior scientists of NIOZ is also appointed as professor at Dutch and foreign universities. Facilitary services: NIOZ invites marine scientists from Dutch and foreign institutes and universities to write scientific proposals involving the institute’s research vessels, laboratories, and the large research equipment, which is often designed and built by the institute’s own technical department. The basi c oceanographic disciplines studied at NIOZ are physics, chemistry, biology and geology. Multidisciplinary research is regarded as one of the main strengths of NIOZ.

More information on www.nioz.nlFinal report of the land

- based testing of the Hyde

- GuardianTM

- System 10 Figure 1 : aerial view of the NIOZ harbour (lower rig ht), NIOZ laboratories (upper left) and TESO ferry (top).Final report of the land

- based testing of the Hyde

- GuardianTM

- System 11 

## 6.2 **Portrait of HYDE** 

**- MARINE/LAMOR (producer of**  **the HYDE** 

**- GUARDIANTM** 

**- ballast water management**  **system)**

More information on: www. hydemarine .com Ballast water treatment

- The Hyde

- Gu ardianTM

- system Hyde Marine, Inc. traces its origins back to the Hyde Windlass Company, established in Bath, Maine in 1865. Hyde Windlass was the largest supplier of anchor windlasses, winches, capstans, steering gear and specialty deck machinery in the Un ited States before, during and after the First and Second World Wars.

Hyde Marine has been involved in ballast water treatment (BWT) since 1996, when it teamed with the Univ. of Michigan to study potential technologies for BWT, particularly for ships op erating in the Great Lakes St. Lawrence Seaway System. This led to Hyde's participation, beginning in 1997, as the engineering contractor for the Great Lakes Ballast Technology Demonstration Project (GLBTDP). The GLBTDP was one of the first BWT research programs in North America and conducted tests of filtration technology for BWT aboard a Canadian Great Lakes bulk carrier, the Algonorth

. After a season aboard the Algonorth , the test equipment was transferred to a barge in Duluth Superior Harbor (Lake Mi chigan), where additional testing of the filtration system and other BWT technologies was conducted, also under the GLBTDP.
Hyde first became involved in its own BWT equipment in 2000, when it installed its initial full scale first generation system ab oard the US based cruise ship Regal Princess

. In 2001 Hyde installed four additional systems, two on cruise ships and one each on a container ship and chemical tanker. In 2003, after the requirements were better defined, Hyde installed a state

- of

- the

- art filtration and UV disinfection system aboard the Coral Princess

. This system, named the H yde

- GuardianTM

- system , was tested extensively on land based installations and on board the Coral Princess in the fall of 2004. The on

- board tests demonstrated the Hyde Guardian's capability to meet the IMO BWT Convention requirements. This system is now f irst in line for acceptance into the U.S. Coast Guard STEP program. In the fall of 2006 an essentially identical system was installed aboard RCCL's Celebrity Mercury

. It was commissioned early in 2007. The Hyde

- GuardianTM

- systems aboard the Coral Princes s and Mercury were granted interim approval for use in Washington State waters by the State of Washington in 2004 and 2007 respectively. All seven systems were commercial transactions and the newest version, the Hyde

- GuardianTM

- system , has been fully comme rcially available since early 2003.


## 6.3 The test facility

The land

- based tests were carried out at the Royal Netherlands Institute for Sea Research (NIOZ), Landsdiep 4, 1797 SZ 't Horntje, Texel, the Netherlands, from March till July 2007 (www.nioz.nl)

. The NIOZ test site is equipped with 3 coated concrete tanks of 300 m3 volume each to simulate the ballast water tanks of the ship (Figure 2). The tanks were (steam) cleaned after each run. Water samples can be taken from bypasses of the standard piping used to fill and to empty the tanks or directly from the tank at outflow at ca. 1 m from the bottom.Final report of the land

- based testing of the Hyde

- GuardianTM

- System 12 Figure 2 : Inside view of one of three subterranean water tanks

According to the requirements of the Guidelines G8, sampling points are fi tted before the treatment system and directly after the system. Samples varying in volume from 500 mL up to 1 m 3 were taken using clean sampling containers. Sampling containers and all further handling of the samples were separated in a control and a treat ed set to avoid cross contamination by the active substance. The basic handling, such as concentrating, filtration and chemical analysis was done at the test site. Different samples (1 to 10 L) were transported to the institute’s laboratories for further s pecial analysis. For re

- growth experiments 10 L of sample was transported (Nalgene bottle) to a climate room for incubation experiments (ca. 12

– 15 oC; a light;dark regime of 16:8 h and 100 **μ** mol quanta.

m - 2 .s - 1 ) The Hyde

- Guardian TM

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

m 3 /h) which was located in the NIOZ harbour. This is a pristine harbour with a direct access to the Wadden Sea and the origin of the test water changes with the tide. Furthermore, provi sion were made to allow the addition of salt water and / or freshwater in order to adjust the salinity of the natural water of the NIOZ harbour to the required test conditions of brackish water and marine water with a minimum of 10 PSU difference. A detail ed description of the test installation is presented in figure 3.

Figure 3 : P&I diagram of the Ecochlor and Hyde

- Guardian TM

- system installation at NIOZ with points of samplingFinal report of the land

- based testing of the Hyde

- GuardianTM

- System 13 

## 6.4 Technical description of the Hyde

- GuardianTM

- System

The Hyde

- GuardianTM

- system operates in

- line during the uptake of the ballast water. It is based on a modular concept which includes a physical se paration as well as disinfection with a UV

- light source resulting in permanent deactivation of organisms.

The Hyde

- GuardianTM

- system used during the tests at NIOZ has been build to the same standards as the double 250 m3/h containerised Hyde

- GuardianTM

- syst em that is installed on the test ship (fig. 4).

Figure 4: The Hyde

- Guardian TM skid mounted unit

The system consists of a UV

- reactor (BersonInLine), a medium

- pressure ultraviolet light disinfection system with a typical output of UV

- C (Fig 5; 200

– 300 nm wavelength) combined with a pre

- filter. The advantage of a pre

- filter ( Spin Klin® discs) is to reduce the number of larger organisms of which is known that UV is not a very effective way of deactivation (Raikow et al. 2007) . In addition filtration reduces the load of (larger) particles and increases therefore the transparency of the water, hence the effectiveness of the UV irradiation.

Figure 5: spectr al composition of the medium pressure (MP) lamp of the BersonInLine UV

system used in the Hyde

- GuardianTM

- system

.

Unlike active substances the working of ultraviolet radiation (UV) is based on transferring electromagnetic energy to molecular components of organisms, resulting e.g. in DNA damage. This will prevent cell replication (growth and reproduction) but also causes (immediate) disintegration of cells, morphological changes, discoloration, cellular membrane leakage and cell damage.Final report of the land

- based testing of the Hyde

- GuardianTM

- System 14 To disinfect mu nicipal water UV is commonly used because in general it does not significantly contribute to D isinfection B y
- P roducts (DBP; (Malley et al. 1995 ; Chin & Bérubé 2005)

. The UV lamps in the Hyde

- GuardianTM

- system are of the medium pressure type and run at a nominal of 100 W per centimeter in length. This produces an average intensity of 140 mW/cm 2 . The end of lamp life is not when a lamp fails to light but rather when its output has decreased by 30%. The physics inside a UV lamp do es not change over time, i.e. at the heart of the lamp the produced UV remains constant. As the amount of UV that is emitted decreases it is necessary to increase the power applied to the lamp to keep the UV intensity above the minimum levels. The Hyde

- GuardianTM

- system uses a UV sensor to continuously monitor the output of the lamps. Besides monitoring the output of UV

- C (200

– 280 nm) the UV sensor can also monitor the cleanliness of the sleeves and indirectly the water quality.

Because the power delivered to the UV lamp is not infinitely adjustable the majority of the time the system will be overdosing the UV. This is probably the only disinfection technology that can safely be overdosed. The control system will keep the amount of energy used to a minimum but given the discrete steps available for power control a new lamp will be running about 130% of the required UV.

The Hyde

- GuardianTM

- system differs from other BWT

- systems as the system i s not only treating during intake ( disc filter and UV

- reactor) but also at discharge (only UV

- reactor). Assessment of the efficacy of the BWT system was therefore immediately at intake (T0 ), at discharge after a holding period of 5 days (T5) and 5 days after discharge (T10) In addition to sampling the large holding basins for a period of 5 days , subsamples ( with a volume of 10 L) of the reference and treated tank were taken at intake and di scharge and incubated in a climate room under optimal growth conditions for the plankton community present in the test water. In case the treatment is insufficient and residual viable organisms remain present, or there is germination of resting stages or c ysts, plankton growth will be stimulated under these favourable conditions. This outgrow experiment also allows studies on the effect of the treatment over a period longer than 5 days (up to 20 days). A sufficiently high level of nutrients will be maintain ed, favouring phytoplankton growth and possible cyst germination throughout the incubation experiments. The applied test protocols were communicated with the Maritime Coastguard Agency (MCA) as well as with the German Administration ( Federal Maritime and H ydrographic Agency of Germany ; BSH ) and a brief description of the various methods is included in the next section. During the certification process the whole practical procedure of intake and discharge has been witnessed on 4 occasions (intake and dischar ge) by a classification society (Lloyds Register , London and Rotterdam ) as well as national and international agencies.

NIOZ is currently acting as an official certification organization for land

- based tests for the German and the UK administration accor ding to approved protocols and final reports will be made public at the moment of awarding the certificate.Final report of the land

- based testing of the Hyde

- GuardianTM

- System 15 

## 7 Requirements to meet the D2

- Standard

According to the D2

- Standard of the IMO/MEPC Convention of 2004 ( (Anonymous 2005) ) ships that meet the requirements of the Convention by meeting the ballast water performance standard must discharge :

The present D2

- Standard is defined as a standard for the water characteristics at discharge

. Furthermore, with exception of some indicator microbes (point 3) organisms < 10 **μ** m are completely excluded. This is certainly an omission since this size class in particular includes numerous phytoplank ton species characterized as Harmful Algal Blooms (HABs). Nevertheless, the standard is clear with respect to the maximum number of organisms remaining present. On the other hand a proper definition of the dimensions of organisms is still subject of (acad emic) discussion. Moreover, as an operational definition for viable organisms the IMO is using: “ organisms and any life stages thereof that are living ” , but a more adequate (scientific) definition is: an organism that is able to complete it’s life

- cycle, i ncluding reproduction (DNA replication). In the case of UV it is known that for certain organisms cell may be (genetically) damaged but remain active for a considerable period. The organisms however, lack the ability to reproduce.
In addition to the (bas ic) requirements for the D2

- Standard we have adopted a variety of methods and techniques to determine abundance, sizes and the viability status of different types of organisms. This includes also plankton < 10 μm other than bacteria (i.e. phytoplankton and viruses). Moreover, we extended our research effort to examine not only the fate of the organisms in the large

- scale holding tanks but also took subsamples for incubation under optimal growth conditions in our to study the growth potential of remaining (v iable) organisms and/or survival stages such as eggs, cysts or dormant cells over a longer period than the recommended 5 days. These later experiments allowed us also to study potential latent toxicity effects (environmental acceptability).



## 7.1 Requiremen ts to meet: guideline G8

Next to the D2

- Standard two guidelines were developed by the IMO/as a framework for approval of ballast water treatment systems (G8) and approval of the use of active substances in ballast water treatment systems (G9). For land

- ba sed testing MEPC 53/Annex 3 Anonymous 2005 ; and the revision of MEPC 58) was compiled of which the most relevant parts will be presented below. The se guidelines were generically designed to meet the conditions of a broad range of potentially effective treatment techniques to be tested in typical port and environmental conditions found across the globe. Most test protocols therefore require extensions of the test design to cover the specific aspects of the treatment. The land

- based testing serves to determine the biological efficacy of the BWT systems under consideration for Type Approval under more or less controlled and replicable

1) Less than 10 viable organisms per cubic metre greater than or equal to 50 micrometers in minimum dimension; 2) Less than 10 viable organisms less than 50 micr ometers in minimum dimension and greater than or equal to 10 micrometers in minimum dimension and 3) Less than the following concentrations of indicator microbes, as a human health standard: 1. Toxicogenic Vibrio chlorerae (serotypes O1 and O139) with less than 1 colony forming unit (cfu) per 100 milliliters or less than 1 cfu per 1 gramme (wet weight) of zooplankton samples: 2. Escherichia coli less than 250 cfu 100 milliliters; 3. intestinal Enterococci less than 100 cfu per 100 milliliters.Final report of the land

- based testing of the Hyde

- GuardianTM

- System 16 conditions. This is intended to ensure that the efficacy of the equipment is consistent and can be shown repeatedly. The test set

- up should therefore be representative of the characteristics of the arrangements used and the type of environment the BWT system was designed for

. One of the main criteria in the G8 test requirements is the salinity range and related to this the differences in Total Suspended Solids (TSS), Particulate Organic Carbon (POC) and Dissolved Organic Carbon (DOC). This resulted in three main categories o f test conditions (Table 1).

Table 1: Three different salinity ranges and minimum concentrations of TSS, POC and DOC in the water.
Salinity
Parameter > 32 PSU 3

– 32 PSU < 3 PSU unit Total Suspended Solids > 1 > 50 > 50 mg/L Particulate Organic Ca rbon > 1 > 5 > 5 mg/L Dissolved Organic Carbon > 1 > 5 > 5 mg/L

In general UV

- reactors are not sensitive to changes in the salinity of the water except that water with a high alkalinity, iron and/or manganese will increase fouling of the UV

- light

sour ce (Gundry 2007) . Historically UV

- reactors are widely used in treating (fresh) drinking water and sewage water and it s use as a tool for disinfection is well studied. It was for this reason that the Type Approval tests were conducted at the intermediate (3

– 32 PSU) and high salinity (>32 PSU) regions. Moreover, the only difference in composition of the test water betwee n the freshwater and intermediate salinity water is the presence or absence of (sea)salt. All other minimum requirements for TSS, POC and DOC for these two water types were identical (Table 1). A further requirement is that the difference between the two s alinity regimes should be at least 10 PSU. The test water, originating from the Wadden Sea, and the actual sampling did vary with the tide and as a result salinity was subject to variations. To assure the 10 PSU salinity difference it was decided to have t he possibility of adding fresh water and upgrade coastal water of the North Sea water by enhancing the salinity (brine solution of commercially available sea salt; ca. 18%). As target number the freshwater addition was adjusted to a salinity of ca. 23 PSU for the low salinity regime and ca. 33 for the high salinity regime. In practice ca. 15 % (v/v) of freshwater was added during the low salinity tests and about 4% of brine solution (Instant Ocean ® ), during the high salinity test runs. In order to compensat e the dilution of the TSS by the freshwater some extra sediment (taking from a nearby mudflat) was added as well. These additions were made close at the pump site, to ensure proper mixing, with a constant flow rate and done during filling of the control an d the treated ballast tank.
Biology

The guideline G8 also defines criteria for the number and diversity of the organisms to be met during Type Approval testing (Table 2). These criteria should be met for all three salinity regions.Final report of the land

- based testing of the Hyde

- GuardianTM

- System 17 Table 2: Minimal nu mbers and species diversity required at intake for different size classes and groups of organisms.

Influent test water
Parameter unit Remarks organisms ≥ 50 micron > 10 5 / m 3 at least 5 species from at least 3 different phyla/divisions

10 ≤ organism size ≤ 50 micron > 10 3 / mL at least 5 species from at least 3 different phyla/divisions

heterotrophic bacteria > 10 4 / mL not further defined

The test water should contain minimum densities of plankton which are typical densities encountered in the Wadden Sea during the annual spring bloom in April/May. With respect to the species diversity, the Wadden Sea is known for its natural richness in organ isms and during the test period (April

– July) indeed a large diversity in organisms, adults, juveniles, eggs, etc. was encountered. An important aspect, so far not recognized in the guidelines (G8), when dealing only with natural populations of organisms in the influent of the test water is the natural seasonality of species and blooms. The actual onset of the spring bloom is characterized by a dominance in phytoplankton, but usually lacks high zooplankton abundance. Only at a later stage zooplankton start s to increase in abundance, subsequently due to predation it will diminish the numerical abundance of (smaller size) phytoplankton component. Furthermore, for the high salinity range, the composition of the organisms in the water resembles that of a typic al oceanic environment. This implies an increase of smaller sized cells, down to the micrometer scale, and also a dramatic decline in the number of larger (>10 μm) organisms. So far this shift in community structure has not been accounted for when using na tural plankton for testing.
Human pathogens

Table 3: Maximum allowed numbers of 3 groups of indicator microbes in the effluent test water on discharge. cfu: colony forming units

Effluent test water

Parameter unit Remarks
Toxicogenic Vibrio chlorerae < 1 cfu/100 mL or < 1 cfu/ g wet weight of zooplankton serotypes O1 and O139

Escherichia coli <250 cfu/ 100 mL
intestinal Enterococci <100 cfu/ 100 mL

Within the group of prokaryotic microbes only bacteria and more specifically the heterotrophic g roup (Table 2) has been defined by the standard but for completeness this should include all bacteria and presently also Archeae. While these microbes are part of the natural community in the aquatic environment the indicator microbes (Table 3), i.e. the h uman pathogens, are introduced as part of human activity and often associated withFinal report of the land

- based testing of the Hyde

- GuardianTM

- System 18 sewage discharge. In the present research all microbes have been included as a bulk parameter, the number of heterotrophs as a viable component as well as the viability of t he whole microbial community has been determined. Within the whole microbial community the number of heterotrophic bacteria was determined as well as E. coli and total enterococci. The test area of the institute is part of a tidal estuary of the Wadden Se a, which is essentially a pristine environment. Moreover, waste water treatment is highly developed in the Netherlands. Therefore, numbers of these human pathogens during the tests were to be expected to be low for most of the sampling period. On the other hand during the different treatment steps a significant amount of particulate organic material is transferred into dissolved organic carbon (DOC) which acts as an excellent substrate stimulating growth of (heterotrophic) bacteria.



