GSI/LB/F/A/1 March 15, 2010 Page 1 of 58



# Report of the Land-Based Freshwater Testing of the Siemens SiCURETM Ballast Water Management System

March 15, 2010

Principal Investigator:

Allegra Cangelosi, NEMWI

Research Team:

Lisa Allinger, NRRI, UMD Mary Balcer, PhD, LSRI, UWS Nicole Mays, NEMWI Tom Markee, LSRI, UWS Christine Polkinghorne, LSRI, UWS Kelsey Prihoda, LSRI, UWS Euan Reavie, PhD, NRRI, UMD Donald Reid, Consultant Heidi Saillard, LSRI, UWS Tyler Schwerdt, AMI Engineering Heidi Schaefer, LSRI, UWS Matthew TenEyck, LSRI, UWSGSI/LB/F/A/1 March 15, 2010 Page 2 of 58



# Report of the Land-Based Freshwater Testing by the Great Ships Initiative
of the Siemens SiCURE TM 

# Ballast Water Management System for Type Approval According to Regulation
D-2 and the Relevant IMO Guidelines

March 15, 2010

Allegra Cangelosi, Principal Investigator

Great Ships Initiative
Northeast-Midwest Institute 50 F St. NW, Suite 950 Washington, DC 20001
Phone: 202-464-4007
Email: acangelo@nemw.org

Approved for Release:

___________________________
PI Signature

March 15, 2010.GSI/LB/F/A/1 March 15, 2010 Page 3 of 58



## EXECUTIVE SUMMARY

The Great Ships Initiative (GSI) provides independe nt no-cost performance verification testing services to developers of ballast treatment systems and processes at a purpose-built, land-based ballast treatment test facility located in the Dulu th-Superior Harbor of Lake Superior. GSI test protocols are consistent with the requirements of t he International Convention for the Control and Management of Ships Ballast Water and Sediments (International Maritime Organization, 2004). GSI procedures, methods materials and findin gs are publicly accessible on the GSI website ( www.greatshipsinitiave.org ).

In August through October 2009, the GSI conducted l and-based tests on the SiCURE TM 

### Ballast Water Management System in cooperation with German Bundesamt für Seeschifffahrt und Hydrographie (BSH), i.e., the German Federal Mariti me and Hydrographic Agency. During the series of five consecutive valid trials, the SiCURE TM 

### Ballast Water Management System was evaluated for its ability to: (a) successfully trea t ballast water without interruption, (b) meet IMO D-2 discharge standards after a five-day holding ti me, and (c) discharge water after the five day retention period that is environmentally benign (i. e., no residual toxicity) pursuant to United States Environmental Protection Agency water qualit y criteria. It should be noted that because freshwater zooplankton are in general smaller than their salt and brackish water counterparts, the larger regulated size category (greater than 50 μ m in minimum dimension) did not incorporate all live zooplankton that were present in the sourc e water assemblage.

The Siemens SiCURE TM 

### Ballast Water Management System functioned properly during the five consecutive trials, and was highly effective at red ucing live organism densities in the fresh water ambient conditions of Duluth-Superior Harbor, as am ended in these tests to achieve IMOconsistent challenge conditions. Live organisms in the regulated size classes were discharged in
densities below the IMO D-2 standard. Microbial an alyses showed system performance in keeping with IMO requirements for bacteria. Chemis try data generated across trials indicated the post-retention discharge to have well less than 0.1 mg/L total residual chlorine (TRC) under
ambient conditions. Ambient water collected immedia tely after treatment and held in a cold environment had TRC and total residual oxidant (TRO ) levels which slightly exceeded this level. However, in a real world application, the intake wa ter would also be cold, and developers claim that the test system is designed to respond to this circumstance (reflected in oxidation-reduction potential, or ORP) with a reduction in chlorine gen erated and injected into the intake stream.

There were no acute toxic effects of treated discha rge on any test species across assays and trials. Chronic toxicity effects in 100 % effluent were det ected in one out of two trials for test species of zooplankton and phytoplankton. There were no chr onic toxicity effects across organisms and
trials in 50 % or lower effluent dilutions.GSI/LB/F/A/1 March 15, 2010 Page 4 of 58



## ACKNOWLEDGEMENTS

The authors would like to express our sincere grati tude to the Great Ships Initiative (GSI) Advisory Committee which provides invaluable input to the GSI Principal Investigator. We also
wish to thank the ten United States and Canadian Gr eat Lakes Ports which launched the GSI, and the Great Lakes Protection Fund which supported the initial scoping exercise. We sincerely thank the United States Department of Transportatio n, Maritime Administration, and National Oceanic and Atmospheric Administration for their su bstantial financial and in-kind support for
the construction of the state-of-the-art GSI land-b ased testing facility. We thank the United States and Canadian St. Lawrence Seaway organizatio ns, the Legislative Citizens Commission on Minnesota Resources, the University of Wisconsin -Superior, and the City of Superior for their active financial and/or in-kind support for G SI operations. We are grateful to the German Bundesamt für Seeschifffahrt und Hydrographie, BSH, (the German Federal Maritime and Hydrographic Agency), for visiting the GSI land-bas ed facility and agreeing to accept data from the GSI in support of its type approval determinati ons, and the Maritime Environmental Resource Center for active collaboration as we pion eer ballast treatment evaluations in the
United States.GSI/LB/F/A/1 March 15, 2010 Page 5 of 58



## CONTENTS

Executive Summary 3

Acknowledgements 4

Contents 5

1.0. Introduction 7

1.1. The Great Ships Initiative 7 1.2. The GSI Land-Based RDTE Test Facility 8
1.3. The Siemens SiCURE TM 

### Ballast Water Management System 11 1.4. Treatment Performance Requirements in Regula tion D-2 11 1.5. Relationship of GSI Testing to G8 and G9 Req uirements in IMO
Convention 12

2.0. Methods 14

2.1. Experimental Goals and Design 14 2.1.1. Treatment System and Test Facility Calibra tion Trials 14
2.1.2. Valid Trial Series 15 2.1.3. Challenge Conditions 16 2.2. Water Chemistry Analysis 17 2.2.1. Total Residual Chlorine (TRC) and Total Re sidual Oxidant (TRO) Sample Collection 17 2.2.2. Total Residual Chlorine (TRC) Determinat ion 18 2.2.3. Total Residual Oxidant (TRO) Determinatio n 18 2.2.4. Selected Disinfection By-Products (DBPs) Determination 19 2.3. Water Quality Analysis 19 2.3.1. Analysis of Total Suspended Solids (TSS) 19 2.3.2. Analysis of Non-Purgeable Organic Carbon (NPOC) and

Dissolved Organic Carbon (DOC), and Dete rmination of Particulate Organic Carbon (POC) Concent rations 20 2.4. Viable Organism Analysis 20 2.4.1. Organisms Greater than 50 μm in Minimum Dim ension 21 2.4.1.1. Sample Collection 21 2.4.1.2. Live/Dead and Size Analysis 21 2.4.2. Organisms 10 – 50 μm in Minimum Dimension 22 2.4.2.1. Sample Collection 22 2.4.2.2. Sample Analysis 23
2.4.3. Bacteria 23 2.4.3.1. Sample Collection 24 2.4.3.2. Sample Analysis 24 2.5. Ambient Physical/Chemical Water Conditions Analysis 25GSI/LB/F/A/1 March 15, 2010 Page 6 of 58



### 2.6. Whole Effluent Toxicity Analysis 25 2.6.1. Standard Whole Effluent Toxicity Tests 25 2.6.2. Cold Whole Effluent Toxicity Tests 26 2.6.3. Determination of Total Residual Chlorine (TRC) and Total Residual Oxidant (TRO) Levels for WET Analysis 26 2.6.4. Statistical Analysis for WET Assay 26 2.6.5. Determination of Quality of Test Organism s for WET Assay 27 2.7. Data Management 27 2.7.1. Data Recording 27 2.7.2. Data Processing and Storage 28 2.8. Quality Assurance/Quality Control 28

2.8.1. Quality Assurance/Quality Control Activitie s 29 2.8.2. Standard Operating Procedures (SOPs) 29
2.8.3. Documents and Records 29
2.8.3.1. Notebooks 29 2.8.3.2. Safety, Operation and Maintenance Reco rds 30

3.0. Results 30

3.1. Challenge Conditions 30

3.1.1. Physical/Chemical Challenge Conditions 30 3.1.2. Biological Challenge Conditions 31 3.2. Viable Organisms in Treated Discharge 33 3.2.1. Regulated Plankton 33 3.2.2. Regulated Microbes 34 3.3. Physical/Chemical Water Conditions 3 5 3.4. Residual Toxicity 37
3.4.1. TRC and TRO Concentrations in WET Tests 37 3.4.2. Whole Effluent Toxic Effects 39
3.4.2.1. Standard WET Results 39
3.4.2.2. Cold WET Results 39

3.4.3. Disinfection By-Products (DBPs) 42

4.0. Discussion of Results 44

5.0. Conclusions 45

References 45

Appendix 1. GSI Land-Based RDTE Facility: Technical Information 46 Appendix 2. GSI SOPs Relevant to SiCURE TM 

### Tests 49 Appendix 3. Species List of Phytoplankton and Zoop lankton Present 51 Appendix 4. Data Quality Objectives, Criteria and Results 52 Appendix 5. Operational Data Summary 58GSI/LB/F/A/1 March 15, 2010 Page 7 of 58



## 1.0. INTRODUCTION

1.1. The Great Ships Initiative

The Great Ships Initiative (GSI) 1 

### is regional effort devoted to ending the problem o f shipmediated invasive species in the Great Lakes-St. La wrence Seaway System and globally. In support of that goal, the GSI established superlati ve freshwater ballast treatment evaluation capabilities at three scales—bench, land-based, and on board ship.

The GSI awards its independent status-testing servi ces to developers of ballast treatment systems and processes determined to be promising. GSI statu s-testing is performed at the scale appropriate to the state of development of the targ et treatment system, with the goal of facilitating the rapid progression of meritorious b allast treatment systems through the research and development and approval processes to a market- ready condition.

GSI has no involvement, intellectual or financial, in the mechanics, design or market success of the actual treatment systems it tests. To ensure th at GSI tests are uncompromised by any real or perceived individual or team bias relative to test outcomes, GSI test activities are subject to rigorous quality assurance, quality control (QA/QC) procedures and documentation. This QA/QC attention also assures high quality and credi ble evaluation findings.

GSI has worked to standardize and intercalibrate it s protocols to evaluate the performance of ballast water treatment systems with IMO guidelines , United States Environmental Protection Agency Environmental Technology Verification draft protocols, and other test facilities. GSI test protocols are as consistent with the requirements o f the International Maritime Organization’s (IMO) Convention for the Control and Management of Ships Ballast Water and Sediments (IMO, 2004) and federal requirements as practicable . In particular, GSI testing directly supports IMO G8 and G9 evaluations. GSI procedures, methods materials and findings are also not proprietary, and are publicly accessible on the GSI website ( www.greatshipsinitiave.org ).

Ms. Allegra Cangelosi of the Northeast-Midwest Inst itute is the Principal Investigator and Manager of the GSI. Researchers from the University of Wisconsin-Superior's Lake Superior Research Institute (LSRI), and the University of Mi nnesota-Duluth's Natural Resources Research Institute, among others, provide critical scientifi c and technical expertise and implementation services to GSI's biological research activities, a nd the GSI generally. Dr. Mary Balcer is the project’s lead zooplankton ecologist. Dr. Euan Reav ie leads all phytoplankton analysis. Mr. Matthew TenEyck, leads the bench-testing and Whole Effluent Toxicity (WET) tests. AMI Consulting Engineers provide engineering expertise in support of GSI testing activities. A GSI Advisory Committee comprising top-level officials o f key stakeholder groups helps steer the GSI providing crucial assistance in making GSI award de cisions and fund-raising. The GSI Advisory Committee includes elected leadership, environmenta l organizations, port directors and federal officials from the United States and Canada, and in dustry representatives. The American Great Lakes Ports Association advises the project, assuri ng that the GSI is meeting the needs of the maritime industry; and coordinating maritime indust ry and supply chain outreach.

1 www.greatshipsinitiative.orgGSI/LB/F/A/1 March 15, 2010 Page 8 of 58



### To date, all GSI tests are supported by general pro ject funds which derive from federal, state and port grants and contributions, and in-kind contribu tions by industry, local government and universities. Over time, GSI will begin to charge t reatment developers for a portion of the testing costs associated with type approval testing for Uni ted States regulatory purposes. The largest contributor of GSI operating funds is the United St ates Department of Transportation, including its Maritime Administration, and the Saint Lawrence Seaway Development Organization. GSI also receives significant funds and in-kind contrib utions from the National Oceanic and Atmospheric Administration, the Canadian St. Lawren ce Seaway Management Corporation, the City of Superior, Wisconsin, and approximately ten U.S. and Canadian ports in the Great Lakes.

In September and October 2009, the GSI conducted la nd-based tests on the SiCURE TM 

### Ballast Water Management System in cooperation with German Bundesamt für Seeschifffahrt und Hydrographie (BSH), i.e., the German Federal Mariti me and Hydrographic Agency. During the series of five consecutive valid trials, the SiCURE TM 

### Ballast Water Management System was evaluated for its ability to: (a) successfully trea t ballast water without interruption, (b) meet IMO D-2 discharge standards after a five-day holding ti me, and (c) discharge water after the five day retention period that is environmentally benign (i. e., no residual toxicity) pursuant to United States Environmental Protection Agency water qualit y criteria.

1.2. The GSI Land-Based RDTE Test Facility

GSI tests reported here evaluated the biological ef ficacy of a ballast water treatment system at its purpose-built, land-based ballast treatment test fa cility located in the Duluth-Superior Harbor of Lake Superior (figure 1). The facility draws raw i ntake water from Duluth-Superior Harbor at
400 m 3 

### /hr to 680 m 3 

### /hr. This main flow of intake water can be amended with TSS and endemic Harbor algae just prior to entering the facility.GSI/LB/F/A/1 March 15, 2010 Page 9 of 58

Figure 1. The GSI’s Land-Based RDTE Facility in Sup erior, Wisconsin.



### A Y-split in the intake piping at the facility simu ltaneously channels one half of the flow (200

m 3 

### /hr to 340 m 3 

### /hr) to a treatment track and the other half (also 200 m 3 

### /hr to 340 m 3 

### /hr) to a
matched control track. The treatment track directs water through the experimental treatment system and into a 200 m 3 

### cylindrical retention tank. The control track by-p asses the treatment system and channels water directly into a matched c ontrol retention tank. After storage, water is discharged sequentially from the treatment and cont rol retention tanks at 340 m 3 

### /hr to the harbor, or to a second set of facility retention tanks wher e it is then transferred to a wastewater treatment facility. A detailed design of the facility can be found in Appendix 1 and information on the facility’s validation can be found at http://www.nemw.org/GSI/documents.htm

.

Water is sampled continuously throughout ballasting functions (intake or discharge) through inline sample points. Each sample point is made up of one to three identical sample ports with a center-located elbow-shaped pitot tube (90 o 

### ) bent towards the direction of water flow used to sample the water. This pitot design is based on a d esign developed and validated by the U.S. Naval Research Laboratory in Key West, Florida, and empirically at GSI. Intake sampling uses sample ports at paired intake sample points (SP#2 a nd SP#3) on the control and treatment tracks (figure 2). Discharge sampling occurs through sampl e ports at sampling points SP#9 or SP#10 (figure 2). All four SPs are made up of three sampl e ports.GSI/LB/F/A/1 March 15, 2010 Page 10 of 58

Figure 2. Simplified Schematic of the GSI Land-Base d RDTE Facility Showing Location of Sample

Points, Injection Points, Retention Tanks, and Trea tment and Controls Tracks.



### Sample water at a given sampling point (i.e., intak e line of the control track, intake line of the treatment track, or the discharge line for the cont rol and treatment tracks) is transferred simultaneously and continuously throughout ballasti ng operations (intake or discharge) from replicate sample ports to replicate 3.8 m 3 

### sample collection tubs via clean 3.8 cm ID flexibl e hoses and automated flow-controlled pneumatic diaph ragm valves.

An on-site mobile field laboratory (figure 3a) and stationary structure (figure 3b) provide benchscale facilities to support time sensitive assays a ssociated with the GSI land-based tests, including live analysis of phytoplankton and zoopla nkton. The laboratories are climatecontrolled, and have enough bench space to allow fo r simultaneous microscopic and analytical analysis of samples by multiple analysts.GSI/LB/F/A/1 March 15, 2010 Page 11 of 58

Figure 3a. The GSI Mobile Field Laboratory



### . Figure 3b. The GSI Stationary Laboratory.



### 1.3. The Siemens SiCURE TM 

### Ballast Water Management System

Siemens Water Technologies developed the SiCURE TM 

### Ballast Water Management System based on the maritime industry-proven Chloropac® El ectrochlorination system for ships’ cooling water piping. This latter system was first develop ed in the early 1970s and has been operated onboard over 2,000 vessels.

SiCURE TM 

### has several unique features designed to provide ef fective treatment of ballast water while minimizing risk to the environment, the ship, and its crew. SiCURE TM 

### is based on electrolysis of seawater and use of hypochlorite as an Active Substance at a viable, “meet the demand” dose. The system is designed to inject only as much Active Substance into ballast water as required to achieve the necessary level of disin fection. This approach is aimed at eliminating
over-chlorination and associated risks of corrosion and generation of disinfection by-products.

The SiCURE TM 

### test system evaluated at GSI in August - October 2 009 used 5.5 m 3 

### /hr side flow that, by passing through an electro-chemical genera tor, was enriched with about 200 mg/L of
chlorine to treat 200 m 3 

### /hr water flow. The system was designed to produce a maximum of 6 mg/L of chlorine, however the actual dose level was defined by an oxidation-reduction potential of the treated water. This control logic was desig ned to ensure a low biocide residual in the water to prevent any corrosion and environmental is sues while meeting the IMO Convention’s ballast water management standards.

1.4. Treatment Performance Requirements in Regulation D- 2

The International Convention for the Control and Ma nagement of Ships Ballast Water and Sediments was adopted by consensus at a Diplomatic Conference at IMO in London on Friday 13 February, 2004. Annex D-2 of the Convention rela tes to ballast water performance standards for ships conducting ballast water management, incl uding use of a ballast water treatment system to effectively treat the ballast water. The regulat ion states that ships conducting ballast water management shall discharge:GSI/LB/F/A/1 March 15, 2010 Page 12 of 58



### • Less than 10 viable organisms per m 3 

### greater than or equal to 50 μ m in minimum dimension;

• Less than 10 viable organisms per mL less than 50 μ m in minimum dimension and greater than or equal to 10 μ m in minimum dimension; and

• Discharge of the indicator microbes shall not excee d the specified concentrations. The indicator microbes, as a human health standard, inc lude, but are not be limited to:

o Toxicogenic Vibrio cholerae (O1 and O139) with less than 1 colony forming unit (cfu) per 100 mL or less than 1 cfu per 1 gram (wet weight) zooplankton samples;

o Escherichia coli - less than 250 cfu per 100 mL;

o Intestinal Enterococci - less than 100 cfu per 100 mL.