## 7.2 **Experimental desi gn** 
A variety of methods was applied to examine the biological efficacy of the Hyde

- GuardianTM

- system for the different categories of organisms during the two test series. For detailed description we refer to read the outline of the official test protocols for the Hyde

GuardianTM

- system (Anonymous 2008) . Sample handling and volumes were according to the description of the guideline for BWT testing (G8) or described in detail when these guidelines were insufficient or other considerations were taken into account, e.g. in the case of sampling and incubation of samples at discharge. Subsamples were taken at randomly or th roughout the whole filling procedure of the tanks. As indicated previously there was great emphasis on analysing of the freshly taken samples and having multiple methods to examine numerical abundance and viability. Besides various biological samples there was also a basic set of physical and chemical parameters which were monitored prior, during and after discharge. A short description of each parameter and how it has been analysed is given below.
Physical and chemical properties of test water

Temperatur e
The water temperature was measured using a calibrated thermometer.

pH

The pH

- level is measured using a calibrated pH

- meter.
Salinity

For salinity ca. 250

- 500 mL water is sampled and stored at room temperature (glass bottles) until a nalysis by direct measurement in the laboratory at NIOZ. Salinity of the water was measured after each test cycle using a refractometer (Atago) calibrated against 0 and 33 PSU standard (sea)water. The accuracy of the salinity measurement is 0.5 PSU.

Diss olved Oxygen

The spectrophotometric method of the Winkler method (Winkler, 1888 ; Pai et al. 1 993 ; Reinthaler 2006) was used to determine the oxygen concentration in the water. Samples were taken using gastight tubing which was specially fitted to the sampling tubin g that was used to sample the ballast simulating tanks. The coded glass bottles are flushed at least three times their volume (ca. 120 mL) with water. The sample bottles were stored in a dark container filled with water of the same temperature as the samp les until further analysis at the laboratory. In the laboratory 1 mL H 2 SO 4 is added prior to measuring the OD at 456 nm with a Hitachi U

- 3010Final report of the land

- based testing of the Hyde

- GuardianTM

- System 19 Spectrophotometer. The oxygen concentration was calculated using standards and expressed as **μ** M O 2 /L (or mg O 2 /L; = **μ** M O 2 * 0.032)

DOC
The concentration of dissolved organic carbon (DOC) was measured according to Reintaler & Herndl (Reinthaler & Herndl 2005) . Samples for DOC (15 mL) were filtered through GF/C filters and sealed in pre

- combusted glass ampoules after adding 50 **μ** l of phosphoric acid (H 3 PO 4 ). Sealed ampoules are stored at 4 °C. The DOC concentration was determined in the laboratory by the high temperature combustion method using a Shimadzu TOC

- 5000 analyzer. Standards were prepared with potassium hydrogen phthalate (Nacalao Tesque, Inc, Kioto, Japan). The mean con centration of triplicate injections of each sample (three in total) is calculated. The average analytical precision of the instrument is < 3 %.
TSS / POC (total suspended solids and particulate organic Carbon)
For TSS/POC pre

- weighted glass fibre filters (GF/C) are used. Each filter was coded and stored in a clean Petri dish. The filtered volume was dependent on the particle load and concentration and type of organisms present in the water. The higher the total particle load in the sample, the smaller was the volume that could be filtered before the filter clogs. Practical volumes were between 100 and 1000 mL per sample. After filtration the filter was rinsed with fresh water (MiliQ) to remove sea salt. Filters were dried overnight at 60 °C and allowed to cool in a vacuum exicator before weighing. The total amount of suspended solids was calculated from the weight increase of the filter and averaged for the three replicates (mg/L). Next, the filter is combusted at 500°C (overnight) and allowed to cool in a vacuum exicator and weighted again. The POC was calculated from the weight decrease between this measurement and the TSS weight.

Figure 6 : Sampling point at tank 3

Biology

The majority of the large size fraction (>50 μm) consists of z ooplankton, while the majority of the small size fraction (10

- 50 μm) consists of phytoplankton. Organisms > 50 μm are retained as recommended in MEPC 54/Inf.3 (using a Hydrobios net).

Samples for the 10

- 50 μm fraction were collected as whole undisturbed samples. These samples were then filtered over a 50 and a 10 μm sieve and fixated. A second set of samples for this size class was taken and not separated from the organisms < 10 μm in order to include the fate of the smaller sized (phyto)plankton communi ty as well and to avoid further damage of the plankton. The results of these samples were comparedFinal report of the land

- based testing of the Hyde

- GuardianTM

- System 20 to the ones from the double filtered samples to evaluate the loss of organisms caused by processing the samples.
Sample sizes

During the land

- based tests c ontainers from 1 to 1000 L were used for sampling and/or storage. Samples were taken continuously and evenly during the whole process of filling or emptying the ballast water tanks. These containers were thoroughly rinsed or heat

- treated prior to use. Samp les for the human pathogens were taken in sterile (bar

- coded) bottles provided by the bacterial test laboratory.

Figure 7 : 1000 L container with a 50 **μ** m Hydrobios sampling net

Organisms > 50 μm

The samples are pre

- concentrated over a Hy drobios 50 **μ** m net resulting in an end volume of approximately 100 to 200 mL. The samples were transferred to the lab directly after sampling and Neutral Red was added in a ratio that yields an end concentration of 1:50,000. Staining time is 2+ hours. Neutr al Red stains living organisms (Crippen 1974 ; Fleming & Coughlan 1978) distinctively and quite rapidly (less than one hour, figure 6). Therefore the

v iability assessment remains unaffected by the possible death of organisms during the staining or during sample analysis. It is assumed that dead but physically intact organisms will also be found. Consequently a detailed inspection of each intact individua l is needed to assess viability. This includes the staining as well as the detection of internal (heart, gills) movement. Organisms which were not intact are assumed to be dead.
Neutral Red is a reliable staining method for all major groups of organisms but inconsistent staining was found for bivalves. For this latter group movement (including internal such as heart and gills for juvenile mussels) has to be used obligatorily to determine viability. This is dependent on the expertise of the person analyzin g the samples. Therefore the same person analyzed all samples.Final report of the land

- based testing of the Hyde

- GuardianTM

- System 21 Figure 8 : (top) live copepod (left) and nauplia (right) from a Neutral Red stained sample. (bottom) dead copepod (left) and nauplia (right) from a NR stained sample. Pictures made with the FlowCam.

The samples are analyzed manually using a binocular with a 20x magnification for counting and up to 50x for species identification and measurements when necessary. For inter comparison a subset of samples was also analyzed using a semi

- automated tool (FlowCam, Fluid Imaging Technologies; (Anonymous 2001) ). Organisms need to be counted according to their size. Here organisms of 50 **μ** m in minimum dimension are relevant. Several tests have shown that a single size bar is not efficient as viable organisms m ove in the counting chamber. Better results are achieved when the entire field of view is equipped with a size grid. The minimum dimension to measure will be adjusted to the specific organism groups.

Organisms < 50 **μ** m
Samples for visual inspection of sp ecies number and diversity were pre

- concentrated using a sieve made of a Hydrobios 10 **μ** m mesh net using the 50 **μ** m prefiltered sample (effective size range is 10

– 50 **μ** m). The retained organisms were flushed into 50 mL Greiner tubes using filtered seawater and fixed with Lugols solution. Sample analysis was conducted by microscopic count with an inverted microscope at 200x magnification (method by Utermöhl). Since the Utermöhl is not suitable to asses viability counting was restricted to the structural integ rity of organisms and therefore the presence of intact cells (Paerl 1978) . This method works for both zoo

- and phytoplankton. This size fraction was also covered by flow cytometry on basis of a single cell measurement (Veldhuis & Kraay 2000) and PAM fluorometry, as a bulk parameter (Schreiber et al. 1993) , using the intact and undisturbed samples. Besides numbers and sizes these two methods can be used to assess t he cellFinal report of the land

- based testing of the Hyde

- GuardianTM

- System 22 viability (Veldhuis et al. 2001 ; Veldhuis & Brussaard 2006) or in case of the PAM fluorometry also the photosynthetic efficiency of the phytoplankton.

Flow cytometry: For total organisms counts 3 mL of unfilte red sample water (reference and treated, each in triplicate) were analyzed using a calibrated flow cytometer. This yields the total number of particles (dead and live organisms as well as detritus) as well as their size range and the presence or absence of chlorophyll. For the counts exactly 1 mL was analyzed. The size of the plankton was determined by comparison to standardized beads (10 and 50 **μ** m). These beads were also used as standards to calibrate the performance of the flow cytometer. For organism via bility testing, on the level of the individual cell, SYTOX Green was added to 1 mL of sample water (control and treated, each in triplicate). After 15 minutes samples were analyzed using the flow cytometer for the presence of dead and/or life organisms (cf

. Veldhuis et al, 2001).



### nucleus B A

Figure : 9(A) Epifluorescence microscopic picture of a life phytoplankton cell. The red signal is due to the presence of chlorophyll and (B) a dead phytoplankton cell with a yellow/green fluorescence of the nucleus after stainin g with SYTOX Green.
PAM fluorometry: The photochemical efficiency of photosystem II of phytoplankton (providing an estimate of the general health of the algae) can be addressed using Pulse

Amplitude Modulated fluorometer (PAM

- fluorometry) WALTZ

- water PA M (Schreiber et al. 1993) . For this 3 mL of unfiltered sample water (control and treated, each in triplicate) are filled into a glass cuvette and analysed using the Pu lse

- Amplitude Modulated fluorometer. The instrument was calibrated against filtered seawater and a healthy fast

- growing population of phytoplankton.
Next to cell specific analysis Plant

- pigments and chlorophyll a were determined to assess the fate of the whole phytoplankton community (Jeffrey et al. 1997 ; Kraay et al. 1992) . For this purpose water samples of 0.2 to 1.5 L (GF/C filters) were taken. The samples were frozen until further analysis. The system used is a Dionex HPLC system equipped with a C18 separation column. The different algal pigments can be separated according to their polarity. The following solvents are used as elutes in t he HPLC gradient: A 0.5M ammoniumacetate in methanol and water (85:15), B acetonitril and water (90:10) and C ethylacetate 100%.

Bacteria

The classical method for counting bacteria in many applications is based on plating on selective media. Unfortunatel y, for studies in the aquatic environment this approach is by far insufficient for various reasons (Gasol & Giorgio 2000) . As a result total bacteria were now determined by flow cytometry, using DNA

- specific stains to get a more accurate bacteriaFinal report of the land

- based testing of the Hyde

- GuardianTM

- System 23 number. In addition samples were taken at discharge for specific human pathogens and heterotrophic bacteria using a plate method. A 1.5 mL water sample was taken and pipetted in a Cryovial (in triplicate) and formaldehyde was added as a preservative. Samples were frozen and stored at

- 50 oC until further analysis. Upon analysis the sample is allowed to thaw completely. A subsample of 100 **μ** l is taken, diluted with a TE

- buffer, and the nucleic acid dye PicoGreen (MP) was add ed. Within 5 to 15 minutes after the addition of the stain the sample is analyzed using a flow cytometer (cf. Gasol & Del Giorgio 2000 ; Veldhuis et al. 1997) . A known bacterial standard is used for calibration and counting. The number of total heterotrophic bacteria was determined using a plate method as the number of colony formi ng units (cfu’s) after incubation of the water at intake and discharge according to an international standard (NEN

- EN

- ISO 6222:1999).
Human pathogens

The samples for microbiological analysis are taken in special bottles of 600 mL and send to a special la boratory for further analysis. This laboratory was “Vitens laboratory bv” at Leeuwarden (accreditation certificate: NEN/ISO/IEC 17025; lab. no. L043). All analysis’ are carried out according to NEN

- EN

- ISO standards. These samples are sent to the laboratory immediately after sampling using a cooled transport container (4 ° C). The analysis is carried out according NEN

- EN

- ISO 7899

- 2 for intestinal enterococci and NEN

- EN

- ISO 9308

- 1 for E. coli and related bacteria of the coli group as adopted for surface and wa ste water analysis in the Netherlands.Final report of the land

- based testing of the Hyde

- GuardianTM

- System 24 

## 8 Results

The present section is a compilation of all relevant information needed for Type Approval Certification tests according to the Guidelines (G8), but also includes some relevant results of experiments condu cted to assess the environmental acceptability of the applied UV

reactor and/or its potential by

- products in the environment upon discharge. Data are presented as averages or ranges separated for the two salinity regimes tested. In Annex 1 a detailed speci es list of observed organisms is presented.

Figure 10 : overview of the Hyde

- GuardianTM

- Ballast Water Treatment System in the NIOZ harbour. In front of the container are the three IBCs with the 50 μm inline sampling nets.

The tests were carried out at two different salinity regimes (Tables 4 and 5) hereafter referred to as low and high salinity test series.



## 8.1 **Physical and chemical**  **parameters**

Table 4: Average salinity and temperature of water at intake during the first low salinity tests of control and treated tanks for test runs 1

– 5.

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

1

- 5 22.1 0.92 8.5

- 10.4
To the low salinity test cycles brackish water from the NIOZ harbour was collected during low tide and fresh water was added to a maximum of 15% (v/v).Final report of the land

- based testing of the Hyde

- GuardianTM

- System 25 Table 5: Average salinity and temperature of water at intake during the high salin ity tests of control and treated tanks for test runs 6

– 10.

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

6

- 10 31.9 1.3 14.3

- 17.5

To the high salinity test cycles coastal water from the North Sea was collected during high tide and a brine solution made from natural sea salt was added to a maximum of 4 % (v/v).

In terms of difference in salinity the variation between both salinity regimes is slightly less than 10 PSU. However, given the standard variation and the fact that tidal effects and prevail ing winds showed occasionally disturbed the ideal situation the minor deviation from the ideal situation is considered to be of minor importance. Moreover, the present configuration of the Hyde

- Guardian

- BWT

- system is not affected by changes in the salinity

. Low salinity

A summary of the results of the basic parameters (oxygen concentration, pH, TSS, POC and DOC) is presented in table 6 for the reference and treated water sample at intake and discharge.

Table 6: average or range of o xygen, pH, inorganic n utrients, TSS, POC and DOC concentration of 5 test series run at low salinity at intake and discharge of the reference and treated tank; 1 : range of values

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

O 2 reference 1 9.3

- 12.9 7.9

- 10.7 mg/L

O 2 tr eated 1 9.4

- 13.0 8.1

- 11.5 mg/L pH reference 8.17 0.10 8.19 0.12 μM pH

- treated 8.12 0.12 8.09 0.08 μM
NO 3 reference 42.5 10.3 41.5 11.1 μM
NO 3 treated 41.5 8.00 36.8 10.7 μM
NO 2 reference 0.48 0.07 0.51 0.07 μM
NO 2 treated 2.99 1.13 4.62 0.52 μM
N H 4 reference 2.8 1.03 7.63 1.15 μM
NH 4 treated 2.9 1.24 5.38 1.63 μM
PO 4 reference 0.16 0.03 0.22 0.07 μM
PO 4 treated 0.24 0.03 0.17 0.05 μM TSS

- reference 42.5 21.90 9.60 2.30 mg/L
TSS

- treated 29.9 11.80 11.10 5.70 mg/L
POC

- reference 16.1 6.80 5.40 1.30 mg/L
POC

- treated 10.5 3.21 5.80 2.63 mg/L
DOC

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

- treated 4.87 2.97 3.36 0.37 mg - C/LFinal report of the land

- based testing of the Hyde

- GuardianTM

- System 26 Table 6 clearly demonstrates that as far as the basic parameters are concerned the values were in accordance with the crit eria as indicated in the guidelines (G8, Table 1). As far as the oxygen values of the reference tanks are concerned values at intake were closely corresponding to a saturation values (100%) for the given salinity and temperature. Since the water temperatur e increased steadily during the test series values are presented as a range and not averaged. At discharge, on day 5, the oxygen concentration in the reference tank declined by as much as 20% compared to intake values. In contrast oxygen concentration in t he treated tank was less affected, due to reduced bacterial activity, resulting in higher values (14% decline relative to intake) as compared to the reference tank. The total suspended solids (TSS) and particulate organic carbon (POC) concentrations were sufficiently high at intake but these values varied considerably among the different test runs. At discharge sediment concentration declined largely, mainly because of sedimentation in the storage tank during the holding period of 5 days. The applied filtr ation step effectively reduced the amount of TSS at intake and partly also the POC, on average by 30 and 35%, respectively. The remaining suspended material remained in suspension at discharge and half of its weight consisted of organic material. In contr ast to the particulate fraction the amount of dissolved organic carbon (DOC) was hardly affected by the ballast water treatment system. Mainly due to microbial mineralisation of this water for 5 days resulted in a reduction of the DOC concentration in the reference and treated tank by as much as 48% and 40%, respectively. With respect to the concentration of inorganic nutrients (NO 3 , NO 2 , NH 4 and PO 4 ) there were with a few exceptions, only minor differences between the reference and treated ballast water t ank. At the low salinity test series only the nitrite ( NO 2 ) concentration increased by a factor 6.2 after the UV

- reactor. It is known that in presence of nitrate U V

- photolysis produces nitrite (Buchanan et al. 2006) . The observed e levated concentration was nevertheless within the range typical for the challenge water used and therefore not excessively high. For comparison the observed concentrations of nitrite were lower than the US (EPA) and EU maximum allowed concentration for dri nking water of 71.4 and 7.9 μM (1000 μg

- N/L a n d 110 μg

- N/L), respectively (Sharpless et al. 2003)

. In this respect it should be noted that the prolonged dark incubation also increased the nitrite as well as the ammonia concentration, in both the reference an d treated tank.Final report of the land

- based testing of the Hyde

- GuardianTM

- System 27 High salinity

Table 7: average or range of o xygen, pH, inorganic nutrients, TSS, POC and DOC concentrations of 5 test series run at high salinity at intake and discharge; 1 : range of values

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

O 2 reference 1 4.7

- 9.2 2.6

- 6.1 mg/L

O 2 treated 1 4.2

- 8.8 2.9

- 6.7 mg/L pH reference 8.07 0.05 8.12 0.09 μM pH

- treated 8.05 0.09 8.10 0.12 μM
NO 3 reference 0.46 0.12 0.45 0.12 μM NO3 treated 0.84 0.40 1.33 0.63 μM
NO 2 reference 6.87 4.5 5 6.28 4.63 μM
NO 2 treated 6.85 4.09 6.00 3.96 μM
NH 4 reference 7.33 4.67 6.74 4.75 μM
NH 4 treated 7.69 4.48 7.32 4.59 μM
PO 4 reference 0.41 0.10 0.56 0.29 μM
PO 4 treated 0.48 0.18 0.82 0.25 μM TSS