1.5. Relationship of GSI Testing to G8 and G9 Requiremen ts in IMO
Convention

The fundamental approach of GSI is to conduct indep endent, scientifically-sound, rigorous, and quality assured evaluations of ballast water treatm ent systems. At the same time, GSI seeks
immediate relevance of its freshwater land-based te sting to regulatory processes such as those
outlined in the IMO Convention and those under deve lopment domestically in the United States and Canada. To that end, GSI protocols are rooted i n the essential features of the IMO G8 guidelines for testing, and the draft Environmental Technology Verification protocols under development by the United States Coast Guard and Un ited States Environmental Protection Agency. All aspects of the testing infrastructure ( e.g. flow rate, retention tank size, sample size, sample collection and analysis equipment and data l ogging) are directly consistent with these requirements. It formally partners with the Marylan d-based Maritime Environmental Resource Center (MERC) to assure that GSI freshwater land-ba sed testing can be directly complemented by comparable brackish/salt water testing.

With respect to physical/chemical and biological ch aracteristics of the intake stream, GSI is fortunate in that its feed water source naturally m eets many of the IMO G8 requirements for intake organism densities and physical/chemical con ditions during the testing season (table 1). Prior to the tests reported here, GSI did not amend water quality in any way in order to avoid associated experimental artifacts. In particular, r apid changes in physical conditions (such as salinity or total suspended solids) as ambient orga nisms are being brought in with ballast water may cause significant mortality in ambient species, while spiked non-native species may be poorly adapted to ambient conditions and decline ra pidly as well.

However, ambient conditions do fluctuate in all nat ural systems and the time window for this specific evaluation of the SiCURE TM 

### ballast water management system was limited. Ther efore, for the tests reported here, GSI agreed to augment intake water to better assure that initial challenge water conditions would meet requirements in the G8 guidelines throughout the trial series. During initial filling of control and test retention tanks, fine grade Arizona Test Dust, and concentrated algae harvested from the Duluth-Superi or Harbor were metered into the intake stream before the flow split to the control and tre atment tracks, to assure adequate concentrations of Total Suspended Solids (TSS) and live phytoplank ton in intake and control discharge. DetailsGSI/LB/F/A/1 March 15, 2010 Page 13 of 58



### on these processes are provided below. Target intak e levels of these parameters appear in Table 1. Actual levels during testing of the SiCURE TM 

### system are reported in the results section of this
document.

All current protocols, guidelines and requirements are open to interpretation especially in these early stages of implementation, and few if any faci lities meet all requirements in the strictest sense. Accordingly, it is ultimately up to an Admi nistration to decide if the system meets their requirements for Type Approval Certification. GSI, along with its sister facility, MERC, sought and received German Administration acceptance of it s protocols prior to initiating this set of

tests.

Table 1. Ranges of Various Physical and Biological Parameters in Ambient Water from DuluthSuperior Harbor (June – September) in Comparison to ETV/U.S. Coast Guard and IMO G8 Recommended Challenge Conditions.

**† Generic Protocol for the Verification of Ballast W**  **ater Treatment Technologies: Draft v4 2008, U.S. EP A**  **Environmental Technology Verification (ETV) Program under contract to U.S. Coast Guard.**  **‡ IMO Guidelines for the Approval of Ballast Water Ma**  **nagement Systems (G8), October 2008, Annex 4 Resolu tion**  **MEPC.174(58).** Parameter Proposed ETV/ U.S. Coast Guard † Recommended

IMO G8 ‡ Historic Ranges

Duluth/Superior Harbor Target Values for Amended

Duluth-Superior Water for SiCURE TM Tests

**Temperature ( o**  **C) 10 – 35**  **– 9 – 22**  **9 - 22**

**Salinity (psu) 0 – 31**  **Two salinities, >10 psu difference**  **0 – 1 0 - 1** 

**Total Suspended Solids (TSS) (mg/L)**  **> 15 > 50**  **2 – 21 50** 

**Particulate Organic Carbon (POC) (mg/L)**  **> 1 > 5**  **0.5 – 2.1 <1** 

**Dissolved Organic Carbon (DOC) (mg/L)**  **> 3 > 5**  **6 – 22 6 - 22** 

**Zooplankton (> 50**  μ **m/m 3**  **) > 10,000**  **> 100,000 100,000 - 3,000,000**  **100,000 – 3,000,000**

**Phytoplankton (10 - 50**  μ

**m/mL) > 100**  **> 1,000 25 – 1,200**  **> 1,000**

**Heterotrophic Bacteria (CFU/mL)**  **> 1,000 > 10,000**  **> 1,000 MPN/mL 1,000 MPN/mL** GSI/LB/F/A/1 March 15, 2010 Page 14 of 58



## 2.0. METHODS

The GSI land-based evaluation of the SiCURE TM 

### Ballast Water Management System was carried out in keeping with the methods summarized in the M ERC-GSI Test Plan (hereafter referred to as Test Plan) submitted to and approved by the Germ an BSH and Siemens Water Technologies
and available at http://www.nemw.org/GSI/MERC+GSITestPlan.pdf . Some refinements were necessitated by circumstance or opportunity. The fo llowing sections describe how each parameter and variable was sampled/analyzed during the SiCURE TM 

### trials at GSI. Additional
details can be found at www.nemw.org/GSI/SOPS.htm . All SOPs relevant to the SiCURE TM 

### tests, as amended, also are presented Appendix 2. A ny deviations from these SOPs during the performance of the SiCURE TM 

### tests were minor and did not affect data quality. More detail on these deviations is available upon request.

2.1. Experimental Goals and Design

The SiCURE TM 

### performance evaluation involved physical and biolo gical characterization of water upon ballasting (uptake/intake of water) and comparisons of organisms in control versus treated water immediately following treatment and a fter a five-day in-tank holding time. Biological characterizations supported direct compa rison with the IMO D-2 organism categories and standards. During a series of five consecutive valid trials, the treatment system was tested for its ability to: (a) successfully treat ballast water without interruption, (b) meet IMO D-2 discharge standards after a five-day holding time, and (c) discharge water after the five day retention period that is environmentally benign (i. e., no residual toxicity) pursuant to United States Environmental Protection Agency water qualit y criteria. A valid trial was one in which control discharge densities of live organisms were at least 10 times the IMO D-2 standard, consistent with IMO G8, and in which the facility o perated properly.

2.1.1 Treatment System and Test Facility Calibrat ion Trials

As provided in the Test Plan, GSI first conducted t wo calibration test runs of the SiCURE TM 

### system. The calibration runs were undertaken to ass ure the facility and the treatment system were operating properly. During these calibration trial s, adjustments to the system were documented only for internal reference by the treatment develo per.

Necessary adjustments to the facility during this c alibration period are documented here for the record. During the two calibration runs, line cont amination by zooplankton was suspected which could have resulted in overestimates of live organi sms in treated discharge. To assure facility performance and trial independence during the valid type approval test sequence, a thorough cleaning of the pipes leading from the control and treatment retention tanks to the sample collection tubs was undertaken well prior to the fi rst discharge operation. This cleansing process involved filling the pipes with chlorinated water ( 4 September 2009), and retaining the chlorinated water in the pipes for the retention pe riod. Just prior to Trial A discharge, water was drained from the pipes and then the pipes were thor oughly flushed with unchlorinated potable water (8 September 2009). After this flush, the dis charge valves were closed, the sample collection tubs were partially filled with potable water, and a zooplankton sample was collectedGSI/LB/F/A/1 March 15, 2010 Page 15 of 58



### from each tub and analyzed to assure no further org anism contamination. In addition, potable water for cleaning was obtained from a tanker truck while the potable water reservoir tank at the GSI facility was thoroughly cleaned and chlorinated and checked for residual organisms.

2.1.2. Valid Trials

Once the two calibration trials were complete, the SiCURE TM 

### treatment system was set for BSH type approval testing by the treatment developer, a nd the facility physical parameters were finalized and set for testing by the GSI Facility M anager. Any further adjustments to either component of the testing (the treatment system or t he facility) were carefully noted and subject to QA/QC documentation. Five valid trials of the Si CURE TM 

### system immediately followed the
calibration runs.

Because the SiCURE TM 

### tests at the GSI facility began later in the summe r than anticipated, there was a need to complete the testing in the shortest span of time possible to assure adequate densities of organisms. To that end, GSI used its capacity to run concurrent tests to overlap trials condensing the period required for five trials to 2 2 days. Consistent with the Test Plan, treatment and control intake operations within a given trial were always simultaneous, and treatment and control discharge operations were always sequential . Table 2 shows the schedule of the five valid tests, including the sequence of intake and dischar ge operations.

Table 2. Timing of Intake and Discharge Operations for the Five Valid Siemens SiCURE TM Trials at the GSI Land-Based Facility in 2009.

Trial Treatment Timing of Operation

Intake Discharge

**A Treatment**  **4 September 09:48-10:45**  **9 September 12:41-13:44** 

**Control 15:15-16:08** 

**B Control**  **11 September 09:52-10:47**  **16 September 08:58-09:53** 

**Treatment 11:51-12:44** 

**C Treatment**  **12 September 09:58-10:56**  **17 September 09:03-9:56** 

**Control 11:56-12:51** 

**D Control**  **20 September 09:35-10:32**  **25 September 08:47-09:41** 

**Treatment 11:06-11:59** 

**E Treatment**  **21 September 09:38-10:36**  **26 September 09:02-09:56** 

**Control 11:38-12:38** GSI/LB/F/A/1 March 15, 2010 Page 16 of 58



### To assure that discharging the control tank first i n no way resulted in contamination of the treated discharge, the following steps were taken:

• Prior to Trial B, the PVC pipes leading from the in take/discharge line to active sample collection tubs were replaced with clean, fl exible PVC tubing, which were removed, cleaned and inspected prior to each remain ing treatment discharge operation.

• During all Test Trials A-E the interior of the rete ntion tanks and sampling equipment (sample collection tubs, drain spout hose and nozzl e, plankton nets, etc.) were cleaned according to GSI/SOP/LB/G/O/3 – Procedure for Cleaning the Retention Tanks and Other Equipment at the GSI Land-Based RDTE Facility

.

2.1.3. Challenge Conditions

Ambient conditions were employed as the physical/ch emical challenge conditions, except that consistent with the Test Plan, Fine Test Dust was a dded to the facility intake to assure TSS levels were in keeping with IMO G8 guidelines. The solids injection procedure is detailed in
GSI/SOP/LB/G/O/5 – Procedure for Injecting Organisms and Solids into t he GSI Land-Based RDTE Facility . Fine Test Dust (ISO 12103-1, A2; nominal 0-80 μ m particle size; Powder Technology Incorporated; Burnsville, MN) was pre-we ighed at LSRI, and sterilized by baking in an oven at 190 °C for one hour. One day prior to t he test trial, ambient TSS was measured in the Duluth-Superior Harbor. On the day of the test tri al, the volume of harbor water to be used in the Solids Injection System (SIS) tank was determin ed in order to augment the intake water to 60 mg/L TSS, and the SIS tank was filled. The prepare d Fine Test Dust was poured into the SIS tank slowly to prevent clumping, and the dust was m ixed for a minimum of 20 minutes prior to the start of the trial. The test dust was injected into the intake water for the entire duration of t he fill at a constant rate using a peristaltic pump lo cated at Injection Point A (figure 2).

Biological challenge conditions were ambient as wel l except that organism densities in the smaller of the two plankton size classes (i.e., 10 - 50 μ m) were enhanced to assure consistency with IMO G8 required thresholds. The solids and ph ytoplankton injection systems were kept separate to reduce the risk of interference. The p hytoplankton injection procedure is detailed in
GSI/SOP/LB/G/O/5 – Procedure for Injecting Organisms and Solids into t he GSI Land-Based RDTE Facility . One to two days prior to the test trial, phytopl ankton entities from the DuluthSuperior Harbor were collected and concentrated usi ng 20 - 50 μ m plankton nets. The concentrated phytoplankton entities were stored at the GSI Land-Based RDTE Facility in holding ponds. Prior to injection, the water conta ining concentrated phytoplankton was mixed, sampled, and analyzed for viable cell density. In a ddition, a sample of Duluth-Superior Harbor
water was collected to determine the ambient viable cell density. Based on the density of cells in the phytoplankton concentrate and ambient intake wa ter, the volume of spiked concentrate that
would be needed to achieve a concentration of 1500 cells/mL in intake water was calculated. This volume was added to an Organism Pressure Injec tion System (OPIS) vessel. The OPIS vessel was pressurized to 25 psi greater than the s ystem pressure. The phytoplankton concentrate
was added at a constant rate to the intake water vi a the pressure differential for the entire duration of the intake procedure via Injection Poin t B (figure 2).GSI/LB/F/A/1 March 15, 2010 Page 17 of 58



### A static mixer, installed in the main intake line j ust after the two metering systems (SIS and OPIS) and prior to the main system “Y split” (figur e 2), assured that the concentrations of these additives was equivalent in the control and treatme nt tracks of the facility. At the end of the five day retention period, and just prior to discharge, the tanks were mixed manually with a small section of pipe which was pulled along the bottom o f the retention tank for a period of 20 minutes to assure that live organisms, especially s piked algal particles, that may have settled to the bottom of the tank during the retention period were accounted for to the greatest extent possible in the discharge water analysis.

2.2. Water Chemistry Analysis

2.2.1. Total Residual Chlorine (TRC) and Total Resi dual Oxidant (TRO) Sample Collection

Samples for total residual chlorine (TRC) and total residual oxidants (TRO) analysis were collected during intake, retention and discharge. At intake:

• A 1 L whole water sample was collected from the con trol intake collection tub;

• A 1 L whole water sample was collected from the pre -treatment intake collection tub;

• Three 1 L whole water samples were collected from t he filter backwash line at approximately 10, 30, and 50 minutes after the star t of the intake procedure;

• Three 1 L post-treatment whole water samples were c ollected from a sample port directly downstream from the treatment system (SP # 16) at approximately 10, 30, and 50 minutes after the start of the intake procedure;

• Three 1 L whole water samples were collected from a post-treatment sample point in the treatment system complex (for use exclusively b y Siemens) at approximately 10, 30, and 50 minutes after the start of the intake pr ocedure, and

• A 1 L whole water sample was collected post-treatme nt from a sample collection tub (#6). The post-treatment sample from collection tu b #6 indicated how much the chlorine concentration had degraded over the approx imately 45 minutes required to
fill the collection tub.

During tank retention:

• A 1 L whole water sample was collected from the tre atment tank on days 3, 4, and 5.

During discharge:

• A 1 L whole water sample was collected from the con trol discharge sample collection tub (#1), and

• Three 1 L whole water samples were collected from t reatment discharge sample collection tubs (#’s 4, 5, and 6).GSI/LB/F/A/1 March 15, 2010 Page 18 of 58



### 2.2.2

. Total Residual Chlorine (TRC) Determination

Samples were analyzed for all forms of chlorine, i. e., free chlorine, hypochlorites, and chlorine bound to nitrogenous compounds. Analysis took place within 30 – 120 seconds after collection
to minimize loss of chlorine due to reaction with o xidizable species in the sample.

For analysis, 100 mL of sample water were transferr ed from the sample collection container into a 150 mL beaker. 1.0 mL of potassium iodide reagent and 1.0 mL of acetate buffer reagent were added to each sample. Analysis was conducted with a Thermo Orion Model 97-70 Residual
Chlorine Electrode connected to an Orion Model 290A pH/mV/ISE meter.

A 1000 mg/L iodate stock solution (1000 mg/L as chl orine) was used to prepare analytical standards, ranging in concentration from 0.100 to 1 0.0 mg/L daily. The standards were prepared in deionized water by making dilutions of the 1000 mg/L iodate stock. Potassium iodide reagent
and acetate buffer were added to the iodate contain ing analytical standards. Iodate or chlorine present in the standards or samples oxidizes iodide to iodine in an acidic solution. The iodine concentration after the reaction was equal to the i odate or chlorine concentration present before the reaction. A calibration curve plotting log of t he chlorine concentration (x-axis) versus the mV response from the Residual Chlorine Electrode (y-ax is) was used to determine total residual chlorine concentrations in the samples.

Quality control sample analysis consisted of analyz ing duplicate samples and samples spiked with known amounts of chlorine. Approximately 10 % of the samples were analyzed in duplicate. Spiked samples were only analyzed for th e discharge samples because chlorine in the intake samples was reduced very rapidly and led to inaccurate spike recoveries. A TRC reference sample (QCI-780, ULTRA Scientific) was analyzed dai ly to confirm the accuracy of the calibration standards and samples.

2.2.3. Total Residual Oxidant (TRO) Determination

Total Residual Oxidant (TRO) analysis was conducted on the same samples as for TRC analysis with the exception of the system backwash samples d ue to high turbidity of these samples. As for TRC, TRO analysis took place within 30 to 120 secon ds of sample collection to avoid sample degradation.

For analysis, 10 mL aliquots (or smaller amounts if sample concentration was above the concentration of highest calibration standard) of s ample water were transferred from the sample
containers into 30 mL beakers. The contents of a Ha ch DPD Total Chlorine Reagent packet were added to each sample. Between 3 and 6 minutes after the addition of the reagent the absorbance of the sample was determined using a Spectronic 20D set at a wavelength of 515 nm. The absorbance of an aliquot of each sample with no rea gent added was also measured and the
absorbance value was subtracted from that of the sa mple containing the reagent. This was to correct for the background absorbance of each sampl e.

A TRO calibration curve was prepared using standard s prepared in deionized water utilizing a 100 mg/L chlorine stock solution. The standards, ra nging in concentration from 0.50 to 3.0GSI/LB/F/A/1 March 15, 2010 Page 19 of 58



### mg/L, were analyzed in the same manner as described for the samples. The calibration data was not linear to the concentration of the upper standa rd so the calibration curve used a second order polynomial fit.

Quality control sample analysis consisted of analyz ing duplicate samples and samples spiked with known amounts of chlorine. Approximately 10 % of the samples were analyzed in duplicate. Spiked samples were only analyzed for t he discharge samples because chlorine in the intake samples was reduced very rapidly. A TRC refe rence sample (QCI-780, ULTRA Scientific) was analyzed daily to confirm the accur acy of the calibration standards and samples.