- reference 33.9 37.10 9.70 3.10 mg/L
TSS

- treated 14 3.60 10.00 5.10 mg/L
POC

- reference 10.2 5.30 4.80 1.30 mg/L
POC

- treated 7.2 1.58 4.50 1.29 mg/L
DOC

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

- treated 4.3 0.83 4.1 0.93 mg - C/L

The results of the basic parameters at the high salinity range were, with a few exceptions, on the whole the same as for the low salinity range (Table 7). At intake the oxygen concentrations were lower than observed for the low salinity test runs. This was mainly due to the temperature increase. Nevertheless, the ambient water wa s nearly saturated with oxygen and at discharge oxygen concentrations declined but they were still far from depleted. Partly because of the increase in seawater from the open North Sea sediment, POC and DOC values were lower as compared to the low salinity test series but still considerably higher than the minimum requirements according to the G8 guidelines. Again the filtration step turned out to be an effective manner of reducing the particle load. Both the TSS content and the POC content declined by a 59 % and 30%, respectively. Other differences were in the concentrations of the inorganic nutrients and in particular those containing nitrogen. While the nitrate concentration was very low, the level of nitrite and ammonia were elevated already in the water at intake. In fact these natural concentrations were much higher than the enhanced nitrite concentration caused by the UV

- reactor. In contrast to the observations at the low salinity test runs the ammonia and nitrite concentration remained virtually const ant during the 5 days of dark incubation.Final report of the land

- based testing of the Hyde

- GuardianTM

- System 28 

## 8.2 **Biology**

Organisms > 50 μm

For the land based tests natural plankton was used and the required diversity of organisms (5 species of at least 3 different phyla) was easily fulfilled for all tests (Table 8). O n average at least 10 different species of 4 to 5 different phyla were present in each sample (full details on species present see Annex 1). Regarding the required minimal numbers of organisms per volume (Table 2), the values for the low salinity series (t est run 1

- 5) were all well above the minimum requirements. On average numbers in the >50 **μ** m size fraction were 25 times higher than the minimum required number of 100,000 m - 3 . At the high salinity range the water at intake was on average for the 5 test ru ns above the minimum numbers of organism but contained in two cases not sufficient numbers (test run 9 and 10). Test run 8 was slightly less than the required minimum of 100,000 organisms per m - 3

. As explained earlier this is mainly due to the fact that n atural seawater was used without any addition of (surrogate) organisms and that on average numbers of larger sized organisms are much lower in high saline water than brackish water. On discharge the number of organisms had declined dramatically in the trea ted tank, to an average value for the low and high salinity test series of 2.9 and 2.4 organisms m - 3

, respectively. It should however, be noted that the observed organisms, although still seemingly alive did receive prior to analysis a second UV

- treatment. Since the applied BWT technology is known to have a delayed effect the organisms may be dying in the following day(s). Due to the very low concentration of organisms this could not be tested with sufficient statistical accuracy. In fact during most test s eries at direct inspection immediately after intake already virtually no living organisms were observed. On average numbers in the treated tank were on discharge at day 5 well below the D2

- standard of 10 organisms m - 3 . In contrast numbers of organisms in t he reference tank were still significantly above the G8 requirement and visual inspection and viability measurement indicated that these organisms were both intact and viable.

Table 8: Number of organisms ( > 50 **μ** m ) of individual test runs and averages for the salinity range at intake (T0) and on discharge at day 5 (T5) of the reference (C) and treated (HG) tank. For test run 4 the same control was used, since this was a combined test run. All numbers are presented per m 3

.

total plankton > 50 micron numb ers/m 3 low salinity intake reference treated
C

- T0 C

- T5 HG

- T5
I 1.24E+06 1.08E+04 3.0
II 4.83E+06 6.17E+02 1.7
III 1.01E+06 2.18E+03 0.0
IV 3.12E+06 2.18E+03 2.7
V 7.3
average 2.55E+06 3.95E+03 2.9 s.d. 1.79E+06 4.65E+03 2.7Final report of the land

- based testing of the Hyde

- GuardianTM

- System 29 Table 8: contin ue d

total plankton > 50 micron high salinity intake reference treated
C

- T0 C

- T5 HG

- T5
VI 4.11E+05 1.31E+04 2.7
VII 2.75E+05 1.67E+04 0.3
VIII 8.57E+04 2.45E+03 1.7
IX 5.15E+04 1.73E+04 1.7
X 6.85E+04 2.76E+04 5.7
average 1.78E+05 1.54E+ 04 2.4 s.d. 1.58E+05 9.05E+03 2.0

Organisms 10

– 50 μm

This size class was dominated by phytoplankton, although heterotrophic organisms like ciliates and flagellates were occasionally present

. For that reason the flow cytometric analysis included chlo rophyll a fluorescence as an extra selection parameter. Moreover, the photosynthetic efficiency of the phytoplankton community was measured, as a tool to determine the efficacy of the treatment more specifically, on this group of organisms. Finally, a 10 L subsample of both the reference and treated water was collected at intake and for the treated water also after the second UV

- irradiance disinfection at discharge and incubated under optimal growth conditions. Results of plankton numbers in the 10

– 50 **μ** m of both reference and treated incubations are included in the table for comparison with the actual measurement of the tanks (Table 9). Table 9 shows that regarding the number of organisms at intake for both salinity regimes the test water contained suffici ent plankton numbers, on average 50% higher numbers than required according to the Guidelines G8. Prolonged incubation of the plankton community in the dark (5 days) resulted in a considerable reduction in the number of plankton in the 10

– 50 μm size rang e, on average by a factor 10. Nevertheless, the plankton community of the reference tanks still contained sufficient numbers of viable organisms to meet the G8 requirements for discharge of the reference tank. Moreover, the bottle incubations, which

were r an parallel to the tank incubations, showed, with two exceptions (series 3 and 4) a considerable increase in numerical abundance of the plankton within 5 days, i.e. clearly indicative of the presence of viable population of mainly phytoplankton.

Figure 11 : internal view of Hyde

- GuardianTM

- system showing the UV

- reactor. The UV

- light is clearly visible in the open sampling line (yellow/greenish).Final report of the land

- based testing of the Hyde

- GuardianTM

- System 30 In contrast, treatment resulted in an efficient removal of plankton although in terms of numerical ab undance numbers remained fairly high, certainly in the test series at the high salinity range, after the first treatment. After 5 days in the tanks residual plankton cells were still detected flow cytometrically. However, analysis of the viability using SY TOX Green and the photosynthetic efficiency measurements clearly demonstrated that these cells, although intact, were no longer viable. The incubated experiments confirmed the low abundance of intact cells and on average the number of viable cells after th e first treatment step was < 1 cell/mL. Because the Hyde

- GuardianTM

- system also included a second UV

- irradiance treatment at discharge this water was incubated for another period of 5 to 7 days to determine the efficacy of the second passing of the UV

- react or over a longer period. Measurement showed that there was indeed a delayed effect. The original numbers in the tank at discharge (HG

- T5) were further reduced to average values of 2.3 and 1.0 (HG

- Dis

- Tx) for the low and high salinity ranges, respectively. Also compared to the bottle incubations of the intake samples the second UV

- irradiance disinfection resulted in a further decline in numbers of organisms.

Table 9: number of t otal plankton ( 10

– 50 **μ** m size range ) of each test run and average for the sali nity range at intake (T0), reference (C) and treated tank (HG), reference and treated incubated samples of the intake (Inc.) at day 5 (T5) during discharge and of the incubated discharge samples (Dis

- Tx). Test run 4 and 5 share the same reference since thi s was a combined test run. All numbers are presented per mL as total counts or of viable cells. Tx= varying sampling day (5 to 7 days).

10

- 50 μm cell numbers Reference Treated cells/mL

low salinity total viable total viable x=5

- 7 days
test run C

- T0 C

- T5 C

- inc

- T5 HG

- T0 HG

- T5 HG

- T5 HG

- inc

- T5 HG

- inc

- T5 HG

- Dis

- Tx
I 1103 140 11184 677 242 <10 31.8 <1 0.0
II 1719 156 18706 836 21.5 <10 1.5 <1 10.9
III 1530 144 1149 483 396 <10 28.9 <1 0.0
IV 1784 147 765 304 103 <10 4.4 <1 0.0
V 309 169 <10 6.7 <1 0.7
average 1534 147 7951 522 186 <10 15 <1 2.3 high salinity
VI 1657 121 9391 1091 53.3 <10 11.1 <1 0.0
VII 1657 149 6007 1453 68.8 <10 3.7 <1 2.2
VIII 2090 149 7774 2111 106 <10 6.7 <1 2.2
IX 100 8 130 5754 514 7.4 <10 5.2 <1 0.7
X 1148 151 5941 1056 44.4 <10 3.7 <1 0.7
average 1512 140 6973 1245 56 <10 6.1 <1 1.0

In order to have a more complete insight of the fate of all organisms, i.e. also the planktonic fraction < 10 **μ** m in size diameter, a single species of phytoplankton in the size range of ca. 6 **μ** m ( Phaeocystis globosa) was also monitored in the control and treated water (Table 10). Although no clear criteria are, yet, defined for this size class it was clear that the Hyde

GuardianTM

- system was also effective in reducing organisms in this size range. In general the response of Phaeocystis in terms of numbers and viable cells showed great similarity with the plankton in the 10 to 50 μm size class. Flow cytometry showed th e presence of intact plankton in relatively high numbers in the treated tanks even after 5 days. This was confirmed by classical analysis of the chlorophyll and associated pigment content (data not shown). Viability tests indicated that the remaining cells were non

- viable. This was alsoFinal report of the land

- based testing of the Hyde

- GuardianTM

- System 31 demonstrated in the incubation experiments. These bottle experiments showed that with exception of the first test run (204 cells/mL) numbers were already very low after the first treatment series. As with the 10 to 50 μm siz e range (Table 9), numbers of Phaeocystis cells also declined further after applying a second UV

- irradiance disinfection step.

Table 10: number of a dominant phytoplankton species present ( Phaeocystis globosa ; ca. 6 **μ** m in size) at each test run and ave rage for the salinity range at intake (T0), reference (C) and treated tank (HG), reference and treated incubated samples of the intake (Inc.) at day 5 (T5) during discharge and of the incubated discharge samples (Dis

- Tx). Test run 4 and 5 share the same re ference since this was a combined test run. All numbers are presented per mL as total counts or of viable cells. Tx= varying sampling day (5 to 7 days).
Phaeocysti s ~ 6 μm Reference Treated cells/mL

low salinity total viable total viable x=5

- 7 days
test run C

- T0 C

- T5 C

- inc

- T5 HG

- T0 HG

- T5 HG

- T5 HG

- inc

- T5 HG

- inc

- T5 HG

- Dis

- Tx
I 7807 179 10170 13646 5865 <10 204 <1 0
II 2661 62 17175 235 88 <10 0.7 <1 57.7
III 5460 178 3314 6850 1559 <10 0 <1 0
IV 7738 1939 4491 3832 332 <10 0 <1 0
V 322 425 <10 9.6 <1 2.2
average 5916 589 8788 4977 1654 <10 43 <1 12 high salinity
VI 3754 632 9458 3475 493 <10 8.1 <1 0.7
VII 2227 204 4364 2611 463 <10 6.7 <1 3.7
VIII 2038 204 6650 3015 957 <10 3.0 <1 3.7
IX 1252 236 21473 119 4 158 <10 1.5 <1 1.5
X 1395 173 14440 2705 665 <10 4.4 <1 0.1
average 2133 290 11277 2600 547 <10 4.7 <1 1.9
Photosynthetic efficiency

Another, fast and reliable approach to gain insight in the physiology and growth response of the whole phytoplankton community (2 to 50 μm in cells size) is by measuring the photosynthetic efficiency (Fv/Fm) of the phytoplankton cells. This can be done for the whole community or after filtration for different size classes when needed. For a clear interpretation it must be noted that values of Fv/Fm > 0.4 are indicative of a healthy phytoplankton population; a Fv/Fm < 0.4 indicates that the phytoplankton community is experiencing severe stress and a value < 0.1 is typically observed in decaying phytoplankton populations. Table 11 shows that during intake the whole phytoplankton community of the Wadden Sea (low salinity range) and coastal waters of the North Sea (high salinity range) was physiologically in a healthy condition, i.e. containing mostly photosynthetic active a nd therefore viable cells. Only at test run 7 the phytoplankton population showed some indication of stress. Prolonged incubation in the dark (5 days of dark stress) of phytoplankton in the reference tanks resulted in a severe reduction in the photosynthet ic efficiency. Although the phytoplankton community did not enter the decay phase the majority of cells were far from healthy. In contrast the photosynthetic efficiency of the phytoplankton from the same water mass but incubated under optimal growth condit ions showed that the cellsFinal report of the land

- based testing of the Hyde

- GuardianTM

- System 32 possessed a healthy status (C

- inc

- T5). This is not surprising since phytoplankton also increased in numerical abundance (Tables 9 and 10). After the first treatment, at intake, most of the phytoplankton community was immediately dead resulting in Fv/Fm values of 0 (HG

- T). A holding time of 5 days in the tank did not change this pattern. Also in the incubated sample no recovery of the phytoplankton population was observed. This results did not change after the second UV

- irradiance disinfection step at discharge and a second incubation period of 5 to 7 days (HG

- Dis

- Tx).

Table 11: Photosynthetic efficiency (F v /F m ) of the whole phytoplankton community (2

- > 50 **μ** m size range) at each test run and average values for the salinity range at intake (T0), reference (C) and treated tank (HG), reference and treated incubated samples of the intake (Inc.) at day 5 (T5) during discharge and of the incubated discharge samples (Dis

- Tx). Test run 4 and 5 share the same reference since this was a co mbined test run. All numbers are presented per mL as total counts or of viable cells. Tx= varying sampling day (5 to 7 days). nd: not determined

low salinity Reference Treated
F v /F m C

- T0 C

- T5 C

- inc

- T5 HG

- T0 HG

- T5 HG

- inc

- T5 HG

- Dis

- Tx
I 0.65 0.16 0. 69 0.00 0.01 0.00 nd
II 0.51 0.19 0.62 0.00 0.00 0.00 0.00
III 0.58 0.10 0.70 0.00 0.00 0.16 0.00
IV 0.51 0.12 0.81 0.00 0.00 0.00 0.00
V 0.01 0.01 0.00 0.00
average 0.56 0.14 0.71 0.00 0.00 0.03 0.00 s.d. 0.07 0.04 0.08 0.00 0.00 0.07 0.00 hi gh salinity
VI 0.59 0.13 0.63 0.00 0.02 0.00 0.00
VII 0.44 0.11 0.65 0.00 0.04 0.00 0.00
VIII 0.67 0.16 0.60 0.00 0.00 0.01 0.05
IX 0.62 0.28 0.72 0.00 0.00 0.00 0.00
X 0.66 0.13 0.64 0.03 0.01 0.00 0.00
average 0.60 0.16 0.65 0.01 0.01 0 .00 0.01 s.d. 0.09 0.07 0.04 0.01 0.01 0.01 0.02

Bacteria
For the microbial community the presence/absence of two types of human pathogens was monitored prior to and after treatment, while the response of the whole microbial community was also assesse d. Table 12 shows that even during intake the number of the target microorganisms was well below the standard as indicated in the D2

- Standard. Only in three cases the numbers of human pathogens were above the detection limit (test runs 7, 8 and 9). The rea son for this is that the NIOZ harbour is located in a pristine environment with little or no urban activity. Subsequently, also the number of human pathogens in the reference and treated tanks were below detection limit during discharge.Final report of the land

- based testing of the Hyde

- GuardianTM

- System 33 Table 12: plate c ounts of the human pathogens E. coli and total enterococci of each test run and averages for the salinity range at intake (T0), reference (C) and treated tank (HG), reference and treated incubated samples of the intake (Inc.) at day 5 (T5) during dischar ge and of the incubated discharge samples (Dis

- Tx). Test run 4 and 5 share the same reference since this was a combined test run. All numbers are presented per mL as total counts or of viable cells. Tx= varying sampling day (5 to 7 days).

human pathogens Reference Treated counts/mL

C

- T0 C

- T0 C

- T5 C

- T5 HG

- T5 HG

- T5 HG

- Dis

- Tx HG

- Dis

- Tx low salinity E.coli Enterococci E.coli Enterococci E.coli Enterococci E.coli Enterococci

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

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

III <0 .1 <1 <0.1 <1 <0.1 <1 <0.1 <1

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

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

average <0.1 <1 <0.1 <1 <0.1 <1 <0.1 <1 high salinity

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

VII 1 <1 <1 <0.1 <1

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

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

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

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

In contrast to the almost complete absence of human pathogens, the typical marine microbial community was abundantly present. T he total bacteria community is well studied in the Wadden Sea, using a method based on staining the nucleic acid of the cells, for many years. The currently observed numbers ranging from 0.72 to 6.6 10 6 per mL (variation of factor 9.1) are typically observ ed in spring and early summer (Table 13). Of this total bacterial population only a very small fraction could be identified as heterotrophic using the plate assay method (<10 to 60 per mL). Therefore, if only plating would be used as criteria for heterotro phic bacteria numbers this would result in a severe underestimation of the actual bacteria numbers. After 5 days of incubation total bacteria numbers on average declined in the control tank but there was considerable variation between the different test

ru ns (C

- T5). As opposed to the tank total bacterial numbers increased, with exception of test run 2, in the incubated water of the reference tank (C

- inc

- T5) for both salinity regimes. This indicates that a significant fraction of the bacteria was viable. In the treated tanks and in the incubation bottles of the same water the total bacteria numbers at discharge varied from the original numbers during intake in an inconsistent manner. In nearly half of the test runs there was an actual increase whereas in the others there was a decrease from the numbers at intake. These variations were nevertheless moderate and never exceeded the original values by more than a factor of 1.9. In a similar manner the numbers of total bacteria of the incubated sample were compara ble with th ose of the holding tank samples. A clear reduction in bacteria number was observed after the second UV

- disinfection step during discharge (HG

- Dis

- Tx) resulting in a decline in number by one order of magnitude relative to the number measured in t he tank at day 5.Final report of the land

- based testing of the Hyde

- GuardianTM

- System 34 In terms of cultivable heterotrophic bacteria the treated water showed some remarkable differences with the water at intake and the reference. During the low salinity test runs the numbers of colony

- forming heterotrophs increased on avera ge by a factor 5 after 5 days of incubation but the second UV

- irradiance disinfection step reduced the number of cfu’s to below the detection limit. At all test runs at the high salinity range there the number of colony forming units was roughly identical to the water at intake. In contrast to the low salinity test runs the number of cfu’s increased in 3 out of 5 test runs at the high salinity tests after the second UV

irradiance disinfection step.