2.2.4. Selected Disinfection By-Products (DBPs) Det ermination

Samples were collected during intake and discharge for analysis of selected disinfection byproducts (DBPs). On intake:

• 

### Two 1 L whole water samples were collected from the filter backwash line, and

• 

### Two 1 L samples were collected from the post-treatm ent sample collection tub (#6).

On discharge:

• 

### Two 1 L whole water samples were collected from the control sample collection tub (#1), and

• 

### Two 1 L whole water samples were collected from the treatment sample collection tub (#6).

Upon collection samples were stored in coolers with ice packs and transferred to the appropriate sample container by the GSI Senior Chemist. The sa mples were preserved appropriately, based on the analysis method, and were shipped cold to An alytical Laboratory Services in Middletown, Pennsylvania for analysis of DBPs that might likely be formed through the electrolytic chlorination process. The following analyses were conducted: trihalomethanes (THMs), haloacetic acids (HAAs), tribromoacetic acids (TBAs ), and haloacetonitriles (HANs). In addition, sodium chlorate, sodium bromade, and sodi um were analyzed

2.3. Water Quality Analysis

2.3.1. Analysis of Total Suspended Solids (TSS)

Samples for TSS analysis were collected during inta ke only as follows:

• Three 1 L whole water samples were collected from t he pre-treatment line (SP #3) at approximately 10, 30, and 50 minutes after the star t of the intake procedure, and

• Three 1 L whole water samples were collected from t he post-treatment line at approximately 10, 30, and 50 minutes after the star t of the intake procedure (SP #16).GSI/LB/F/A/1 March 15, 2010 Page 20 of 58



### Samples were collected in-line rather than from the sample collection tubs to avoid settling of suspended solids. This approach assured a more accu rate measurement of solids and organic
carbon in the intake water.

For analysis, the samples were vacuum filtered thro ugh pre-washed, dried, and pre-weighed Whatman 934-AH glass fiber filters. After the samp le was filtered it was dried in an oven and brought to constant weight. TSS values were determ ined based on the weight of particulates on
the filter and the volume of water filtered.

Quality control sample analysis consisted of analyz ing approximately ten percent of the samples in duplicate. A TSS reference standard (QCI, 711, ULTRA Scientific) was analyzed on multiple occasions along with TSS samples to confirm the acc uracy of the data being generated.

2.3.2. Analysis of Non-Purgeable Organic Carbon (N POC) and Dissolved Organic Carbon (DOC), and Determination of Particul ate Organic Carbon (POC) Concentrations

In these tests, NPOC was measured as a surrogate fo r total organic carbon (TOC), though it may be a slight underestimate of TOC. The analytical in strument used to measure NPOC purges the sample with air to remove inorganic carbon before m easuring organic carbon levels in the sample. Thus, the NPOC analysis does not incorpora te any volatile organic carbon which may be present in the sample.

Aliquots of the same samples that were analyzed for TSS were also analyzed for NPOC and DOC. Before the TSS analysis was conducted, aliquot s of approximately 50 mL of the sample were transferred to glass bottles and acidified wit h hydrochloric acid for NPOC analysis. An aliquot of the filtrate from the TSS analysis was t ransferred to a glass bottle and acidified for analysis of DOC. A Shimadzu Total Organic Carbon An alyzer (Model TOC-5050A) was employed for analysis of both NPOC and DOC. Concent rations of NPOC and DOC were determined based on a calibration curve developed o n the instrument using organic carbon standards prepared from potassium hydrogen phthalat e. Reported particulate organic carbon concentrations (POC) were determined as the differe nce between the NPOC and DOC values for a sample.

Quality control sample analysis consisted of analyz ing approximately 10 % of the samples in duplicate. A reference standard (#516 Demand, Envir onmental Resource Associates) was analyzed daily to confirm the accuracy of the data being generated.

2.4. Viable Organism Analysis

Sample water for analysis of viable organisms was s imultaneously collected from replicate sample ports into identical 3.8 m 3 

### collection tubs during each intake or discharge op eration. Volumes retained varied with the operation (intake versus discharge) and treatment (control versus treatment), depending upon anticipated organ ism concentrations, but were always greater than IMO guideline volumes. The water in each colle ction tub constituted an independent time integrated replicate sample of the 200 m 3 

### experimental water mass.GSI/LB/F/A/1 March 15, 2010 Page 21 of 58



### 2.4.1. Organisms Greater than 50 μm in Minimum Dimension

2.4.1.1. Sample Collection

During the intake operation, i.e. the filling of th e treatment and control 200 m 3 

### retention tanks, the following time-integrated sample volumes were c ollected by continuous flow from the intake lines simultaneously:

• 2 - 4 m 3 

### from the pre-treatment intake line,

• 2 - 4 m 3 

### from the control intake line, and

• 2 - 4 m 3 

### from the immediate post-treatment intake line.

During discharge:

• One 2 - 4 m 3 

### time-integrated sample was continuously collected from the control discharge, and

• Two to three replicate time-integrated samples of 2 - 4 m 3 

### each (total volume 4 to 9

m 3 

### ) were continuously collected from the treatment di scharge.

Flow control valves and system logic assured that s ample flow rates were equivalent and proportional to intake and discharge flow rates thr oughout each operation. Immediately after filling, the entire sample volumes were drained fro m the sample collection tubs and concentrated through 35 μ m (50 μ m diagonal dimension) plankton nets into 1 L cod-en ds for microscopic
examination. See GSI/SOP/LB/RA/SC/6 - Procedure for Zooplankton Samp le Collection

.

2.4.1.2. Live/Dead and Size Analysis

Live/dead analysis took place within two hours of c ollecting and concentrating the individual samples. Microzooplankton (e.g., rotifers, copepod nauplii, veligers, etc.) and macrozooplankton (e.g., crustanceans), all generally greater than 50 μ m in minimum dimension (with the exception noted below) were analyzed simultaneously by separa te taxonomists. Microzooplankton subsamples were analyzed in a Sedgewick Rafter coun ting chamber by examination under a compound microscope at a magnification of 40X to 10 0X. Macrozooplankton were analyzed in a Ward’s Counting Wheel at a magnification of 20 to 30X using a dissecting microscope. Due to high densities, quantification of zooplankton in in take and control discharge samples required analysis of sub-samples and extrapolation to the en tire sample volume. For these samples, a subsample was removed for analysis using a Henson-S tempel pipette. The treatment discharge samples had lower densities allowing analysis of a greater proportion of the sample. Treatment discharge samples were split in half using a Folsom Plankton Splitter. Half of the sample was analyzed for macrozooplankton and the other half wa s examined for microzooplankton. The proportion and total concentration of live versus d ead organisms was determined using standard movement and response to stimuli techniques.

Quality assurance measures included live/dead analy sis of at least 10 % of the samples by two separate taxonomists. The average percent similari ty of taxonomic identification (live organismsGSI/LB/F/A/1 March 15, 2010 Page 22 of 58



### only) and the average relative percent difference o f the number of live organisms counted were calculated for all second analyses. These data qua lity measurements were compared against the data quality objectives outlined in the GSI Quality Assurance Project Plan (GSI, 2009), and the percentage of data quality measurements meeting the data quality objectives was determined for microzooplankton and macrozooplankton separately.

Because freshwater zooplankton are in general small er than their salt and brackish water counterparts, the larger regulated size category (g reater than 50 μ m in minimum dimension) does not incorporate all live zooplankton that may be pr esent in a freshwater assemblage. This freshwater phenomenon raises special issues with re spect to assessing zooplankton densities for the purpose of comparison with the IMO D-2 standard . It cannot be assumed that every zooplankton individual meets the size criterion in the strictest sense. Moreover, the smaller species must be analyzed using a compound microscop e on a slide under a cover slip as noted above. Normally, this approach at times precludes d irect sizing of an organism’s minimum dimension during live analysis. During these tests , the Lecanid rotifer figured prominently in treated discharge samples. The minimum dimension o f this saucer-shaped organism (depth) was never visible to the microscopist for direct measur ement. To accommodate to this situation, following live analysis, samples were preserved wit h 10 % Lugol’s solution and numerous Lecanid rotifer specimens were extracted and direct ly measured in all dimensions. This evaluation showed the organisms in this taxonomic g roup to be generally less than 50 μ m in
actual minimum dimension. Live individuals in the L ecanidae family present in treated discharge were therefore not added to the tally of regulated live organisms, but were counted and documented as live zooplankton for consideration by the regulatory authority. See GSI/SOP/LB/RA/SA/2 - Procedure for Zooplankton Samp le Analysis

.

2.4.2. Organisms 10 – 50 μm in Minimum Dimension

2.4.2.1. Sample Collection

For live analysis of organisms 10 – 50 μ m in minimum dimension, one sample of 1 L was collected immediately after filling from the pre-tr eatment sample collection tub and one sample of 1 L was collected from the immediate post-treatm ent sample collection tub. During discharge, one sample of 1 L was collected from the control ta nk via sample collection tub, and three samples of 1 L each were collected from the replica te treatment sample collection tubs. Analysis occurred on-site within 1.5 hours of sample collect ion, with samples stored in coolers during the interim. Prior to analysis, samples were concentrat ed through a 10 μ m plankton net and stored in a 25 mL sample container. See GSI/SOP/LB/RA/SC/3 - Procedure for Algae/Small Prot ozoa Sample Collection

.GSI/LB/F/A/1 March 15, 2010 Page 23 of 58



### 2.4.2.2. Sample Analysis

For analysis, a 1.5 mL subsample of the concentrate d sample was transferred to a 2 mL sample container, with 4 μ L of FDA stock solution added. The subsample was th en allowed to incubate in the dark for 5 minutes. the 1.5 mL incubated alg ae sample was mixed and 1.1 mL was immediately transferred to a Sedgwick-Rafter cell, covered and placed on the stage of a microscope that was set for simultaneous observatio n using brightfield and epifluorescence. At least two horizontal transects were counted (an are a known to reflect greater than 1 mL of original sample water). If time permitted, addition al transects were counted to increase statistical power. This resulted in greater than 100 live cells counted from the pre-treatment intake and control discharge samples, and often fewer than 10 live cells counted in two trans ects for posttreatment intake and treatment discharge samples. Single cell entities and cells comprising
colonial and filamentous entities were characterize d as follows: alive = cells showing obvious green fluorescence from cell contents; dead = cells showing no or very little evidence of green fluorescence from cell contents; and ambiguous = ce lls or entities that cannot be clearly identified as alive or dead (should be uncommon). R ecords were kept of transect lengths and
widths so that the total counted area and volume an alyzed could be calculated later.

Entities less than 10 μ m in all visible dimensions or greater than 50 μ m in minimum dimension were not counted. Counting and measurement of all o ther entities followed standard procedures for individuals (length and width), colonies (e.g., number of cells, cell length and width) and filaments (e.g., number of cells, cell length and w idth or total filament length if cells could not be discerned). The remaining concentrated sample in th e 25 mL bottle was archived using a preservative (formalin or Lugol’s) for long-term st orage.

Quality assurance measures included analysis of at least 10 % of the samples by two separate taxonomists using a dual-headed microscope (i.e., b oth taxonomists analyzed the same sample at the same time). The average percent similarity of taxonomic identification and the average relative percent difference of the number of live o rganisms counted were calculated for all second analyses. These data quality measurements we re compared against the data quality objectives outlined in the GSI Quality Assurance Pr oject Plan (GSI, 2009), and the percentage of data quality measurements meeting the data quality objectives was determined. See GSI/SOP/LB/RA/SA/1 - Procedure for Algae/Small Prot ozoan Sample Analysis

.

2.4.3. Bacteria

Control and treatment samples were collected and an alyzed for heterotrophic bacteria, two specific indicator pathogens: E. coli and enterococci, and viable toxigenic Vibrio cholerae

.GSI/LB/F/A/1 March 15, 2010 Page 24 of 58



### 2.4.3.1. Sample Collection

One liter whole water samples were collected as fol lows:

• On intake, three were collected immediately after f illing from the pre-treatment sample collection tubs, and three were collected fr om the post-treatment sample
collection tubs.

• On discharge, three were collected from a control s ample collection tub and three were collected from a treatment sample collection t ub.

All samples were collected according to GSI/SOP/LB/RA/SC/4 – Procedure for Microbial Sample Collection , and were immediately transported in an insulated cooler to the LSRI and analyzed as individual replicates.

2.4.3.2. Sample Analysis

Viable heterotrophic bacteria were enumerated accor ding to GSI/SOP/BS/RA/MA/1 – Procedure for Quantifying Heterotrophic Plate Counts (HPCs) u sing IDEXX’s SimPlate® for HPC Method

. This method utilizes the IDEXX SimPlate® for HPC Me thod (IDEXX Laboratories, Inc.; Westbrook, Maine), which is based on IDEXX Laborato ries’ patented multiple enzyme technology.

The presence and abundance of E. coli ( GSI/SOP/BS/RA/MA/4

- Procedure for the Detection and Enumeration of Total Coliforms and E. coli Using ID EXX's Colilert® ) and enterococci ( GSI/SOP/BS/RA/MA/3 - Procedure for the Detection an d Enumeration of Enterococcus using
EnterolertTM ) were determined using Colilert® and EnterolertTM, respectively, which are both based on IDEXX’s patented Defined Substrate Technol ogy (DST®).

RNA and DNA colony blots were prepared at the LSRI following GSI/SOP/LB/RA/SA/3 - Procedure for the Colony Blot Preparation for Enume ration of Culturable Vibrio chloreae , a procedure in which the RNA or DNA of potential Vibrio Cholerae, and a limited number of additional species which may grow on the selective media, is fixed to a filter. Filters which exhibited colony growth were then shipped to the Ma ryland Pathogen Research Institute at the University of Maryland (College Park, MD) for analy sis of potential viable toxigenic V.
cholerae . Viable toxigenic V. cholerae is assayed with a commercial DFA kit specific for serogroup O1 (New Horizons Diagnostics) using monoc lonal antibodies tagged with fluorescein isothiocyanate (FITC) (Hasan et al ., 1994).

Quality control samples included a media blank and a positive control for E. coli /total coliforms
and Enterococcus spp .; a media and peptone-saline diluent blank for het erotrophic bacteria; and a thiosulfate citrate bile salts sucrose (TCBS) aga r blank, and DNA, and RNA blanks for Vibrio

spp. Quality assurance measures included analysis of at least 10 % of the samples in duplicate. The average relative percent difference of all dupl icates analyzed during the test trials was calculated separately for E. coli

, Enterococcus spp ., heterotrophic bacteria, and Vibrio spp . InGSI/LB/F/A/1 March 15, 2010 Page 25 of 58



### addition, at least 10 % of the samples were counted by two separate analysts and the average relative percent difference for all second counts w as determined. These data quality measurements were compared against the data quality objectives outlined in the GSI Quality Assurance Project Plan (GSI, 2009), and the percent age of data quality measurements meeting the data quality objectives was determined.

2.5. Ambient Physical/Chemical Water Conditions A nalysis

Temperature, salinity, dissolved oxygen, chlorophyl l fluorescence, turbidity and pH were measured every 15 minutes during the test trials by two identical multi-parameter probes (calibrated according to manufactures specification s) placed, one each, into the control and test tanks. A calibrated, hand-held instrument was used to measure temperature, salinity, and dissolved oxygen from the control sample collection tub, the pre-treatment sample collection tub, and post-treatment sample collection tub during int ake. In addition, temperature, salinity, and dissolved oxygen were measured during discharge fro m one control sample collection tub and two or three treatment sample collection tubs. See GSI/SOP/LB/RA/SC/8 - Procedure for Collecting Physical/Chemical Data and Samples at th e GSI Land-Based RDTE Facility

.

2.6. Whole Effluent Toxicity Analysis

GSI’s whole effluent toxicity testing of the SiCURE TM 

### system involved tests for chronic toxicity involving three freshwater species as arrayed in ta ble 3. Toxicity tests were conducted on treated
water from all five test trials.

Table 3. Standard Operating Procedures Relative to Whole Effluent Toxicity Testing.



### 2.6.1. Standard Whole Effluent Toxicity Tests

One set of tests—Standard Whole Effluent Toxicity T ests (Standard WET)—measured toxicity following five days storage in the land-based facil ity’s 200 m 3 

### retention tanks. For these tests, samples were collected for analysis of residual tox icity at discharge. Sample water, stored in large HDPE containers, was immediately transported to the LSRI and was used immediately upon arrival to set up the Standard WET tests. Fol lowing initial set up of the tests, the remaining sample water was held at 4 °C in the dark to retain as much of the initial toxicity as possible, and GSI SOP Code Test Type Test Species Test Endpoint

**GSI/SOP/BS/RA/WET/1 Chronic**  **Cladoceran (**  Ceriodaphnia dubia) **Survival and Reproduction** 

**GSI/SOP/BS/RA/WET/2 Chronic**  **Fathead Minnow (**  Pimephales promelas) **Survival and Growth**

**GSI/SOP/BS/RA/WET/3 Chronic**  **Green Alga (**  Selenastrum capricornutum) **Growth**GSI/LB/F/A/1 March 15, 2010 Page 26 of 58



### portions of the discharge sample water was warmed t o 25 °C each day to serve as renewal water for the bioassay. A dilution series, using Duluth-S uperior Harbor water, was run for each species. All tests were conducted in temperature-c ontrolled incubators, water baths, or at ambient room temperature following the SOPs listed in table 3.

2.6.2. Cold Whole Effluent Toxicity Tests

A second set of trials—Cold Whole Effluent Toxicity (Cold WET) tests—was conducted to estimate the TRC, TRO and toxicity effects on organ isms under cold water conditions. Though informative, it should be noted that in these assay s the SiCURE TM 

### system’s internal logic that self-regulates chlorination of intake water based o n oxidation-reduction potential (ORP) was not engaged resulting in higher chlorine levels in disc harge samples than the treatment developer
believes will occur under actual cold ambient condi tions. Treated water was
collected continuously from a sample port just downstream of the treatment system (SP #15) and diverted into a sample collection tub during th e filling of the treatment retention tank. A 50 L whole water subsample was extracted and placed in a dark, refrigerator set at 4 o 

### C for five days, thus simulating cold temperature tank retenti on. A portion of the sample water was warmed to 25 °C prior to initial set up of the Cold WET assay, and was warmed prior to daily
renewal as described above for the Standard WET ass ay. There was no dilution series used for the Cold WET assay; test organisms ( Selenastrum capricornutum, Ceriodaphnia dubia, and Pimephales promelas ) were exposed to 100 % sample water. The Cold WET assay was conducted concurrently with the Standard WET assay following the SOPs listed in table 3.

2.6.3. Determination of Total Residual Chlorine ( TRC) and Total Residual Oxidant (TRO) Levels for WET Analysis

Samples for TRC and TRO were collected from the sto ck exposure solutions daily. Analysis of these samples was conducted in the same manner as for the samples from the testing at the landbased facility (see sections 2.2 – 2.3) with the ex ception that smaller sample volumes (20 mL) were used due to the limited volume of sample avail able. Amounts of reagents added to the samples were adjusted so that a constant ratio of r eagent to sample volume was used.

Standards were prepared, analyzed and subjected to quality control in the same manner as described for the land-based analysis (see sections 2.2 – 2.3). Spikes were only performed on deionized water due to the rapid degradation of chl orine in Duluth-Superior Harbor water.