Table 13: Total bacteria number (numbers per mL) and col ony forming heterotrophs (cfu/mL) of each test run and average for the salinity range at intake (T0), reference (C) and treated tank (HG), reference and treated incubated samples of the intake (Inc.) at day 5 (T5) during discharge and of the incubated disc harge samples (Dis

- Tx). Test run 4 and 5 share the same reference since this was a combined test run. All numbers are presented per mL as total counts or of viable cells. Tx= varying sampling day (5 to 7 days). nd: not determined

total bacteria numbers/mL heterotrophic bacteria Reference Treated cfu/mL low salinity C

- T0 C

- T0 C

- T5 C

- inc

- 5 C

- T5 HG

- T0 HG

- T5 HG

- inc

T5 HG

- T5 HG

- Dis

Tx HG

- Dis

- Tx

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

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

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

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

IV 1.3E+06 30 0.7E+06 4.9E+06 <10 1.4E+06 3.8E+05 8. 8E+05 375 9.4E+04 <10

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

average 3.9E+06 32 2.1E+06 5.1E+06 43.0 2.8E+06 2.4E+06 1.6E+06 234 3.0E+05 <10
s.d. 2.3E+06 16.4 1.2E+06 2.4E+06 30.6 1.4E+06 2.4E+06 1.5E+06 132.6 3.1E+05 high salinity

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

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

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

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

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

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

- >1000
s.d. 2.1E+06 15.2 2.0E+ 06 1.9E+06 21.6 1.1E+06 4.2E+05 1.6E+06 30.0 5.0E+05Final report of the land

- based testing of the Hyde

- GuardianTM

- System 35 

## 9 Environmental acceptability

Ballast water treatment system s applying active substances, but also systems that do not use active substances should demonstrate according to the rev ised guidelines G8 (MEPC58, October 10 th ) that the treated water upon discharge is not harmful to the environment and organisms. Although officially not obligatory for the Hyde

- GuardianTM

- system a series of studies were conducted to examine long

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

- term incubation experiments, one at each salinity regime, were conducted with treated water which was collected in a clean container (10 L) and incubated under optimal growth conditions in a climate room. The experiments were done for treated water collected at intake (pre

- filtration and UV

- reactor) and at discharge (second UV

- irradiance disinfection step). The second series of (discharge) samples were stored in the dark tank for 5 days prio r to the incubation. To stimulate regrowth of planktonic organism extra nutrients were added ( nitrate ; 30 μM, phosphate; 2 μM, silicate; 20 μM). In particular at the peak of the spring bloom and thereafter the ambient nutrient concentration (mainly silicat e and phosphate) can be extremely low, which may prevent growth of phytoplankton and also bacteria.



### time [days] 0 2 4 6 8 10 12 14 16 18 20 22 24 26phytoplankton 10-50**μ**m [cells/mL]



### 1e-2 1e-1 1e+0 1e+1 1e+2 1e+3 1e+4 1e+5

control Hyde-Guardian intake Hyde-Guardian discharge

Figure 12: time course of phytoplankton numbers (10

– 50 μm size range) during a 25 day incubation period, control and treated water incubated at intake and treated water also incubated at discharge. Test run X; values are mean of three replicates. Line marks the D2

- Standard value of 10 cells/mL.
Figure 12 shows that when incubated under optimal growth conditions the total cell numbers of phytoplankton in the 10

- 50 μm declined in the first three days. The same was observed for the smaller phytoplankton (data not shown)

. Regrowth of phytoplankton only occurred after 7 days of incubation. This observation indicates tha t the treatment did not deactivate the entire plankton community. Some cells survived as resting stages, cysts or did recover from the UV

- irradiance disinfection step. Although figure 12 only shows the results for the larger fraction a similar trend was ob served for the smaller phytoplankton as well (data not shown). Nevertheless, even under favourable conditions recovery, resulting in cell number exceeding the minimum number of 10/mL, was observed after 7 days. For this reason a second UV

- irradiance disinf ection step was applied at discharge. This resulted in a further reduction of the total number of particles by an order of magnitude.Final report of the land

- based testing of the Hyde

- GuardianTM

- System 36 Similar to the first UV

- disinfection treatment there was a delayed effect of ca. 3 days (figure 12). After this period th e plankton community stabilized and regrowth started 14 days after the initial intake of the water. The second UV

- irradiance disinfection step was certainly effective in this respect but the this water was still not entirely free of living plankton. In t erms of vitality it should be noted that regrowth of phytoplankton was observed and the final numbers achieved in these experiments, exceeded even the numbers of the control bottles (Figure 9). Therefore, potential by

- or end

- products generated by the UV

- reactor did not affect the water quality in such a manner that growth of phytoplankton in the receiving environment would be inhibited.



## time [days] 0 2 4 6 8 10 12 14 16 18 20 22 24 26bacteria [cells/mL]



### 1e+5 1e+6 1e+7

control Hyde-Guardian intake Hyde-Guardian discharge

Figure 13: time course of bacteria numbers during a 25 day incubation period, co ntrol and treated water incubated at intake and treated water also incubated at discharge. Test run X; values are mean of three replicates.

With respect to total bacteria numbers the control and the two treated water samples showed a trend over time whic h was basically similar to that of development of the phytoplankton community over time (fig. 13). In the control bottle bacteria increased on the first day, remaining at a constant level for 8 days and declining gradually till the last day of sampling (da y 20). The final numbers at the end were one order of magnitude lower than the original values at intake. In contrast bacteria numbers in the treated water at intake declined rapidly by a factor 10 in the first 5 days of incubation. From day 7 onwards ther e was rapid growth and at the day 20 numbers had increased to a factor 2 higher than the initial numbers. Similar to the phytoplankton (figure 9) the second treatment with the UV

- reactor at discharge resulted in a further reduction of the total amount of bacteria present. A recovery, in terms of bacterial growth was delayed until day 11. The bacterial growth rate was reduced, compared to that of the bacteria at intake, and final numbers after 25 days of incubation were only 50% of the original bacteria num bers at intake.Final report of the land

- based testing of the Hyde

- GuardianTM

- System 37 

## 10 Discussion and evaluation of results

The presented data show that the experimental design, type of test protocols used and additional experiments provide a solid data base of information on the performance of the Hyde

- GuardianTM

- system under semi

- in situ conditions . The initial idea of a treatment systems based on the (permanent) deactivation of organisms through ultraviolet light disinfection, both at intake and discharge turned out to be effective even in surface water with an extremely high density of mucilage containing organisms and a high turbidity. As a first step to reduce the particle load the coarse filter effectively removed the larger fraction including organisms. With only some minor deviations the pres ent 10 test runs were conducted according to the guidelines G8 of the IMO. The basic chemistry and physical properties of the water were not altered by the treatment and no factors could be detected effecting the vitality of the treated water upon discharg e. The treated water remained stable for most of the parameters over the whole holding period of 5 days. Concentrations of dissolved oxygen (DO) and dissolved organic carbon (DOC) hardly varied. Only the concentration of total suspended solids and the par ticulate organic carbon declined slightly mainly as a result of sedimentation. In this respect it should be noted that the application of the self

- cleaning filter effectively removed larger particles and the load of sediment in the treated tanks was only m inimal compared to that in the control tank. With respect to the water chemistry a slight increase in nitrite concentration was observed during the low salinity test runs in the treated water (cf. Sharpless & Linden 2001) . This apparent increase was still 50% of the natural nitrite concentrations observed during the second test series at high salinity and should therefore be considered as non hazardous. Moreover, the values were still lower than the maximum allowed concentration for drinking water (Sharpless et al. 2003)

. The stable DO and DOC over time was indicative of a reduced biological activity during the holding period of 5 days. Number s of organisms were in compliance with the D2

- Standard at discharge but the second UV

- disinfection effectively reduced the number of viable organism further. The treated water did not contain toxic or growth inhibiting substances. Neither were chemicals produced as by

- products. On the cont rary this water was rich in growth stimulating nutrients (nitrogen, phosphorus and silicate) for phytoplankton and bacteria. With apparently some living cells present and ample growth stimulating resources present , phytoplankton and bacteria could start b looming in the presence of light. Still even under favourable conditions growth was delayed by a much as 10 days.
During the testing period we encountered some issues which need attention for future testing and legislation. 1. The number of organisms in the > 50 **μ** m fraction, and to some extend also in the 10

– 50 **μ** m size range, cannot (easily) be met in the salinity range > 32 PSU. The addition of cultured organisms is very difficult and poses a variety of problems. With increasing salinity the seawater will have the characteristics of typical open ocean water. This implies low to extremely low numbers of the organisms in the above indicated size ranges. For that reason we have extended our focus to the organisms < 10 **μ** m since there is no biologically relevant reasoning why tests should exclude this size fraction. 2. The long

- term incubations (ca. 20 days) and the environmental acceptability studies provide good insight in the response of the whole community to the growth potential/limitation of the treated water when released into environment. These experiments should be conducted next to the standard set of toxicology and residual chemistry tests of the treated ballast water. 3. Testing for the presence of human pathogens strongly depends on the natural abundance of these microbes in the natural environment. Since these pathogens cannot be supplemented for safety reasons accurate testing is therefore not possible.Final report of the land

- based testing of the Hyde

- GuardianTM

- System 38 Moreover, viability tests of the total bacteria community showed that not all bacteria were effectivel y deactivated. Therefore, at least in theory the human pathogens would remain a potential risk. This is a factor of concern for the land

- based tests as well as for the ship

- board trials when the ship remains in fairly clean ports for intake and discharge. Although UV systems , including the one applied in the present BWT

system, in general have been proven to effective in the disinfection of (drinking) water no data test are available. 4. The Guidelines G8 and the update version as adopted at MEPC 58 (10 Octobe r 2008) are incomplete with respect to treatment systems including a second treatment at discharge. In line of the tank and sample volume for treatment at intake a similar protocol should be developed for treatment at discharge. However, considering the

ex pected number of organisms at discharge and statistical errors associated with the actual counts a second sample tank to collect and store the discharge would be required with a volume two orders of magnitude larger than the present tank. i.e. 20,000 m 3 . A s an alternative for these large volume incubations, but restricted to the smaller sized plankton, the bottle (10 L) incubations either in the dark or in a more favourable condition could be applied.

In conclusion, the present configuration of the Hyde

- GuardianTM

- system offers a reliable and environmentally safe cleaning of the ballast water resulting in organism numbers well below the Standard of the IMO Regulation

- D2.Final report of the land

- based testing of the Hyde

- GuardianTM

- System 39 

## 11 References
Anonymous (2001) July 11

- 13, 2001 Cape May Flow CAM Report. Fluid Imaging Technologies, Inc P.O.Box350, East Boothbay, Maine, 054544:pp 12. Anonymous (2005) Guidelines for approval of ballast water management systems (G8). Annex3 Resolution MEPC.125(53) Annex:Parts 1,2,3 and 4 Anonymous (2008) Test prot ocol protocol for the biological efficacy testing of the Hyde

- Guardian

- Ballast water treatnebt system (ECOCHLOR, Inc) as part of the type approval process under the resolution MEPC 125.53 Buchanan W, Roddick F, Porter N (2006) Formation of hazardous by

- pro ducts resulting from the irradiation of natural organic matter: comparison between UV and VUV irradiation. Chemosphere 63:1130

- 1141 Carlton JT, Geller JB (1993) Ecological roulette: the global transport of nonindiginous marine organisms. Science 261:78

- 82 Chin A, Bérubé P (2005) Removal of disinfection by

- production precursors with
ozone
- UV advanced oxidation process. Water Res. 39:2136

- 2144 Crippen RW, & J.J. Perrier (1974) The use of neutral red and evans blue for live

dead determinations of marine plankton. Stain Techn. 40:97

- 104 Drake LA, Choi K

- H, Ruiz GM, Dobbs FC (2001) Global redistribution of bacterioplankton and virioplankton communities. Biological Invasions 3:193

- 1999 Endresen Ø, Behrens HL, Brynestad S, Andersen AB, Skjong R (2004) Ch allenges in global ballast water management. Mar. Pollut. Bull. 48:615

- 623 Fleming JM, Coughlan J (1978) Preservation of vitally stained zooplankton for live/dead sorting. Estuaries 1:135

- 137 Gasol JM, Del Giorgio PA (2000) Using flow cytometry for counting natural panktonic bacteria and understanding the structure of planktonic bacterial communities. Sci. Mar. 64:197

- 224 Gollas c h S, Dammer M, Lenz J, Andres H

- G (1998) Non

- indigenous organisms introduced via ships into German waters. In: Carlton e.d., Balla st water: Ecological and fisheries implications ICES Cooperative Research Report No 224.:50

- 64 Gundry M (2007) Ultraviolet disinfection

- practical aspects. Water Supply June/July:33

- 36 Hallegraeff GM, Valentine JP, Marshall J

- A, Bolch CJ (1997) Temperat ure tolerances of toxic dinoflagellate cysts: application to the treatment of ship's ballast water. Aquatic Ecology 31:47

- 52 Hamer JP, McCollin TA, Lucas IAN (2000) Dinoflagellate cysts in ballast tank sediments: between tank variability. Mar. Pollut. Bu ll. 9:731

- 733 Haskoning R (2001) Global market analysis of ballast water treatment technology. Report committed by Northeast

- Midwest Institute. 42810/001R/HSK/SKO. Hoagland P, Anderson DM, Kaoru Y, White AW (2002) The economic effects of Harmful Algal Bl ooms in the United States: estimates, assessment issues, and information need. Estuaries 25:819

- 837 Jeffrey SW, Mantoura RFC, Wright SW (1997) Phytoplankton pigments in oceanography. In: S.W. Jeffrey, R.F.C. Mantroura and S.W. Wright (eds) Phytoplankton pigments in oceanography: guidelines to modern methods SCOR

- UNESCO, Paris pp. 661. Kraay G, Zapata M, Veldhuis MJW (1992) Separation of chlorophylls c1, c2 and c3 of marine phytoplankton by reversed

- phase

- C18

- high

- perfomance liquid chromatography. J. Phyco l. 28:708

- 712Final report of the land

- based testing of the Hyde

- GuardianTM

- System 40 Malley JJ, Shaw J, Ropp J (1995) Evaluation of by

- products produced by treatment of groundwater with ultraviolet irradiation. AWWA Research Foundation and American Water Works Association Paerl HW (1978) Effectiveness of various counting me thods in detecting viable phytoplankton. N.Z Journal of Marine and Freshwater Research 12:67

- 72 Pai S

- C, Gong G

- C, Liu K

- K (1993) Determination of dissolved oxygen in seawater by direct spectrophotometry of total Iodine. Mar. Chem. 41:343

- 351 Raikow D, Reid D, Blatchley E, Jacobs G, Landrum P (2007) effects of proposed physical ballast water treatments on aquatic invertebrates resting eggs. Environmental Toxicology and Chemistry 26:707

- 725 Reinthaler T (2006) Prokaryotic respiration and production in the open ocean. Ph

D thesis University of Groningen:pp. 128 Reinthaler T, Herndl GJ (2005) Seasonal dynamics of bacterial growth efficiencies in relation to phytoplankton in the southern North Sea. Aquat. Microb. Ecol. 39:7

- 16 Rigby G, Taylor AH (2001) B allast water treatment to minimise the risks of introducing nonindigenous marine organisms into Australian ports. Astrl. Gov, BaWa research Ser. Rpt 13. Ruiz GM, Rawlings TK, Dobbs FC, Drake LA, Mullady T, Huq A, Colwell RR (2000) Global spread of microorg anisms by ships. Nature 408:49

- 50 Schreiber U, Neubauer C, Schliwa U (1993) PAM fluorometer based on medium

frequency pulsed Xe

- flash measuring light: A highly sensitive new tool in basic and applied photosynthesis. Photosynth. Res. 36:65

- 72 Sharpless C, Linden K (2001) UV photolysis of nitrate: effect on natural organic matter and dissolved organic carbon and implication for UV water disinfection. Envir. Sci. Techn. 35:2949

- 2955 Sharpless C, Page M, Linden K (2003) Impact of hydrogen peroxide on nitr ite formation during UV disinfection Water Res. 37:4730

- 4736 Veldhuis MJW, Brussaard CPD (2006) Harmful algae and cell death. Ecological Studies; Springer

- Verlag Berlin Heidelberg Vol. 189 E. Graneli & J.T. Turner (eds) Ecology of Harmful Algae Veldhuis MJW, Cucci TL, Sieracki ME (1997) Cellular DNA content of marine phytoplankton using two new fluorochromes: taxonomic and ecological implications. J. Phycol. 33:527

- 541 Veldhuis MJW, Kraay GW (2000) Application of flow cytometry in marine phytoplankton research: current applications and future perspectives. Sci. Mar. 64:121

- 134 Veldhuis MJW, Kraay GW, Timmermans KR (2001) Cell death in phytoplankton: correlation between changes in membrane permeability, photosynthetic activity, pigmentation and growth. Eur. J. Phycol. 36:167

- 177 Williams RJ, Griffiths FB, VanderWal EJ, Kelly J (1988) Cargo vessel ballast water as a vector for the transportation of non

- indigenous marine species. Estuarine, Coastal and Shelf Science 26:409

- 420Final report of the land

- based testing of the Hyde

- GuardianTM

- System 41 

## Appendix 1

Phytoplan kton Marsdiep April

July 2008 +: present but rare, ++: present; +++: dominant;
data by Jolanda van Iperen ++++: very abundant; +++++: massively present
group species name relative dominance autotrophic flagellate Phaeocystis globosa colony cell +++++ Phaeocystis globosa colonies < 50 μm ++++ Phaeocystis globosa colonies > 50 μm +++++ autotrophic flagellate Phaeocystis globosa flagellate cell +++++ heterotrophic flagellate parasite cyst of Ochromonas group ++++ diatom pennate Pseudoni tzschia delicatissima group ++++ heterotrophic flagellate heterotrophic flagellate indet. <10 μm ++++ autotrophic flagellate Prymnesiales indet. <10 μm ++++ diatom centricate Thalassiosira spp. 10 μm ++++ diatom centricate Chaetoceros socialis ++ ++ autotrophic flagellate Hemiselmis group +++ diatom centricate Skeletonema "costatum" group +++ autotrophic flagellate Plagioselmis group +++ heterotrophic flagellate Paulinella spp. +++ heterotrophic flagellate heterotrophic flagellate indet. 10