2.6.4. Statistical Analysis for WET Assay

Data was analyzed using the Comprehensive Environme ntal Toxicity Information Systems program (version 1.7, Tidepool Scientific Software, McKinleyville, CA). Data analyses included normality, homogeneity of variance, one-way analysi s of variance (ANOVA), and suite of tests for comparison between treatment means. Non-normal survival data was transformed using the natural log (EPA, 2002) to normalize the data. The endpoints of the chronic toxicity tests were:

• Lowest Observed Effect Concentration (LOEC), i.e., the lowest concentration in a test with a statistically significant difference in response from the control response.GSI/LB/F/A/1 March 15, 2010 Page 27 of 58



### • No Observed Effect Concentration (NOEC), i.e., the highest concentration in a test for which there was no statistically significant di fference in response from that of the
control.

• Median Lethal Concentration (LC 50 

### ), i.e., the concentration resulting in death of 50 % of exposed individuals by a predetermined time.

• Effective Concentration (EC 25 

### ), i.e., the concentration resulting in inhibiting a biological function (e.g. growth, reproduction) of 25 % of exposed individuals by a predetermined time.

These measures are extrapolations of statistical re sults to the experimental endpoints. Mean percent survival, mean dry weight values, mean cell density, and mean number of young per female for the laboratory controls and treatments w ere analyzed with a statistical significance
level of 0.05.

2.6.5. Determination of Quality of Test Organisms for WET Assay

Whole Effluent Toxicity tests were initiated with h ealthy, vigorous organisms. To determine the overall health of the test organisms, reference tox icant tests were performed with Ceriodaphnia
dubia and Pimephales promelas prior to the start of each definitive test or at l east once per month. To determine the validity of the Standard an d Cold WET tests, percent survival, dry weights of survivors, mean cell density, and mean n umber of young per female in the controls were compared to the test acceptability criteria pu blished in the U.S. EPA’s Short-term Methods for Estimating the Chronic Toxicity of Effluents an d Receiving Waters to Freshwater Organisms (4 th 

### edition, 2002). Class I standardized weights were used as a check for the organism drying process and the performance of the balance. Daily a nd weekly calibration of test meters ensures optimal performance.

2.7. Data Management

2.7.1. Data Recording

Specific forms (i.e., by size class of organism, by scale of testing, etc.) were used to record sample collection and analysis data. Specific form s were also used to record sample custody, handling and storage information for those samples that were not analyzed on site at the landbased facility or at the LSRI (e.g., disinfection b yproducts and Vibrio cholorae DNA and RNA colony blots). GSI Senior Personnel were responsib le for ensuring that the forms were correctly filled out and timely. They were also responsible f or maintaining the forms on file, creating electronic copies, and posting to the GSI SharePoin t intranet website for storage and archiving. Technical systems audits (TSAs) were conducted duri ng each intake and discharge operation for all test trials. During each TSA the GSI Assistant QA/QC officer observed data recording procedures related to sample collection, zooplankto n analysis, phytoplankton analysis, and/or chemistry analysis and checked for compliance. Prob lems identified during each TSA were documented on the specific Technical Systems Audit Checklist for that trial’s intake or discharge.GSI/LB/F/A/1 March 15, 2010 Page 28 of 58



### Following the completion of SiCURE TM 

### test trials, a thorough review of all data sheets and laboratory notebooks was completed to ensure compli ance with the documentation procedures outlined in all relevant GSI SOPs and in the GSI Qu ality Assurance Project Plan (QAPP). A Technical Systems Audit Checklist was completed dur ing observation of sample collection and analysis activities, and during the data review. A QA/QC Log Book was used to document any
additional data verification and validation activit ies. The TSA checklist and QA/QC log book were scanned to electronic format and posted to the GSI SharePoint website.

2.7.2. Data Processing and Storage

All original datasheets were stored in three-ring b inders, each with a unique identification code specific to the SiCURE TM 

### system tests. All log books were also given a uni que identification code and are specific to SiCURE TM 

### . At least one backup copy (i.e., an electronic co py stored on the GSI SharePoint website) was made of all complet ed datasheets, and in some cases additional hardcopies were also made. The raw data is in the custody of the appropriate GSI Senior Staff Member until the final technical report has been ap proved, after which time it will be archived by the GSI Assistant QA/QC Officer on the Universit y of Wisconsin-Superior campus for a period of at least five years.

A database designed using the Microsoft Access soft ware suite was used to store, manage and process phytoplankton and zooplankton data. Microso ft Excel was used in conjunction with the
database to create various dataset formats for subs equent analysis. Microsoft Excel was also used to store, manage, and process microbial, water chemistry, water quality, and whole effluent toxicity data. Database entry and maintenance was t he responsibility of the GSI database management staff. Regular checks for data entry err ors were conducted by comparing database records and Excel spreadsheets with the original pa per data sheets. This was a manual inspection process and though rather time consuming, was an es sential procedure for discovering errors. After examination and QA analysis, the data distrib ution files from the Access database were posted to the LSRI’s Local Area Network (LAN) in an organized hierarchical folder system. A backup of the database was also made regularly to a void any loss of data following computer/electronic glitches. Files were also poste d to the GSI’s SharePoint website to provide a secondary data backup/storage mechanism.

2.8. Quality Assurance/Quality Control

The GSI’s Quality Assurance Project Plan (QAPP) out lines the management activities the GSI undertakes to assure the credibility of its land-ba sed research activities. The plan covers QA/QC data quality indicators, evaluation processes, perf ormance measures and acceptance criteria; instrument certification and calibration; personnel training requirements; QA/QC of documents and records; data management; and QA/QC assessments and response actions; etc. The plan is updated annually, with a specific process used for review, comment, approval, distribution and posting. It closely follows the format of the U.S. Environmental Protection Agency’s (EPA’s) “EPA Guidance for Quality Assurance Plans”.GSI/LB/F/A/1 March 15, 2010 Page 29 of 58



### 2.8.1. Quality Assurance/Quality Control Activities

The GSI Assistant QA/QC Officer conducted Technical Systems Audits (TSAs) during each Trial’s intake and discharge operation, as well as a thorough data verification and validation following the completion of SiCURE TM 

### tests to ensure that all information was complete, that SOPs were correctly followed, and that QA/QC object ives were met. The results of these observations were documented on a pre-printed TSA C hecklist and in a QA/QC log book specific to SiCURE TM 

### . The GSI Assistant QAQC Officer created an electro nic copy of each TSA Checklist and the QA/QC log book and posted those t o the GSI SharePoint website. Once the final technical report is finalized, the original Q A/QC data will be archived on the UWS campus for a period of at least five years.

2.8.2. Standard Operating Procedures (SOPs )

GSI SOPs were developed by the relevant GSI senior personnel in conjunction with the GSI PI and GSI Senior QA/QC Officer. The SOPs follow a com mon format and all include specific QA/QC procedures and metrics. The Senior QA/QC Offi cer is responsible for distributing the SOPs to the relevant parties for approval. Draft an d final copies of all SOPs relevant to the
SiCURE TM 

### tests were posted to the GSI SharePoint website; t he final version was also posted to the GSI public website ( www.greatshipsinitiative.org ). SOPs were updated on an as-needed
basis.

2.8.3. Documents and Records

The GSI Senior QA/QC Officer is responsible for mai ntaining all electronic documents and records for a period of five years unless custody i s transferred using a chain of custody form.
Electronic versions of all GSI documents and record s are saved to the GSI SharePoint website once complete. Hard copies of GSI documents and rec ords are scanned and also saved to the GSI Sharepoint website. Due care and diligence will be taken to properly dispose of documents and records that are no longer required after the five year period has lapsed. Disposal procedures will
involve electronic deletion of documents and record s from the GSI SharePoint website and the personal computers of GSI personnel, as well as man ual shredding of hard copies.

2.8.3.1. Notebooks

Bound field and laboratory notebooks were used to r ecord observations, sampling details and onsite laboratory and field measurements. These note books were assigned a unique identification code by the GSI Assistant QA/QC Officer, in order t o maintain a record of all laboratory notebooks specific to the SiCURE TM 

### tests. Notebooks were also used to record instrume nt and equipment calibration and maintenance information. All notebooks were examined periodically during the Technical Systems Audits by the GSI QA/Q C officers and were checked for compliance with GSI SOPs following the completion o f testing. Problems identified by the periodic QA/QC review were documented and included in a training/response file.GSI/LB/F/A/1 March 15, 2010 Page 30 of 58



### 2.8.3.2. Safety, Operation and Maintenance Records

The GSI Land-Based RDTE Facility Operations Manager , along with safety staff at AMI Consulting Engineers (Duluth, MN) provided safety t raining to all relevant GSI personnel prior
to the start of the SiCURE TM 

### tests. The GSI Land-Based RDTE Facility Operations Manager ensured that all forms associated with safety, oper ation and maintenance (i.e., confined space entry permit forms) were correctly filled out and m aintained on file.



## 3.0. RESULTS

Five sequential valid tests of the Siemens SiCURE TM 

### Ballast Water Management System were completed consistent with the Test Plan accepted by the German BSH as meeting IMO G8 and G9 guidelines. Standard and Cold WET chemical assay s were conducted in conjunction all of these trials, and valid Standard and Cold WET biolo gical assays were completed with two to five of the these trials, depending upon the test specie s and assay type.