- 30 μm ++ autotrophic flagellate Pyramimonas spp. <10 μm ++ heterotrophic flagellate Choanoflagellatea indet. ++ hetero/auto flagellate Cryptophyceae "light" group ++ autotrophic flagellate Teleaulax acuta group ++ heterotrophic dinoflagellat e Oxyrrhis marina ++ diatom centricate Leptocylindrus minimus ++ diatom centricate Minutocellus group ++ diatom centricate Thalassiosira spp. 10

- 30 μm ++ heterotrophic dinoflagellate Katodinium glaucum ++ diatom centricate Guinardia delicatula ++ freshwater green alga Pediastrum spp. ++ heterotrophic flagellate Ciliophrys group ++ autotrophic flagellate Prasinophyceae indet. <10 μm ++ hetero/auto dinoflagellate Gymnodiniaceae indet. 10

- 30 μm ++ diatom centricate Various species ++ aut otrophic flagellate autotrophic flagellate indet. <10 μm ++ diatom centricate Chaetoceros spp. <10 μm solitairy cells + autotrophic flagellate Chlorophyceae , Telonema spp

. + heterotrophic flagellate Chrysophyceae indet

. + freshwater green alga Cr ucigenia spp. , Chlorophyta + diatom centricate Skeletonema "costatum" lenses + freshwater green alga Oocystis spp. + diatom pennate Asterionellopsis glacialis + autotrophic flagellate Rhodomonas group + heterotrophic flagellate Bodo group +Final report of the land

- based testing of the Hyde

- GuardianTM

- System 42 Species list zooplankton and some larger (atypical) plankton 

## Appendix 1 cont inued
Phylum Class Subclass, Order, etc. species

no. Identified genera m ost likely present

Sarcomastigophora Dinoflagellida 2 Noctiluca, Protoperidinium

Bacillar iophyceae 3+ Bidulphia, Coscinodiscus

Cnidaria Hydrozoa 2+ Obelia Scyphozoa 2 Aurelia, Cyanea

Ctenophora 2+ Pleurobrachia, Beroe, Mnemiopsis

Nemathelminthes Rotatoria 1+ Asplanchna Nemato da 1+

Annelida Polychaeta 2+

Arthropoda Crustacea Order Calanoida 4+ Temora, Acartia, Centropages, Calanus and/or Pseudocalanus Oithona

Order Harpacticoida 2+ Tigriopus Subclass Cirripedia 1+ Semibalanus Suborder Cladocera 2 Podon, Evadne Subclass Malacostraca 2+ Carcinus (zoea larvae)

Mollusca Gastropoda 1+ Littorina Lamellibranchia 2+ Cerastoderma Mya

Echinodermata Ophiuroidea and/or 2+ Ophiothrix, Ec hinocardium Echinoidea Urochordata Larvacea 1 Oikopleura Minimum number of species encountered (10 phyla): 32

### 1 (24)



## Environmental acceptability evaluation of the Hyde GUARDIAN TM 

## Ballast Water Treatment System as part of the Type Approval Process

Version 3 Non-Confidential
20 April 2009

Authors
Dr. Cato ten Hallers, CaTO Marine Ecosystems & Royal Netherlands Institute for Sea Research (NIOZ), Netherlands Professor Emeritus Erkki Leppäkoski, MeriEko Ltd and Åbo Akademi University, Finland

Mr. Jukka Sassi, VTT Technical Research Centre of Finland, Finland

Mr. Matt Granitto, Hyde Marine Inc., US Mr. Tom Mackey, Hyde Marine Inc., US Mr. Mats Björkendahl, Hyde Marine Inc., a Lamor Group Company, Finland

### 2 (24)
20 April 2009

Table of Contents
Glossary 1 Introduction ................................................................................................................................ 5 2 UV light and disinfecting action.................................................................................................. 5 3 Identification of Preparations, Active Substances and Relevant Chemicals ................................. 8 3.1 Preparations (G9: 2.1.3; GESAMP-BWWG 6, Annex IV: 1.1.3)............................................. 8 3.2 Active Substances (G9: 2.1.1; GESAMP-BWWG 6, Annex IV: 1.1.4) ................................... 9 3.3 Relevant Chemicals (G9: 2.1.4; GESAMP-BWWG 6, Annex IV: 1.1.6)................................. 9 3.4 Other components (GESAMP-BWWG 6, Annex IV: 1.1.5).................................................. 10 4 Data on Effects on Aquatic Plants, Invertebrates and Fish, and Other Biota, Including Sensitive and Representative Organisms (G9: 4.2.1.1; GESAMP-BWWG 6, Annex IV: 3.3) .............................. 10 4.1 Environmental effects ........................................................................................................... 10 4.2 Aquatic toxicity .................................................................................................................... 12 4.2.1 Acute Aquatic Toxicity (GESAMP-BWWG 6, Annex IV: 3.3.2) .................................. 12 4.2.2 Chronic aquatic toxicity (GESAMP-BWWG 6, Annex IV: 3.3.3) ................................. 13 4.2.3 Endocrine disruption (GESAMP-BWWG 6, Annex IV: 3.3.4) ...................................... 14 4.2.4 Sediment toxicity (GESAMP-BWWG 6, Annex IV: 3.3.5) ........................................... 144.3 Bioavailability/Biomagnification/Bioconcentration/Food web/Population effects (GESAMPBWWG 6, Annex IV: 3.3.6, 3.3.7) ................................................................................................... 14 5 Data on Mammalian Toxicity (G9: 4.2.1.2; GESAMP-BWWG 6, Annex IV: 3.4) .................... 14 5.1 Data on mutagenicity and genotoxicity (GESAMP-BWWG 6, Annex IV: 3.4.8) .................. 14 6 Data on Environmental Fate and Effect under Aerobic and Anaerobic Conditions (G9: 4.2.1.3; GESAMP-BWWG 6, Annex IV: 3.5)................................................................................................... 15 7 Physical and Chemical Properties for the Preparations, Active Substance and Treated Ballast Water (G9: 4.2.1.4; GESAMP-BWWG 6, Annex IV: 3.6).................................................................... 15 8 Analytical Methods at Environmentally Relevant Concentrations (G9: 4.2.1.5; (GESAMPBWWG 6, Annex IV: 3.7).................................................................................................................... 15 9 Use of Active Substance or the Preparation (GESAMP-BWWG 6, Annex IV: 4)...................... 15 10 Material Safety Data Sheets (GESAMP-BWWG 6, Annex IV: 5)............................................. 15 11 Risk Characterization................................................................................................................ 15 11.1 Evaluation of the treated ballast water (G9: 5.2; GESAMP-BWWG 6, Annex IV: 6.2) ......... 15 11.1.1 Results from the Land Based Tests................................................................................ 15 11.1.2 Results from the shipboard tests.................................................................................... 16 11.2 Prediction of discharge and environmental concentrations (GESAMP-BWWG 6, Annex IV: 6.3.3) 17 11.3 Effects on sediment (G9: 5.1; GESAMP-BWWG 6, Annex IV: 6.3.7) .................................. 17 12 Quality Control/Quality Assurance (G9: 4.2.4) ......................................................................... 17 13 Use of the Active Substance and Preparations........................................................................... 18 14 Risk Assessment (G9: 6, 7)....................................................................................................... 18 14.1 Risk to safety of ship (GESAMP-BWWG, Annex IV: 7.1) ................................................... 18 14.1.1 Ship Integrity and the BWTS ........................................................................................ 18 14.1.2 Hazardous Area............................................................................................................. 19 14.1.3 Corrosion ...................................................................................................................... 19 14.1.4 Fire and Explosion ........................................................................................................ 20 14.1.5 Chemical Storage.......................................................................................................... 20 14.1.6 Accidental Chemical Release ........................................................................................ 20 14.2 Risk to technicians and/or ship’s crew .................................................................................. 20 14.3 Risks to human health (GESAMP-BWWG, Annex IV: 7.2).................................................. 21 14.4 Risks to the aquatic environment (GESAMP-BWWG, Annex IV: 7.3) ................................. 21

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20 April 2009

14.5 Noise .................................................................................................................................... 21 15 Assessment Report (G9: 4.3; GESAMP-BWWG, Annex IV: 8)................................................ 21 16 Appendices............................................................................................................................... 23
List of Figures
Figure 2-1. Formation of thymine – thymine dimer from adjacent thymine residues. .............................. 6 Figure 2-2. Graph depicting the germicidal curve and typical lamp technology outputs.......................... 8

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20 April 2009

Glossary

BWM Ballast Water Management BWTS Ballast Water Management System CAS Chemical Abstracts Service DBP Disinfection By-products DOC Dissolved Organic Carbon FAO Food and Agriculture Organization GESAMP-BWWG Group of Experts on the Scientific Aspects of Marine Protection - Ballast Water Working Group G2 Resolution MEPC.173(58) Sampling for Compliance Control (G2) G8 Resolution MEPC.174(58) Guidelines for Approval of Ballast Water Management Systems (G8) G9 Resolution MEPC.169(57) Guidelines for Approval of Ballast Water Management Systems that Make Use of Active Substances (G9) HAA Haloacetic Acids IMO International Maritime Organization IMO D-2 Regulation D-2 Ballast Water Performance Standard LC 50 

### Lethal Concentration, 50% LPHO Low Pressure High Output (lamps) MEPC Marine Environment Protection Committee (of the IMO) MSDS Material Safety Data Sheet NIOZ The Royal Netherlands Institute for Sea Research NOEC No Observed Effect Concentration NOM Natural Organic Matter Pa Pascals PAH Polycyclic Aromatic Hydrocarbon PSU Practical Salinity Units STEP U.S. Coast Guard Shipboard Technology Evaluation Program THM Trihalomethane TOC Total Organic Carbon TOX Total Organic Halides US EPA U.S. Environmental Protection Agency UV-A Ultraviolet Light, wavelength 315-400 nm UV-B Ultraviolet Light, wavelength 280-315 nm UV-C Ultraviolet Light, wavelength 200-280 nm UV-Vac Ultraviolet Light, wavelength 10-200 nm (UV-V) VOC Volatile Organic Compound WET Whole Effluent Toxicity WHO World Health Organization

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1 Introduction
With this document, accompanying the type approval test reports (land based and shipboard tests) Hyde Marine Inc. whose registered office is at 28045 Ranney Parkway, Cleveland, OH 44145 USA, appliesfor approval of environmental acceptability according to International Ballast Water Management Convention Guideline G8 (Guidelines for Approval of Ballast Water Management Systems) and in agreement with G9 (Procedure for Approval of Ballast Water Management Systems that Make Use of Active Substances),where appropriate. The document is structured to support evidence that no active substances are used or generated for ballast treatment and that relevant chemicals and other compounds (DBPs) are either not produced or not produced at unacceptable levels.
The document first argues the case for absence of active substances by the treatment method (filtration and UV irradiation) and then presents information on potential by- and end products, their toxicological profiles and the actual presence or absence of such products in the treatment process under the different circumstances the system might meet.
Reference is made to the relevant paragraphs of BWM Guideline G9: Resolution MEPC.169(57) – Procedure for approval of ballast water management systems that make use of Active Substances (G9) and the GESAMP-BWWG Methodology as described in MEPC/58/2/7, 6th GESAMP-BWWG Meeting report, Annex IV.
The environmental performance of the Hyde GUARDIAN TM 

### system is being evaluated according to thedefinitions in G9, the guideline for approval of ballast water management systems that make use of active substances, to which G8 (Resolution MEPC.174 (58) - Approval of ballast water management systems) refers in the paragraphs on environmental acceptability, and according to the methodology developed by the GESAMP-BWWG.
This review is based on the existing literature and on the results from land based and shipboard toxicity tests in the application of Hyde GUARDIAN TM 

### in fully marine and brackish water. It is known from e.g. oxidation processes that the formation of disinfection by-products (DBPs) in marine waters may differ considerably from those in the more frequently studied drinking-, process- and wastewater applications 1



### . Therefore it was important to document whether similar problems as those that occur in some of the fresh-water applications may also arise from UV ballast water treatment saline waters.

2 UV light and disinfecting action
Ultraviolet light (UV) is an electromagnetic radiation of shorter wavelengths ( Ȝ ) than visible light, withwavelengths ranging from 10 – 400 nm and energies from 3 eV to 124 eV. The ultraviolet spectre consists of frequencies (

f ) above those of the visible colour violet.
Ultraviolet (UV) wavelengths 10-400 nm lay outside the visible light spectre. UV is classified according to spectral bands (expressed in nanometres – nm):

x UV-A – 315-400 nm

x UV-B – 280-315 nm

x UV-C – 200-280 nm

x UV-Vac – 10-200 nm (UV-V).

1 Kornmüller A (2007) Review of fundamentals and specific aspects of oxidation technologies in marine waters. Water Science and Technology 55, 1-6

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Natural solar UV includes the full spectrum of UV; artificially UV is made by specific devices, including a UV lamp and a generator, which, depending on construction, can be specialised to generate UV of a specific spectral band or of one single wavelength. Artificial UV is used for several purposes in and outside water treatment. This paper will focus on UV as used in water treatment, where UV is mainly used for disinfection and for photolysis of chemical bonds. For optimal action both processes require specific spectral bands that differ from each other. UV-C is optimal for disinfection, while UV-V results in photolysis of chemical bonds. Wavelengths below 200 nm can also result in unwanted by-products due to photolysis, such as ozone.
Physical process UV disinfection is a physical form of disinfection, as opposed to the chemical form of chlorine and other chemicals. Some molecules, when subjected to UV light, will absorb its energy. Once absorbed, theelectronic energy is sufficient to break bonds and promote the formation of new bonds within the molecule, leaving it damaged. For this reason, UV-C light is called phototoxic (toxic light). The most important molecules of living cells, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are very sensitive to phototoxicity. The most common effect of UV-C is the formation of a cyclobutyl ring between two adjacent thymine nucleic acids located on the same strand of DNA/RNA, as shown on Figure 2-1.

Figure 2-1. Formation of thymine – thymine dimer from adjacent thymine residues 2

.


### The resulting structure, called a thymine dimer, locally distorts the helical structure of the DNA/RNA molecule preventing the proper attachment of transcriptional and replicating enzyme complexes. This damage most commonly results in inhibition of the transcription and replication of the genetic molecules within the affected cell, which results in death of that single cell. 3


### Structural damage to DNA material, rendering the DNA unable to replicate, inhibits reproduction of theorganism. For disinfection and germicidal purposes UV-C penetrates through cell membranes and cyto2 New York State Energy Research and Development Authority (NYSERD) (2004) Evaluation of Ultraviolet (UV) radiation disinfection technologies for wastewater treatment plant effluent.). Final Report 04-7. December 2004. 3 New York State Energy Research and Development Authority (NYSERDA) (2004) Evaluation of Ultraviolet (UV) radiation disinfection technologies for wastewater treatment plant effluent.). Final Report 04-7. December 2004.

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plasm and can enter the cell nucleus; in both nucleus and cytoplasm UV-C damages or destroys genetic material, rendering the DNA unable to reproduce. The optimal wavelength for disinfection is 265 nm. The treatment method is nonintrusive and does not alter the chemistry or physical property of the water to be treated. No organisms have been demonstrated to be resistant to UV.
UV-B, of larger wavelengths, penetrates less deep in fluids and is hence less fit for disinfection. UV-Vrisks the formation of by-products due to ionisation and dissociation of chemical compounds by photolysis.
Artificial UV-C is generated by purpose-constructed devices that generate UV-C through different process, pressures and temperature regimes.
Low pressure UV lamps consist of a quartz lamp with attached electrodes. The lamp is filled with inert gas and a minor amount of mercury. For disinfection purposes the quartz should be natural, as artificial quartz causes the generation of not only UV at 254 nm, but also UV-V of 185 nm. The latter bears the risk of generation of ozone as a by-product. A low pressure UV generator has typically a pressure of 1.3 – 0.13 Pa. The temperature in a low pressure UV lamp is 40.5 – 43 o 

### C. The lamp functions optimally in water of around 21 o 

### C, else the UV is less effective. The lamp functions at 120-140 V, with a current of < 500 mA and a power output of 40 - >100 W. The mercury in the lamp is partly vaporised. The lamp typically emits light of one specific wavelength.
Medium pressure UV-C lamps function at much higher pressures (13-1300 kPa). Temperatures inside the UV lamp rise to 500-600 o 

### C, at which temperature the mercury is completely vaporised. The lamp is stable in all temperature conditions and has a broad spectral output

. The high temperature and pressure creates a plasma excitation of the mercury, which collapses emitting characteristic absorption lines. The principle of the construction of the medium pressure lamp that operates with a reactor is similar as thatof the low pressure lamps. Here too the quartz of the lamp should be natural, so as to prevent the formation of potentially harmful by-products.
UV irradiation for disinfection is most effective at germicidal wavelengths (240-280 nm, Figure 2-2).The effectiveness of the UV treatment (adequate destruction of the genetic material) requires that a sufficient energy dose is absorbed by the organism. The energy dose is measured as the product of the lamps intensity (rate at which photons are delivered to the target) and the time of exposure. UV lamps at low pressure and low intensity are most common in disinfection, however for shipboard use, mediumpressure lamps (>3 kW) are better suited for the application due to compact size and lower power consumption. 4


### Basically, low pressure lamps have a mercury vapour pressure of between 0.1 and 10 Pa, whereas medium pressure lamps have a mercury vapour pressure (when hot) of between 50 and 300 kPa.
Low intensity (normal) low pressure lamps have only mercury present (plus an argon buffer gas), whereas high intensity low pressure (usually called low pressure high output or LPHO lamps) either have an amalgam spot (a compound with mercury and another metal such as indium) or a special longelectrode section with a 'cold spot' (Bolton, James R., Bolton Photosciences Inc. 2009. Personal communication).
The low pressure low intensity lamps are typically about 65 W each. The low pressure high intensity lamps can go as high as 300 W. Regardless of the intensity low pressure lamps are monochromatic (Granitto, Matt, Hyde Marine Inc. 2009. Personal communication)

4 Techneau (2007) UV disinfection and UV/H2O2 oxidation: by-product formation and control. Techneau D2.4.1.1, May 2007.

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Figure 2-2. Graph depicting the germicidal curve and typical lamp technology outputs.



### All organisms have different responses to UV irradiation. The way in which UV is measured is called dose. The UV dose is simply a multiplication of the intensity of the UV and contact time. An organism exposed to 140 mW/cm 2 

### radiation (intensity) for 0.5 s would receive a UV dose of 70 mWs/cm 2 

### or 70 mJ/cm

2 

### . As an example of appropriate amounts of UV, the US EPA’s standard for using UV to disinfect drinking water requires a dose of 40 mJ/cm 2



### . The Hyde GUARDIAN TM 

### system is designed to provide a minimum average dose of 200 mJ/cm 2 

### . This average minimum dose to be effective for disinfection is calculated using the expected end of lamp life intensity of the UV lamp and at a water transmission of T10 = 92%.
BWT through the use of UV is repeatedly proven to be effective against zooplankton, phytoplankton, bacteria and viruses (Malmlook et al 2007 5 

### ). In addition, ”UV irradiation is an attractive, rapid means of disinfection, as it leaves no harmful residues 6 

### ”.

3 Identification of Preparations, Active Substances and Relevant Chemicals

3.1 Preparations (G9: 2.1.3; GESAMP-BWWG 6, Annex IV: 1.1.3)
“Preparation” means any commercial formulation containing one or more Active Substances including any additives. This term also includes any Active Substances generated on board for purposes of ballast water management and any relevant chemicals formed in the in the ballast water management system that makes use of Active Substances to comply with the Convention.

5 Malmlook R, Badran O, Abu-Khader MM, Holdo A, Dales J (2008) Fuzzy sets analysis for ballast water treatment systems: best available control technology. Clean Technologies and Environmental Policy 10, 397-407

. 6 Oemcke DJ, Van Leeuwen J (2007) Chemical and physical characterization of ballast water. Part 1: Effects on ballast water treatment processes. Journal of Marine Environmental Engineering 7, 47-64

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Following this definition the Hyde GUARDIAN TM 

### system is not using any preparations other than UV light.

3.2 Active Substances (G9: 2.1.1; GESAMP-BWWG 6, Annex IV: 1.1.4)

”Active Substance” means a substance or organism, including a virus or a fungus that has a general or specific action on or against harmful aquatic organisms and pathogens.
Hyde GUARDIAN TM 

### system does not use or generate Active Substances, but only uses MP UV-light for disinfection; the photons emitted by the UV light are energy packages and therefore not substances or organisms. The process of disinfection is demonstrated by the description of the system in Chapter 2.2.

3.3 Relevant Chemicals (G9: 2.1.4; GESAMP-BWWG 6, Annex IV: 1.1.6)

” Relevant Chemicals ” means transformation or reaction products that are produced during and after employment of the ballast water management system in the ballast water or in the receiving environment and that may be of concern to the ship’s safety, aquatic environment and/or human health.
UV from solar radiation produces short-lived OH radicals that can act as an intermediate to reaction byproducts. In that sense OH radicals may be considered relevant chemicals. The potential to form OH radicals by the Hyde GUARDIAN TM 

### system is discussed below. Ozone is only formed at wave lengths below the spectre of the Hyde GUARDIAN TM 

### system (below 200 nm) and should therefore not be considered a relevant chemical for the Hyde GUARDIAN TM 

### system.
Formation of OH radicals is enhanced by the presence of chlorine radicals in the water, a situation particular for drinking water purification by UV when chlorine is present in the upstream water to be purified 7 

### . Little is known about the fundamental photochemistry of free chlorine. Following photolysis of aqueous free chlorine to HOCl, the HOCl may absorb a photon ( Ȝ < 511 nm), leading to an OH radical and a Cl radical. Although in seawater chlorine is present in NaCl, the situation as described for drinking water is not to be expected to play an important role in seawater. For fresh water shipping areas, NaCl is not present or only at very low levels. The other situation where OH formation is enhanced is in the presence of added hydrogen peroxide (H 2 

### O 2 

### ) 8 

### or added ozone (O 3 

### ). This situation is not to be expected in an aquatic environment. Solar UV OH formation might be promoted by the ozone when that is present in natural circumstances. As neither H 2 

### O 2 

### , not excess chlorine is present in the conditions that the Hyde GUARDIAN TM 

### system operates, the formation of OH radicals is not expected to be at a level that may cause adverse effects; levels may even be too low to be detectable at all, even when measured at the nanosecond life span of OH radicals. By using UV of Ȝ >200 nm only, the potential to form by-products, such as OH and O 3 

### , through photolysis is prevented. Such by-products may be formed at lower wave length, typically at Ȝ

185 nm, a wave length that is emitted when the lamp is made of synthetic (artificial) quartz. Pure quartz (quartz of natural origin) does not emit wavelengths below Ȝ 200 nm. The lamps used in the Hyde GUARDIAN TM 

### system are all made of pure quartz. Furthermore, the UV source in the Hyde GUARDIAN TM 

### system is shielded by double pure quartz layer, since also the quartz sleeve that covers the UV lamp is made of pure quartz.

7 Watts MJ, Linden KG (2007) Chlorine photolysis and subsequent OH radical production during UV treatment of chlorinated water. Water Research 41, 2871-2878
8 Techneau (2007) UV disinfection and UV/H 2 O 2 oxidation: by-product formation and control. Techneau D2.4.1.1, May

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3.4 Other components (GESAMP-BWWG 6, Annex IV: 1.1.5)
“Other components” of a preparation means any other substances in a preparation, other than the Active Substance(s) or Relevant Chemicals, produced during the treatment of ballast water.
For evaluating the presence of other components, the reaction by-products from the Hyde GUARDIAN TM 

### system, we investigate disinfection by- and end products (DBPs), i.e., chemical organic and inorganic substances that can form during a reaction of UV as disinfectant with the treated ballast water and/or with organic matter in the ballast and receiving water 9



### .
4 Data on Effects on Aquatic Plants, Invertebrates and Fish, and Other Biota, Including Sensitive and Representative Organisms (G9: 4.2.1.1; GESAMP-BWWG 6, Annex IV: 3.3)
Disinfection by UV is a physical process, where bonds are broken through photolysis. Such bonds can either be DNA and other cell material in living organisms or bonds of hazardous organic chemicals, such as may be present in wastewater streams. The administered energy should be sufficient to break thebonds. For disinfection this means sufficient energy to destroy the genetic material so as to render it incapable of reproduction (recombining DNA material). When the administered energy is too low DNA material might be damaged only, but still able to recombine, which can result in mutations that might prove harmful 10 

### . In the UV spectral band produced by the Hyde GUADRIAN TM 

### system (200-400 nm) the energy delivered to the ballast water is 200 mJ/cm 2 

### per treated m

3 

### . This minimum dosing, which is considerably higher than the US EPA’s standard for UV to disinfect drinking water (40 mJ/cm 2 

### ), is set sufficiently high to prevent the possibility that DNA can still recombine.

4.1 Environmental effects
Scientific and technical reports on UV disinfection and by- and end products are only partly applicable to screening of environmental and health impacts of ballast water treated by UV. UV disinfection is common in treatment of drinking water purification and wastewater treatment, reducing bacterial andmicroorganism loads as well as breaking down organic contaminants. By-products formed by UV disinfection on waters containing hazardous chemical compounds, not present naturally in an aquatic environment (such as disinfection products of wastewaters containing PAHs ), are mentioned in this report but are not considered relevant for the purpose of environmental evaluation of MP UV in ballast water treatment.
By-products from UV treatment arise (1) directly through photochemical reactions, and (2) indirectly through reactions with products of photochemical reactions. By-products formed at manufacturing of drinking water are rather well documented. It was shown in several studies that UV disinfection at UV doses < 400 mJ/cm 2 

### does not affect formation of trihalomethanes (THMs), haloacetic acids (HAAs) and Total Organic Halides (TOX) 11



### . UV treatment on its own was shown to have very little impact on the organic constituents in raw water. At low irradiation intensities, UV treatment does not change pH, turbidity, dissolved OC level, colour,

9 www.lenntech.com/water-disinfection/disinfection-byproducts.htm 10 Kalisvaart BF (2001) Photobiological effects of polychromatic medium pressure UV lamps. Water Science and Technology 43, 191-197; Taghipour F, Sozzi A (2005) Modeling and design of ultraviolet reactors for disinfection by-product precursor removal. Desalination 176, 71-80; Cooper WJ, Jones AC, Whitehead RF, Zika RG (2007) Sunlight-induced photochemical decay of oxidants in natural waters: implication in ballast water treatment. Environmental Science and Technology 41, 3728-373311Malley J, Show J, Ropp J (1996) Evaluation of the by-products produced by the treatment of groundwaters with ultraviolet radiation. American Water Works Association Research Foundation, Denver, CO; Liu W, Andrews SA, Bolton JR, Linden KG, Sharpless C, Stefan M (2002) Comparison of disinfection byproduct (DBP) formation from different UV technologies at bench scale. Water Science and Technology: Water Supply 2, 515-521; Kashinkunti RD, Linden KG, Shin G-A, Metz DH, Sobsey MD, Moran MC, Samuelson AM (2004) Investigating multibarrier inactivation for Cincinnati-UV, by-products, and biostability. American Water Works Association Journal 96,114-127

.

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nitrite, nitrate, bromide, iron or manganese of the water being treated 11 

### . The maximum UV dosage used (1.6 Ws/cm2) did not result in any significant reduction of TOC (total organic carbon), chloroform or HAA (haloacetic acids) formation potentials 12



### . There is a plethora of applications, other than ballast water treatment, in which UV has been used for water purification for years or even decades. Among these, commonly known uses in e.g. disinfection ofdrinking water and municipal wastewater, and uses in pharmaceutical and cosmetic industries, laboratories, food and brewery industries etc, have been evaluated and accepted by administrations worldwide.Many references focus on disinfection of drinking water (i.e. freshwater), the DBPs reported and discussed being chlorine and different daughter substances formed by chlorination. Often UV treatment of drinking water is combined with ozonisation or chlorination 13 

### . Research has focused on drinking water and on the impact of UV on the formation of halogenated DBPs after subsequent chlorination, and on the transformation of organic material to more degradable components 14 

### . Addition of chlorine may result in the formation of different DBPs, such as trihalomethanes (THM), haloacetic acids (HAAs), carboxylic acids, aldehydes, and increase of TOX (Total Organic Halogen). 15 

### Buchanan et al. (2006) reported on the formation potential of THM, HAA, nitrite and peroxide resulting from UV photo-oxidation followed by biological treatment for the removal of natural organic matter (NOM). Samples exposed to different doses of UV were found to be non-cytotoxic and non-mutagenic 16



### .For groundwater and filtered drinking water, UV disinfection at typical doses did not impact the formation of THMs and HAAs. Several studies have shown the formation of low levels of (in the US nonregulated) DBPs, such as aldehydes. In filtered drinking water no significant change in aldehydes, carboxylic acids, or total organic halides was found (Kashinkunti et al. 2004).
UV-radiation is also used in seawater reverse osmosis (SWRO) desalination plants. Munshi et al. (1999)noted that UV did not cause any phase change in water, which does not lead to any large-scale accumulation of toxic by-products in the water phase; in summary, UV-radiation was found to be a potential alternative to chlorination 17



### . At hospitals water is treated by UV to insure pure water for pathology laboratories and kidney dialysis where bacteria-free water is of utmost importance 18 

### . For water used in the preparation of dialysis fluid, chemical contaminant levels are set very low. The additional treatment ensures that the water meets the more stringent requirements.
Application of UV disinfection for wastewater treatment is considered environmentally positive: (1) no chemicals are added to the effluent stream, reducing the risk of detrimental effects to aquatic life; (2) no trihalomethane (THM) are formed and (3) UV disinfection reduces safety, handling or explosion risks 19



### .

12 Chin A, Berube PR (2005) Removal of disinfection by-product precursors with ozone-UV advanced oxidation process. Water Research 39, 2136-2144 13 E.g., Watts MJ, Linden KG (2007) Chlorine photolysis and subsequent OH radical production during UV treatment of chlorinated water. Water Research 41, 2871-2878 14 Swaim PD, Cotton CA, Jeyanayagam SS (2004) Ultraviolet disinfection. In: Water Treatment Plant Design. McGraw-Hill 15 Kashinkunti RD, Linden KG, Shin G-A, Metz DH, Sobsey MD, Moran MC, Samuelson AM (2004) Investigating multibarrier inactivation for Cincinnati-UV, by-products, and biostability. American Water Works Association Journal 96, 114-12716 Buchanan W, Roddick F, Porter N (2006) Formation of hazardous by-products resulting from the irradiation of natural organic matter: Comparison between UV and VUV irradiation. Chemosphere 63, 1130-1141
17 Munshi HA, Sasikumar N, Jamaluddin AT, Mohammed K (1999) Evaluation of ultra-violet radiation disinfection on the bacterial growth in the Swro Pilot plant, Al-Jubail. Proceedings of the 4th Gulf Water Conference, 1 Bahrain, Feb.13-17, 1999. 17 pp 18 E.g., Hoenich N, Levin R (2008) Water Treatment for Dialysis: Technology and Clinical Implications In: Ronco C, Cruz DN (eds): Hemodialysis - From Basic Research to Clinical Trials. Contribution to Nephrology Basel, Karger, 161, 1-6 19 Lau PJ (1997) Applying disinfection alternatives to wastewater treatment. Pollution Engineering 29, 64-66

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4.2 Aquatic toxicity
The results from land- and ship-based toxicity tests used in this document were performed in full marine (31.9 PSU) and in brackish water (22.1 PSU). It is known from, e.g., oxidation processes that the formation of disinfection by-products (DBPs) may be considerably different in marine waters from fresh water applications 20 

### . Therefore it was important to document whether similar problems may arise also from UV treatment for water of higher salinities.

4.2.1 Acute Aquatic Toxicity (GESAMP-BWWG 6, Annex IV: 3.3.2)
Some few examples only of acute (eco)toxicity of UV-treated were found during the literature search. In an acute immobilisation test, UVC-treated freshwater appeared to be toxic to the water flea Daphnia carinata . The observed toxicity was attributed to photo-oxidative degradation of metal-binding sites in natural organic matter, which resulted in the release of bioavailable copper ions 21



### . Nitrite toxicity in seawater Some changes in nutrient levels were recorded during the shipboard tests 22 

### . For example, both phosphate and nitrate levels at deballasting were higher in treated samples relative to untreated samples at the time ofdeballasting. It was not possible to definitively conclude that these changes directly resulted from UV irradiation: part of the differences could be attributed to an increase in the degradation of planktonic organisms resulting from the treatment process, rather than UV irradiation directly.
Photolysis of nitrate to nitrite has been demonstrated in UV-irradiated drinking water (e.g. Sharpless & Linden 2001) 23 

### . In shipboard tests, the concentration of NO 2 

### (μgN L -1 

### increased from an initial concentration of 0.6 up to 1.75 – 2.73 μgN L -1 

### in treated samples 10 days after treatment. In the land-based tests 24 

### , at the low salinity test series only the nitrite concentration increased by a factor 6.2 after the UV reactor, up to 4.62 μM at discharge (day 5). The observed elevated concentration was nevertheless within the range typical for the test water used and therefore not excessively high. It is also possible that the prolonged dark incubation also increased the nitrite concentration in both the reference and treated tank.
The observed concentrations of nitrite were lower than the US (EPA) and EU maximum allowed concentration for drinking water of 71.4 and 7.9 μM (1000 μg-N/L and 110 μg-N/L), respectively 25 

### . In seawater conditions these nitrite levels seem not to be of any toxicological significance. High concentrations of chloride ions effectively reduce the toxicity of nitrite. Seawater fish are usually less sensitive to nitrite than freshwater fish 26 

### . For example, nitrite was found to be 55 times more toxic to juvenile milkfish ( Chanos chanos ) in fresh water than in brackish (16 psu) water. The 48-h median LC 50 

### values were, respectively, 12 and 675 mg NO 2 

### -N L -1 27 

### . The 96-h LC 50 

### of nitrite-N for European eel (Anguilla anguilla ) can range from 84.0 mg L -1 

### in freshwater to 812.0mg L -1 

### in full strength seawater (salinity = 36.0

20 Kornmüller A (2007) Review of fundamentals and specific aspects of oxidation technologies in marine waters. Water Science and Technology 55, 1-6 21 Parkinson A, Barry MJ, Roddick FA, Hobday MD (2001) Preliminary toxicity assessment of water after treatment with uv-irradiation and UVC/H2O2. Water Research 35, 3656-3664 22 Wright DA (2009) Shipboard Trials of Hyde ‘Guardian’ system in Caribbean Sea and Western Pacific Ocean, April 5 th - October 7th, 2008. Final Report to Hyde Marine and Lamor Corp. University of Maryland Center for Environmental Science, Chesapeake Biological Laboratory, Solomons Maryland, 122 pp 23 Sharpless CM, Linden KG (2001) UV photolysis of nitrate. Effects of natural organic matter and dissolved inorganic carbon and implications for UV water disinfection. Environmental Science and Technology 29, 2949-2955 24 Veldhuis MJW, Fuhr F, Stehouwer P-P (2009) Final report of the land-based testing of the Hyde-GuardianTM -System, for Type Approval according to Regulation-D2 and the relevant IMO Guideline (April – July 2008). Royal Netherlands Institute for Sea Research, Den Burg, Texel, the Netherlands, 42 pp 25 Sharpless C, Page M, Linden K (2003) Impact of hydrogen peroxide on nitrite formation during UV disinfection Water Research 37, 4730 - 4736 26 Avilez IM, de Aguiar LH, Altran AE, Moraes G (2004) Acute toxicity of nitrite to matrinxã, Brycon cephalus (Günther, 1869) (Teleostei-Characidae). Ciência Rural, Santa Maria 34,1753-
1756 27 Spotte SH (1992) Captive Seawater Fishes: Science and Technology. Wiley, 976 pp

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psu) 28 

### . Similar results were obtained when the relative toxicity of nitrite to chinook salmon fingerlings ( Oncorhynchus tshawytscha ) was measured by static bioassay. In freshwater, the 48-h median lethal nitrite concentration was 19 mg L -1 

### . In seawater, 1,070 mg L -1 

### nitrite caused only 10% mortality in 48 h 29



### . Bromate formation in UV-treated seawater through ozoneIt is known that (1) ozone can be produced by ultraviolet light and (2) ozonation or chlorination of bromide-containing water can facilitate the conversion of bromide into bromate (BrO 3 

### (-), which is a carcinogenic chemical 30



### .No data were found on concentrations of ozone produced during short-term exposure of seawater to UVC nor the risk of production of bromate (and its derivates) in UV-treated seawater. In April 2006, a thorough assessment of environmental risks of bromate as a relevant chemical was performed by Japan MEPC 55/2 in connection with basic approval of active substances used by Special Pipe Ballast Water Management System (combined with ozone treatment) in accordance with the Procedure for approval of Ballast Water Management Systems that make use of active substance (G9) 31 

### . The risk evaluation to the environment focused on the bromate ion. It was considered that the short-term toxicity of ballast water discharged after being treated in the system is basically insignificant: acute toxicity of the bromate ion in seawater was low (NOEC 32 mg/L). Since the concentration of bromate ions (oxidant concentration) at the time of discharge is lower than 0.16 mg/L, Long-term effects are unlikely to occur. Therefore the bromate ion to be discharged from this system will not cause any long-term or short-term effects on aquatic organisms. The Japanese system, submitted to MEPC 55 for approval, uses ozone as an active substance whereas in Hyde GUARDIAN TM 

### system ozone is not likely to be produced since the Hyde GUARDIAN TM 

### operates in a higher UV spectral bandwidth (above 200 nm).

4.2.2 Chronic aquatic toxicity (GESAMP-BWWG 6, Annex IV: 3.3.3)
The most applicable and relevant indirect evidence of zero NOEC (No Observed Effect Concentration) of UV-treated saltwater originates from uses in marine aquariums and hatcheries, in which bacteria-free water prevents disease agents (bacteria, fungi) from growing without producing any DBPs harmful to marine organisms. In mussel and oyster culture, UV-treated water is used for rearing all stages of spat, from fertilization to larvae and juveniles 32 

### , which are regarded as the most sensitive life-cycle stages. Ultraviolet treatment is sometimes employed also in water purification systems for tropical freshwater, marine and cool water aquaria and pond systems, not only to reduce microbial levels but also to aid in oxidation of organics, phosphate and nitrogenous compounds through the collateral production of ozone (O 3 

### ) 33



### . In fish aquaculture, the applications include treatment of water in hatcheries, and fry rearing tanks 34 

### . UV is ideally suited for these applications as it uses no chemicals and does not create by-products, which would harm the fish stock, or other aquatic life, on discharge 35 36 

### . However, the results from a 5-month experiment indicate that seawater recently disinfected with ultraviolet radiation (254 nm) can increase

28 Saroglia MG, Scarano G, Tibladi E (1981) Acute toxicity of nitrite to sea bass ( Dicentrarchus labrax ) and European eel ( Anguilla anguilla ). Journal of the World Aquaculture Society 12, 121-126 (quoted in Avilez et al. 2004) 29 Crawford RE, Allen GH (1977) Seawater inhibition of nitrite toxicity to chinook salmon. Transactions of the American Fisheries Society 106, 105-109. 30 Huang X, Gao N, Deng Y (2008) Bromate ion formation in dark chlorination and ultraviolet/chlorination processes for bromide-containing water. J Environ Sci (China) 20, 246-51 31 MEPC 55/2, submitted by Japan in 2006 32 Helm MM, Bourne N, Lovatelli A (eds) (2004) Hatchery culture of bivalves. A practical manual. FAO Fisheries Technical Paper. No. 471. Rome, FAO.177 pp. www.fao.org/docrep/007/y5720e/y5720e06.htm 33 Saltwater Aquariums. http://saltaquarium.about.com 34 Shackley SE, King PE (2006) A sterile sea water circulating system for the jar culture of developing fish eggs. Journal of Fish Biology 12, 235-237 35 Hanovia News (2007) UV water disinfection in fish farms and hatcheries. http://halmapr.com/news/hanovia/2007/09/28/aquaculture/ 36 Øye AK, Rimstad E (2001) Inactivation of infectious salmon anaemia virus, viral haemorrhagic septicaemia virus and infectious pancreatic necrosis virus in water using UVC irradiation. Diseases of Aquatic Organisms 48, 1-5

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the frequency of cataract (cloudiness of the lens of the eye) in juvenile cod. It is hypothesized that ozone or other photoproducts formed by UV-C radiation may cause cataract in fish 37 

### ; however, other studies on this topic found seem to deal with natural exposure to UV-A and UV-B 38 

### , not UV-C.
In 2002, chlorine was replaced by UV treatment for water purification in a dolphinarium in Edmonton, Alberta, Canada 39 

### . A routine water-testing package consisted of a number of common water parameters (sodium, potassium, calcium, magnesium, total nitrate, iron, manganese, sulphate, chloride, carbonate, bicarbonate, fluoride, hardness, alkalinity, and total dissolved solids). The total nitrate level was the onlyparameter that changed with the implementation of the UV system. Other parameters that were measured throughout the implementation process included dissolved oxygen, pH, suspended solids, and temperature. It was found that both prior to and after implementation the aforementioned parameters did not undergo any change.

4.2.3 Endocrine disruption (GESAMP-BWWG 6, Annex IV: 3.3.4)
No evidence has been found that the by-products that could be formed or have been found to be present at low levels (nitrite) can cause endocrine disruption.

4.2.4 Sediment toxicity (GESAMP-BWWG 6, Annex IV: 3.3.5)
No evidence has been found that by-products could result in sediment toxicity.

4.3 Bioavailability/Biomagnification/Bioconcentration/Food web/Population effects (GESAMPBWWG 6, Annex IV: 3.3.6, 3.3.7)

No evidence has been found that short lived nitrites as a DBP of UV-C treatment could result in biomagnification or bioconcentration.

5 Data on Mammalian Toxicity (G9: 4.2.1.2; GESAMP-BWWG 6, Annex IV: 3.4)
Mammalian toxicity studies have not been performed on DBP (nitrite) specifically for Hyde GUARDIAN TM 

### system. There are no scientific data available indicating that nitrite concentrations at the levels found in the Hyde GUARDIAN TM 

### discharge can be expected to be toxic to mammalians

. 40



### 5.1 Data on mutagenicity and genotoxicity (GESAMP-BWWG 6, Annex IV: 3.4.8)
An environmental concern, which has been assumed but not investigated in depth, is the risk of genetic mutation. If the exposure to UV-C is insufficient to kill all of the organisms, the damage to the geneticmaterial might lead to viable mutations and reproduction might still be possible, and hence pose an environmental risk 41 

### . Even if most or all of the surviving organisms with damaged genetic material will failto reproduce, a possibility exists that genetically altered DNA will benefit the surviving (micro)organisms.

37 Björnsson B (2004) Can UV-treated seawater cause cataract in juvenile cod ( Gadus morhua L.)? Aquaculture 240, 187-199 38 Cullen AP, Monteith-McMaster CA, Sivak JG (1994) Lenticular changes in rainbow trout following chronic exposure to UV radiation. Current Eye Research 13, 731 – 737; Sharma JG Masuda R, Tanaka M (2005) Ultrastructural study of skin and eye of UV B irradiated ayu Plecoglossus altivelis . Journal of Fish Biology 67, 1646-1652; Formicki G, Stawarz R (2006) Ultraviolet influence on catalase activity and mineral content in eyeballs of gibel carp ( Carassius auratus gibelio ). Science of the Total Environment 369, 447-450 39 Dombrosky J (2002) The use of ultraviolet irradiation for sole disinfection of an inland marine mammal facility.www.trojanuv.com/en/images/IC/Dolphin%20Lagoon.pdf40 FAO/WHO (2003): Nitrite (and potential endogenous formation of N-nitroso compounds). In: Safety evaluation of certain food additives and contaminants. Geneva, World Health Organization, Joint FAO/WHO Expert Committee on Food Additives. WHO Food Additives Series No. 50; http://www.inchem.org/documents/jecfa/jecmono/v50je05.htm 41 Buchholz K, Tanis D, Macomber S, Farris E (1998) Ballast Water Secondary Treatment Technology Review. Final Report to Northeast-Midwest Institute. www.nemw.org/Balsurv2_UV.htm

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The risk of thriving mutants is prevented by the high minimum dosing of UV (200 mJ/cm 2 

### - 5 times the dose required by the US EPA drinking water disinfection standard), which dose is sufficiently high to ensure the adequate level of DNA damage. As an extra precaution the Hyde GUARDIAN TM 

### system exposes ballast water to UV-C disinfection once more to the same high dose during de-ballasting.

6 Data on Environmental Fate and Effect under Aerobic and Anaerobic Conditions (G9: 4.2.1.3; GESAMP-BWWG 6, Annex IV: 3.5)
Nitrite released with ballast water will enter the natural nitrogen cycle run by the marine ecosystem where NO 2 

### is present in low concentrations, produced by e.g. planktonic algae and through decomposition of fecal pellets and dead zooplankton organisms 42 

### . It is expected that NO 2 

### from BWT will follow the same reduction and oxidation pathways as NO 2 

### from natural sources; NO 2 

### will be oxydised to NO 3 

### and reduced to NH 4 

### .
7 Physical and Chemical Properties for the Preparations, Active Substance and Treated Ballast Water (G9: 4.2.1.4; GESAMP-BWWG 6, Annex IV: 3.6)

Nitrite is the univalent radical NO 2 

### or a compound containing it, such as a salt or an ester of nitrous acid. Its regulatory name is nitrite, molecular formula NO 2- 

### , and molecule weight is 46 g/mol 43 

### . Nitrite CAS number is 14797-65-0. 44



### 8 Analytical Methods at Environmentally Relevant Concentrations (G9: 4.2.1.5; (GESAMPBWWG 6, Annex IV: 3.7)
DBPs (nitrite) had been analysed using standardised methods. The impact of by-products in the discharge was analysed in 20-days incubation studies in a climate room.

9 Use of Active Substance or the Preparation (GESAMP-BWWG 6, Annex IV: 4)
The Hyde GUARDIANTM system does not use or generate Active Substances.

10 Material Safety Data Sheets (GESAMP-BWWG 6, Annex IV: 5)
Material safety data sheet for nitrite is presented in Appendix 1.

11 Risk Characterization Nitrite is not persistent and does not bioaccumulate. Nitrite is not toxic at the concentrations resulting from the Hyde GUADRIANTM system.

11.1 Evaluation of the treated ballast water (G9: 5.2; GESAMP-BWWG 6, Annex IV: 6.2)

11.1.1 Results from the Land Based Tests
The land based test trials of the Hyde GUARDIAN TM 

### system were carried out at the Royal Netherlands Institute for Sea Research (NIOZ), Texel, the Netherlands, from March till July 2008. The System was tested according to the D-2 Standard and the IMO Guidelines for Type Approval testing (G8).

42 Carlucci AF, Hartwig EO, Bowes PM (1970) Biological production of nitrite in seawater. Marine Biology 7, 161-166 43 http://www.lenntech.com/hazardous-substances/nitrite.htm 44 http://www.epa.gov/OGWDW/dwh/t-ioc/nitrates.html

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The main findings from the test trials are summarised below 45 

### :

x The basic chemistry and physical properties of the water were not altered by the treatment and no factors could be detected effecting the vitality of the treated water upon discharge;

x With respect to the water chemistry a slight increase in the nitrite concentration was observed during the low salinity test runs in the discharge. This is in agreement with findings by Sharpless & Linden 46 

### . Yet the increase was only half of the natural nitrite concentrations observed during the second test series at high salinity and should therefore be considered to be within the natural range. The values were also below the maximum allowed concentration for drinking water 47



### x The treated water did not contain toxic or growth inhibiting substances, neither were other chemicals than nitrite found that could have been formed as by-products from the treatment.
The complete report of Veldhuis et al. (2009) has been submitted to Lloyds Register and to Maritime and Coastguard Agency in December 2008.

11.1.2 Results from the shipboard tests
The three shipboard test trials were conducted aboard the Princess Cruise Lines ship M/V Coral Princess in 2008 to test the efficacy of the Hyde GUARDIAN TM 

### system under normal working conditions. The trials took place during the vessel’s regular spring schedule in the Caribbean Sea, the summer schedule in the N.W. Pacific Ocean between Whittier, Alaska and Vancouver, Canada, and during therepositioning cruise from the western Pacific to the vessel’s winter base in Fort Lauderdale, Florida. Trials consisted of determination of water quality parameters and a comparison of biological endpoints in treated and untreated ballast water samples, with reference to both IMO G8 and the U.S. Coast Guard Shipboard Technology Evaluation Program (STEP).
In the report from the shipboard test trials 48 

### the following conclusions can be found:

x While, to date, no potentially toxic chemical changes resulting from UV irradiation have been identified, this resolution adopts an approach requiring tests for residual toxicity that essentially follow IMO G9 guidelines, even for systems not involving the addition of active substances. The resolution was adopted too late for such testing to be incorporated into land based testing of the system, which ended in July 2008. Hence, three tests, one chronic (growth based) and two acutetoxicity bioassays, were incorporated into the third trial in order to provide empirical toxicological evidence on this point.

x Results of Whole Effluent Toxicity (WET) bioassays conducted during trial 3 indicated no significant differences between the toxicity of treated vs. untreated water samples at the time of discharge from the vessel. In invertebrate and vertebrate larval assays undiluted water from both treated and untreated tanks appeared not to show any significant toxicity relative to laboratory controls. In the case of phytoplankton there appeared to be a small degree of toxicity associatedwith water retrieved from ballast waters following the (10 day) residence time, although the toxicity did not differ significantly between treated and untreated water."

x It might be concluded from those data that any toxic element present in discharged water did not

45 Veldhuis MJW, Fuhr F, Stehouwer P-P (2009) Final report of the land-based testing of the Hyde-GuardianTM -System, for Type Approval according to Regulation-D2 and the relevant IMO Guideline (April – July 2008). Royal Netherlands Institute for Sea Research, Den Burg, Texel, the Netherlands, 42 pp 46 Sharpless CM, Linden KG (2001) UV photolysis of nitrate. Effects of natural organic matter and dissolved inorganic carbon and implications for UV water disinfection. Environmental Science and Technology 29, 2949-2955 47 Sharpless C, Page M, Linden K (2003) Impact of hydrogen peroxide on nitrite formation during UV disinfection Water Research 37, 4730 – 4736 48 Wright DA (2009) Shipboard Trials of Hyde ‘Guardian’ system in Caribbean Sea and Western Pacific Ocean, April 5 th - October 7th, 2008. Final Report to Hyde Marine and Lamor Corp. University of Maryland Center for Environmental Science, Chesapeake Biological Laboratory, Solomons Maryland, 122 pp

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result from UV irradiation. Such results have been supported by an extensive literature searchthat has revealed no evidence of residual chemical toxicity in water resulting from prior UV irradiation of that water. While some changes in nutrient levels appeared to be associated with water treatment it was not possible to definitively conclude that these changes directly resulted from UV irradiation. For example, both phosphate and nitrate levels at de-ballasting were higher in treated samples relative to untreated samples at the time of discharge from the ship. It is possible that part of this difference could be attributed to an increase in the degradation of planktonic organisms resulting from the treatment process, rather than UV irradiation directly. Nitrite levelswere also higher in treated samples 10 days after treatment. While UV photolysis of nitrate to nitrite has been demonstrated in UV-irradiated drinking water 49 

### , the levels involved are very small relative to the U.S. drinking water standard of 1000 ȝ g L -1 

### , or the European standard of 100 ȝ g L 1 

### . Nitrate levels reported in this trial were negligible relative to these standards and pose no toxicological threat associated with discharge.
The final report of Wright (2009) has been submitted to Lloyds Register and to Maritime and Coastguard Agency in January 2009.

11.2 Prediction of discharge and environmental concentrations (GESAMP-BWWG 6, Annex IV: 6.3.3)
The nitrite levels measured from the treated ballast water during the land based testing were 4.62 μM in low salinity seawater. In water intake the nitrite concentration was 2.99 μM. The nitrite concentration in the treated water, in high salinity water, was 6.00 μM. In water intake the concentration was 6.85 μM. During the shipboard testing nitrite levels showed a 3-4 fold increase, from 0.8 to 2.73 μg/l in trial #3, in treated samples after 10 days.

11.3 Effects on sediment (G9: 5.1; GESAMP-BWWG 6, Annex IV: 6.3.7)
Particles and organisms larger than 50 μm are removed by the filter. This means that small particles, that may have a higher organic content, will accumulate inside the ballast tanks. Experience from the ships that have had the Hyde GUADRIANTM system installed show little sediment accumulation inside the ballast tank and only a thin layer of silt (< 2 mm) has been observed. The DBP (nitrite) is not expected to have adverse effects on sediment.

12 Quality Control/Quality Assurance (G9: 4.2.4)
The Quality Control and Quality Assurance issues have been addressed in two separate reports. Concerning the shipboard trials of the Hyde GUADRIANTM system the QA/QC protocol has been presentedin the Appendix D and the Standard Operating Procedures (SOP) for UMCES Analytical Services Division in the Appendix E of the reference (Wright, 2009). 50


### Regarding the land based test trials a description of the Test Protocol and Quality Assurance Project Plan (QAPP) has been presented in the reference (NIOZ, 2009) 51 

### . According to the reference it has been approved by Maritime Coastguard Agency (UK) and Lloyds Register (LR) in April 2008. The both reports have been submitted to LR in January 2009.

49 E.g. Sharpless CM, Linden KG (2001) UV photolysis of nitrate. Effects of natural organic matter and dissolved inorganic carbon and implications for UV water disinfection. Environmental Science and Technology 29, 2949-2955 50 Wright, DA (2009). Shipboard Trials of Hyde ‘Guardian’ system in Caribbean Sea and Western Pacific Ocean, April 5th – October 7th, 2008. Final Report to Hyde Marine and Lamor Corp. University of Maryland Center for Environmental Science, Chesapeake Biological Laboratory, Solomons, Maryland 20688. 51 Royal Netherlands Institute for Sea Research (NIOZ) (2009). Test protocol and Quality Assurance Project Plan (QAPP ) for the biological efficacy testing of the Hyde Marine Ballast Water Treatment System (HBWTS, Hyde Marine Inc) as part of the Type Approval Process under Resolution MEPC 125.53

.

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13 Use of the Active Substance and Preparations
The Hyde GUARDIANTM system does not use or generate Active Substances for its treatment.

14 Risk Assessment (G9: 6, 7)

14.1 Risk to safety of ship (GESAMP-BWWG, Annex IV: 7.1)
The potential risk to the safety of the ship and crew raised by the operation of the Hyde GUARDIANTM system is assessed, in sections below.

14.1.1 Ship Integrity and the BWTS
The Hyde GUARDIANTM system is installed in parallel with the ship’s ballast piping system. The system is always installed somewhere down the ballast pipe line after the ballast pump. There are no specific requirements or restrictions as of distance from the ballast pump or any of the associated ballasttanks. The Hyde GUARDIANTM filtration system can be installed separately from the disinfection system. The disinfection system (UV unit) can be installed either vertically or horizontally as this has noaffect on the operation or efficacy of the system. However the multi spine Galaxy filter system is recommended to be installed in a vertical position in order for the back-flush of the filter spines to be efficient. A small inclement (less than 30o) does not have affect but a horizontal (90o) installation position could make the back-flushing to be more inefficient, as the spines would back-flush against each other. This recommendation does not apply for the single spine Crystal filter system.
The Hyde GUARDIANTM system is normally isolated from the ballast piping system by inlet and outletpneumatically actuated butterfly valves which are normally closed when the Hyde GUARDIANTM system is not in operation. For emergency use the inlet and outlet valves can be operated via the fail safe Supervisory Control System, but also mechanically at valve location. Aside from the in-built by pass pipe which by-passes the filter system during de-ballasting but forces ballast water flow through the UV disinfection unit, the entire system can be bypassed in case of emergency by operation of ships valves.
Operation of the system bypass valves should be restricted to emergency operation only as this will violate the ballast water treatment convention. Theses valves should remain locked and their use should require permission of the chief or captain. The bypassing of the treatment system will need to be recorded in the ballast water log book.
The Hyde GUARDIANTM system is fully automatic and the system can be fully integrated to the shipscontrol. If any of the logged operating parameters of the BWMTS are not within the acceptable and predetermined tolerances, it will shut down the Hyde GUARDIANTM system and not allow untreated water to pass.
Generally speaking the Hyde GUARDIANTM system after installed on the ship will be one of the ships equipment that requires least attention by the ships crew in terms of operation of the system (especially if fully integrated into ships control) but also in terms of maintenance. The ships crew can easily betrained on site how to operate the system from the Supervisory Control Board (local control) LCD display and control panel. The control panel, as a standard Hyde GUARDIANTM option, features simple function buttons and a simple and clear LCD text display. The ships crew can start and stop the BWTS system pressing simple function buttons and also control and monitor the BWTS operating parameters, simply by following the clear instructions on the LCD text display.

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If the Hyde GUARDIANTM system is chosen not to fully integrate into ships control, but installed as stand alone equipment. The ships crew need to manually start the BWTS system locally after having started the ships ballasting pump(s). After starting the Hyde GUARDIAN TM 

### at the Control Panel and choosing either ballast or de-ballast mode, the system opens the inlet valve to the system and keeps the delivery valve closed, and waits for the UV unit to heat up to its operational temperature. After the UV unit has reached its operational temperature and is ready for treating the water, only then the deliveryvalve of the system is opened and water flows through the system. The warm up time is slightly dependant on the ambient water temperature, but normally the operating temperature is reached within 2 min from starting of the system. The system will continue to run automatically without input from the ships

crew. When filling or emptying the ballasting tanks (i.e. ballasting procedure), is finished, the crew needs to stop the BWTS system locally. The system will close the delivery valve and conduct a final back-flush cycle of the filters (only if ballasting of tanks has taken place). After this the system will shut down and the UV unit cools down.
If the Hyde GUARDIANTM system is fully integrated into ships control via a digital link, (such as Modbus and Profibus), the controls and monitoring of the BWTS are managed from the station where ballasting the procedure is taking place, without manual interface from the ships crew. In that case the starting signal to the ballast pump and the selection of ballasting or de-ballasting mode, enabled by the ships crew, will give the signal to the Hyde GUARDIANTM Control system to run the system without input of the ships crew.

14.1.2 Hazardous Area
The Hyde GUARDIANTM system can be safely installed onboard ships having hazardous areas, e.g. tankers. However there are limitations what concern the location of the individual system components.
The Hyde GUARDIANTM filter system can be installed safely inside a hazardous area since it’s completely pneumatically operated. The Hyde GUARDIANTM UV disinfection chamber can, with a smallmodification, be made into an explosion proof system by simply purging the UV chamber. This is principally done by pressurizing the internal part of the UV chamber which features the electrical components. Hence no dangerous gases or vapours can enter inside the UV chamber which if penetrated could get in touch with the electrical system and potentially cause an electrical arc and consequential fire/explosion by igniting the gas and/or vapour. By requirement also the areas which are classified as hazardous areas, e.g. a Cargo Pump room features also on-line constant gas and vapour monitoring equipment which potentially would give an alarm even before gas and/or vapour would enter into the UV chamber. However a fail safe pressure switch is installed to the UV chamber which constantly measures the pressure inside the UV chamber. In case of pressure loss inside the chamber, the pressure switch would give a signal disconnect all power to the UV system.
The Hyde GUARDIANTM Power Panel and Control Panel would be installed outside the hazardous area, e.g. machinery room space. All the penetrations through the bulkhead into the hazardous area would have to be done by type approved penetration gland and sealing for hazardous areas. The booster pump, which is only used during the back-flush mode of the filters, would be supplied with an explosion proof electric or hydraulic motor.

14.1.3 Corrosion
The Hyde GUARDIANTM system does not cause corrosion of the ships structure or the ships systems to which the Hyde GUARDIANTM system is in contact with. The Hyde GUARDIANTM filtration stage is

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pure solid liquid separation system and the UV disinfection treatment does not change the treated water by any means which could have an affect on corrosion. If a corrosion problem exist onboard the ship where the Hyde GUARDIANTM system is installed, the system will not enhance nor will it reduce the corrosion rate, since the Hyde GUARDIANTM system has no affect on corrosion.

14.1.4 Fire and Explosion
The Hyde GUARDIANTM system does not possess any risk for explosion. The filters are pressure rated to 10.0 bar and under exceptional circumstances the water pressure of a ships ballast system rarely reaches above 7.0 bar. Under normal circumstances the operating pressure of a ships ballast system is in the range of 1.5 to 5.0 bars. In the case of the Hyde GUARDIANTM system, during the back-flushing and when the delivery valve of the system is closed the pressure is temporarily raised to 5.0 bar pressure by external booster pump to enable efficient back flushing. If the ships own ballast pump deliver 5.0 bar pressure, the external booster pump is not required. The Hyde GUARDIANTM filter system is however equipped with a simple pressure relief valve fitted on the centre manifold which in case of pressure build up would let out excessive pressure and drain small amounts of the ballast water to ships bilge system. There is no risk of explosion in the UV chamber.
The Hyde GUARDIANTM system features an electrically powered UV disinfection unit, power panel, and control cabinet components. These Hyde GUARDIANTM components are encapsulated in steel frames and ingress protected to min IP 54 standard in accordance with general industry standard and in accordance with ships classification society rules. And hence does not possess any higher risk of fire then any other electrical system onboard. In case of fire e.g. short circuited system caused by any of the Hyde GUARDIANTM electrical components or in case of fire from external source which would damage any of the Hyde GUARDIANTM electrical components, the Supervisory Control system will shut down the BWTS.

14.1.5 Chemical Storage
There are no chemicals used in the Hyde GUARDIANTM system.

14.1.6 Accidental Chemical Release
There are no chemicals used in the Hyde GUARDIANTM system.

14.2 Risk to technicians and/or ship’s crew
The Hyde GUARDIANTM system does not possess any risk for the ships technicians and/or ships crew.The system does not feature any mechanically rotating parts which could cause damage or injury to personnel operating, or conducting maintenance to, the Hyde GUARDIANTM system. The Hyde GUARDIANTM system which does not use any active substance nor produce any active substance during the treatment process is completely safe for the crew to operate or inspect during operation and also when the system is idle or when stopped.
As of ships crew conducting routine maintenance to the system, as the Hyde GUARDIANTM system features electrical components, proper safety procedures are to be adhered to. These safety procedures aregeneral and are not different from any other safety procedure for a ships electrical systems or components. All the safety procedures specific to the Hyde GUARDIANTM system are well described in English language in the Owners Operating and Maintenance Manual supplied with each system. Also all theelectrical components on the Hyde GUARDIANTM system are labelled with industry accepted WARNING signs for electrically powered components.

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14.3 Risks to human health (GESAMP-BWWG, Annex IV: 7.2)
There is no direct human health risk associated with the Hyde GUARDIANTM system. The system is very robust, totally isolated from the outside world; the routine maintenance is simple and robust and is easy to adhere to. Only by violating the simple and easy to read safety procedures related to the routine maintenance of the system there might be a risk for human health. Even in such case or in an emergencydirect eye contact with a powered up UV-C lamp should be prevented at all times. Under normal operation there is no possibility for a person to get in contact with UV-C as the UV is well confined inside the UV chamber. And the small amount of UV light which might be visible through drain hose connected tothe UV chamber does not cause any harm to eye sight. The UV-lamps contain a small amount of mercury (< 200 mg/lamp) that that is well enclosed within the device under normal operation. If a lamp breaks during lamp replacement work, the mercury can be rendered harmless by binding it with sulphur powder. Contact with skin and eyes and inhaling vapours should be prevented

14.4 Risks to the aquatic environment (GESAMP-BWWG, Annex IV: 7.3)
With the Hyde GUARDIANTM system there are no currently known risks to the aquatic environment. The Hyde GUARDIANTM system does not use an active substance nor is a active substance producedduring the treatment process so there is no risk of any active substance being released to the aquatic environment which could cause a risk for the same

14.5 Noise
The power cabinet of the Hyde GUARDIANTM does emit a slight hum, <40db from the transformers. The cabinet also has cooling fans will contribute to the ambient noise level.
The treatment part of the Hyde GUARDIANTM system does not generate any sound or noise except ofthe sound of water flowing through a standard ships ballast pipe system, which in the case of a ship installation will be absorbed or is neglected by the ambient noise level existing onboard a ship in a machinery -, a pump room or a cargo pump room space.

15 Assessment Report (G9: 4.3; GESAMP-BWWG, Annex IV: 8)
The Hyde GUARDIAN TM 

### system, using UV ( Ȝ 200-400 nm) does not use or produce active substances for its meant ballast water treatment.
The Hyde GUARDIAN TM 

### ballast water treatment system does not produce any DBPs that would fit into either of the two distinct classes 52 

### of undesirable DBPs - those that affect water’s aesthetic quality and those that are harmful to the environment or human health. Based on literature that is currently available, it was also found that UV treatment produces neither odour nor taste.
As the result from the land-based tests it can be concluded that the present configuration of the Hyde GUARDIAN TM 

### system was found to offer a reliable and environmentally safe cleaning of the ballast water resulting in organism numbers well below the IMO Standard D2.
Additionally, the results from the ship-board tests prove that under the conditions encountered during these trials, the system was found to comply with all IMO G-8 standards relating to the elimination ofbiota and with respect the issue of residual toxicity of treated water related to chemicals generated dur52 Wolfe RL (1990) Ultraviolet disinfection of potable water. Environmental Science and Technology 24, 768-773

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ing treatment.
When the shipboard and land-based tests with the Hyde GUARDIAN TM 

### system were agreed on in early 2008, the valid G8 Guidelines at that time (Resolution MEPC.125(53)) did not include the requirementsfor the environmental acceptability of the discharged water, which are included in the revised G8 Guidelines as adopted in October 2008. The land-based tests were performed in the spring and early summer of 2008. During those tests the discharge water was analysed for potential by- or end products of the UV treatment, as a precautionary action. Only a slight increase in nitrite, formed by conversion of nitrate in the water was found. As stated in this report, nitrite is a constituent of sea and fresh waters.
The revised Guidelines G8 (Resolution MEPC.174(58)) were adopted on 10 October 2008. The revised Guidelines include a reference to environmental acceptability, as introduced in paragraph 1.8 : "TheseGuidelines contain recommendations regarding the design, installation, performance, testing, environmental acceptability and approval of ballast water management systems".
The Resolution also states in paragraph 5.1.5 that “ if it can reasonably be concluded that the treatmentprocess could result in changes to the chemical composition of the treated water such that adverse impacts to receiving waters might occur upon discharge, the documentation should include results of toxicity tests of treated water. The toxicity tests should include assessments of the effects of hold time following treatment, and dilution, on the toxicity. Toxicity tests of the treated water should be conducted in accordance with paragraphs 5.2.3 to 5.2.7 of the Procedure for approval of ballast water management systems that make use of Active Substances (G9), as revised, (Resolution MEPC.169(57)) ”. Resolution MEPC.174(58) was adopted after all land-based tests with the Hyde GUARDIAN TM 

### system were completed in July 2008.
To accommodate the environmental acceptability requirements of the revised G8, all possible effort was made by the testing bodies to provide toxicity data required in Resolution MEPC.174(58). During the last shipboard test trial Whole Effluent Toxicity (WET) tests were conducted on treated and untreated water samples. The objective was to identify any residual chemical toxicity that could have resulted from the Hyde GUARDIAN TM 

### ballast water treatment system. Results of the WET bioassays indicatedno significant differences between the toxicity of treated vs. untreated water samples at the time of discharge from the vessel.
The reviewed literature did indicate that in the UV BW treatment process no relevant chemicals (i.e., metabolites, intermediates, residual substances) are produced at levels that are expected to cause adverse impacts on aquatic life by the discharge of UV-treated ballast water. These findings are also supported by the results of the Hyde GUARDIAN TM 

### land based and shipboard test trials.
Based on the practical operational experience from the first Hyde GUARDIAN TM 

### system installed on board the Coral Princess in 2003, the system has proven its reliability and safety to the ship and its crew. Based on the results from the land-based and ship-board tests, and on the findings from the literature, it can reasonable be concluded that the Hyde GUARDIAN TM 

### Ballast Water Treatment System could not result in changes to the chemical composition of the treated water such that adverse impacts to receiving waters might occur upon discharge.

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16 Appendices

Annex 1.

Technical Factsheet on: NITRATE/NITRITE 53



### As part of the Drinking Water and Health pages, this fact sheet is part of a larger publication: National Primary Drinking Water Regulations Drinking Water Standards (in mg/L) Nitrate- MCLG: 10; MCL: 10; 10-day HAL: 10 Nitrite- MCLG: 1; MCL: 1; 10-day HAL: 1 Total (Nitrate+Nitrite)- MCLG: 10; MCL: 10; 10-day HAL: 10 Health Effects Summary Acute: Excessive levels of nitrate in drinking water have caused serious illness and sometimes death. The serious illness in infants is due to the conversion of nitrate to nitrite by the body, which can interfere with the oxygen-carrying capacity of the child's blood. This can be an acute condition in which health deteriorates rapidly over a period of days. Symptoms include shortness of breath and blueness of the skin. Drinking water levels which are considered "safe" for short-term exposures: For a 10-kg (22 lb.) child consuming 1 liter of water per day, a ten-day exposure to 10 mg/L total nitrate/nitrite. Chronic: Effects of chronic exposure to high levels of nitrate/nitrite include diuresis, increased starchy deposits and hemorrhaging of the spleen. Cancer: There is inadequate evidence to state whether or not nitrates or nitrites have the potential to cause cancer from lifetime exposures in drinking water. Usage Patterns Most nitrogenous materials in an aquatic environment tend to be converted to nitrate, so all sources of combined nitrogen, particularly organic nitrogen and ammonia, should be considered as potential nitrate sources. Primary sources of organic nitrates include human sewage and livestock manure, especially from feedlots.The primary inorganic nitrates which may contaminate drinking water are potassium nitrate and ammonium nitrate. Potassium nitrates are used mainly as fertilizers (85%), with the remainder in heat transfer salts, glass and ceramics, and in matches and fireworks. Ammonium nitrates are used as fertilizers (84%) and in explosives and blasting agents (16%).

Release PatternsThe major environmental releases of inorganic sources of nitrates are due to the use of fertilizers. According to the Toxics Release Inventory, releases to water and land totalled over 112 million pounds from 1991 through 1993. The largest releases of inorganic nitrates occurred in Georgia and California.

53 http://www.epa.gov/OGWDW/dwh/t-ioc/nitrates.html

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Environmental FateDue to its high solubility and weak retention by soil, nitrates are very mobile in soil, moving at approximately the same rate as water, and has a high potential to migrate to ground water. Because it does notvolatilize, nitrate/nitrite is likely to remain in water until consumed by plants or other organisms. Ammonium nitrate will be taken up by bacteria. Nitrate is more persistent in water than the ammonium ion. Nitrate degradation is fastest in anaerobic conditions. Chemical/Physical Properties
CAS Number: Nitrate ion: 14797-55-8; Nitrite ion: 14797-65-0 Color/ Form/Odor: Domestic fertilizer grade ammonium or potassium nitrates are in prilled (beaded) or crystalline forms, usually coated with an anti-caking agent and adsorbed fuel oil. Solubilities: Nitrates and nitrites are highly soluble in water Soil sorption coefficient: N/A

Bioconcentration Factor: N/A Trade Names/Synonyms: Potassium salt: Potnit, Hitec, Niter, Nitrate of potash, Saltpeter.Ammonium salt: German or Norway saltpeter, Varioform I, Merco or Herco prills, Nitram. Other Regulatory Information
Monitoring: -- For Ground Water Sources: Initial Frequency-Nitrate: 1 sample annually Nitrite: 1 sample during first 3-year compliance period Repeat Frequency-Nitrate: 1 sample annually Nitrite: determined by State -- For Surface Water Sources: Initial Frequency-Nitrate: 1 sample each quarter Nitrite: 1 sample during first 3-year compliance period Repeat Frequency-Nitrate: 1 sample annually Nitrite: determined by State --Triggers - If detect at > 5 mg/L nitrate, sample quarterly. If detect at > 0.5 mg/L nitrite, sample quarterly. If detect total nitrate + nitrite > 5 mg/L, sample quarterly 

### Analysis

Reference Source Method Number

EPA 600/4-79-020 353.1; 353.2; 353.3; 300.0; 354.1 

### Standard Methods 418C; 418F
ASTM D3867-85A; D3867-85B Treatment/Best Available Technologies : Ion exchange; Reverse osmosis; Electrodialysis (nitrate only)

For Additional Information: Sources of toxicological and environmental fate data include: Toxic Substance Control Act Information Line - 202/554-1404 Toxics Release Inventory, National Library of Medicine - 301/496-6531 Agency for Toxic Substances and Disease Registry - 404/639-6000