3.1. Challenge Conditions

3.1.1. Physical/Chemical Challenge Conditions

Dissolved organic carbon (DOC) and total suspended solids (TSS) values were in the range or
well in excess of the IMO recommended minimum. Inta ke water salinity during these tests was always less than 1 PSU. Ambient particulate organi c carbon (POC) was consistently below the IMO guideline across trials, probably due to the la te-season timing of the tests. A summary of physical/chemical conditions of intake and discharg e water along with the recommended ranges in the IMO G8 guidelines appear in table 4.GSI/LB/F/A/1 March 15, 2010 Page 31 of 58

Table 4. Average Concentration (± Std. Dev.) of To tal Suspended Solids (TSS), Dissolved Organic Carbon (DOC), and Particulate Organic Carbon (POC) in Challenge Water During Five Trials of the

Siemen’s SiCURE TM Ballast Water Management System.

Parameter IMO G8

Guideline Sample Trial A Trial B Trial C Trial D Trial E

**TSS (mg/L) >50**  **Intake 34.2 ± 9.4**  **53.1 ± 0.8 54.7 ± 3.2**  **53.0 ± 2.6 54.4 ± 1.1** 

**Post-Treatment 36.5 ± 5.9**  **48.1 ± 1.6 47.0 ± 0.9**  **49.2 ± 2.7 47.2 ± 0.8** 

**DOC (mg/L) >5**  **Intake 7.8 ± 0.03**  **8.4 ± 0.3 8.5 ± 0.2**  **8.8 ± 0.2 8.9± 0.2** 

**Post-Treatment 7.7 ± 0.2**  **8.9 ± 1.0 8.6 ± 0.2**  **9.1 ± 0.2 8.3 ± 0.1** 

**POC (mg/L) >5**  **Intake 0.3 ± 0.4**  **0.0 ± 0.4 0.1 ± 0.2**  **0.5 ± 0.5 0.6 ± 0.6** 

**Post-Treatment 0.7 ± 0.5**  **0.0 ± 1.0 0.2 ± 0.4**  **0.2 ± 0.4 1.3 ± 0.1** 

**Salinity (PSU) <3**  **Intake and Discharge**  **<1 <1**  **<1 <1**  **<1**



### 3.1.2 Biological Challenge Conditions

Densities of live organisms on intake and control d ischarge were consistent with or exceeded IMO G8 guidelines during all five trials. In the g reater than 50 μ m size class, the intake density of live organisms ranged 287,920 to 1,037,822 with an average of 656,824, meeting the required density of 100,000 live organisms per m 3 

### . The density of live organisms in the control dis charge samples ranged from 321,058 to 742,741, with an ave rage of 463,141, well exceeding the required density of 100 per m 3 

### . Intake concentrations of the smaller planktonic o rganisms (10 -
50 μ m) also were consistently greater than the required 1000 cells/mL (ranging from 1179.6 to 1832.5 per mL with an average of 1580.1). The contr ol discharge densities of these smaller organisms also exceeded the G8 requirement of 100 l ive cells per mL (with the exception of the Trial E for which the value was 95.6) with an avera ge of 150.3. Table 5 summarizes these
values.GSI/LB/F/A/1 March 15, 2010 Page 32 of 58

Table 5. Live Plankton Concentrations in Test Faci lity Intake Water and in Control Discharge Across Trials.

Live Organism Size Category IMO G8

Guideline Sample Trial A Trial B Trial C Trial D Trial E

**> 50** μ **m (#/m 3**  **) >100,000**  **Intake 769,661**  **627,566 1,037,822**  **287,920 561,153** 

**>100 Control** 

**Discharge 321,058**  **486,506 330,787**  **434,611 742,741** 

**10 – 50** μ **m (#cells/mL)**  **>1000 Intake**  **1179.6 1832.5**  **1563.1 1683.2**  **1642.1**

**>100 Control** 

**Discharge 178.8**  **204.9 136.9**  **135.4 95.6** 



### Plankton diversity also met IMO requirements. The z ooplankton community at the test site was dominated by the rotifers Keratella (loricate) and Polyarthra and Synchaeta (illoricate or softbodied) which comprised over 80 % of total density. Copepod nauplii, dreissenid mussel veligers, copepod copepedites, and bosminid cladoce rans were also common in the zooplankton community. The density of live soft-bodied rotifers decreased considerably over the five day holding time of some of the trials, and was extreme ly low upon discharge. Densities of loricate (hard bodied) rotifers decreased little or in some cases increased during the holding period in the control water mass. The smaller regulated size clas s (10 – 50 μ **m** 

### ) was dominated by phytoplankton entities of diatoms, green algae, blu e-green algae, chrysophytes and cryptophytes. Protozoans, including ciliates and flagellates, wer e also present, comprising up to 3 % of the assemblages in intake samples. Dominant taxa during these trials were Aulacoseira spp. (filamentous diatom), Melosira spp. (filamentous diatom), Cyclotella spp. (single-celled centric diatom), Asterionella formosa (colonial pennate diatom), Fragilaria spp. (filamentous diatom),
Oscillatoria limnetica (filamentous blue-green), Pandorina morum (colonial green), and
Rhodomonas spp. (single-celled cryptophyte).

Microbial organism concentrations in the intake and control discharge samples during the
SiCURE TM 

### trials are provided in table 6.GSI/LB/F/A/1 March 15, 2010 Page 33 of 58

Table 6. Microbial Organism Concentrations in Test Facility Intake Water and Control Discharge Across Trials.

Microbial Organism IMO G8

Guideline Sample Trial A Trial B Trial C Trial D Trial E

E. Coli **(MPN/100 mL) N/A**  **Intake 31**  **213 51**  **9 8** 

**NA Control** 

**Discharge < 1**  **1.0 1.0**  **< 1 1.0** 

**Toxigenic V. cholerae**  **(CFU/100 mL) N/A**  **Intake < 1**  **< 1 < 1**  **< 1 < 1** 

**N/A Control** 

**Discharge < 1**  **< 1 < 1**  **< 1 < 1** 

**Enterococci (MPN/100 mL)**  **N/A Intake**  **86 434**  **640 660**  **148**

**N/A Control** 

**Discharge 35**  **2275 295**  **149 54** 

**Total Heterotrophic**  **Bacteria (MPN/mL)**  **N/A Intake**  **1217 8133**  **3667 2900**  **467**

**NA Control** 

**Discharge 1650**  **1200 500**  **88 900** 



### 3.2. Viable Organisms in Treated Discharge

3.2.1 Regulated Plankton

Using the methods noted in the methods section for determining the numbers of live organisms that fit within the regulated size categories of pl ankton, organism densities in discharge water from the five consecutive trials consistently met t he IMO D-2 standard 2 

### . The density of live organisms > 50 μ m size class decreased dramatically immediately pos t-treatment (> 99.9 % reduction), and ranged from 38 to 193 individuals p er m 3 

### . Bosminids, copepod copepodites, chironomids, dreissenid veligers, and a few Keratel la and Lecanid rotifers were observed in the post-treatment samples. Soft-bodied rotifers were e ntirely absent from the subsamples analyzed. In post-retention discharge samples, the average de nsity of live organisms in the > 50 μ m size class ranged from 1 to 7 individuals per m 3, . 

### meeting G8 requirements (table 7).

In the smaller size class, numbers of live organism s in the immediate post-treatment samples also were much lower than at intake, ranging from 1 to 1 3 per mL. Concentrations in discharge samples after the five day retention period were co nsistently less than 10 cells/ml, meeting G8 requirements for treatment.

2 As noted in the methods section, not all zooplankto n in the natural freshwater assemblage meet the str ict definition
of 50 μ m in minimum dimension. The total average density o f live zooplankton in discharge samples ranged from 2 to 20 per m 3. The live organisms in these discharge samples were chironomid larvae, copepod nauplii, and Lecanid rotifers, with the latter being generally below the regulated size range as noted above. A list of all live zooplankton by taxonomic category and size class appear in Appe ndix 3.GSI/LB/F/A/1 March 15, 2010 Page 34 of 58

Table 7. Live Plankton Densities within Regulated Size Categories in Test Facility Post-Treatment Intake and Discharge Across Trials.

Live Organism Size Category IMO G8

Guideline Sample Trial A Trial B Trial C Trial D Trial E

**> 50** μ **m (#/m 3**  **) NA**  **Post Treatment**  **193 38**  **151 176**  **53**

**<10/m 3**  **Discharge 5**  **7 4**  **4 1** 

**10 – 50** μ **m (#cells/mL)**  **NA Post**  **Treatment 6.8**  **1 5.8**  **4.8 13.5** 

**<10 /mL Discharge**  **0.0 0.0**  **0.0 6.8**  **2.9**



### 3.2.2. Regulated Microbes

Immediate post-treatment, and post-treatment post-r etention microbial samples were consistently
below the IMO G8 standard for E. Coli and enterococcus , though intake levels and control discharge levels of E. coli were also below G8 discharge limits for all trials (table 8).
Enterococcus levels in treated discharge were always 1 to 3 log s lower than control discharge and never above 10 MPN per 100 mL (table 8). Althou gh heterotrophic plate counts immediately following treatment averaged an MPN fewer than ten per mL, post-treatment post-retention MPN average increased to 4.1E+05 per mL (table 8). This increase was not observed in the post retention control samples.GSI/LB/F/A/1 March 15, 2010 Page 35 of 58

Table 8. Microbial Organism Concentrations in Test Facility Post-Treatment Intake and Discharge Across Trials.

Microbial Organism IMO G8

Guideline Sample Trial A Trial B Trial C Trial D Trial E

E. Coli **(MPN/100 mL) N/A**  **Post Treatment**  **Intake < 1**  **< 1 < 1**  **< 1 < 1** 

**< 250 CFU/ 100 mL**  **Discharge < 1**  **< 1 < 1**  **< 1 < 1** 

**Toxigenic V. cholerae**  **(CFU/100 mL) N/A**  **Post Treatment**  **Intake < 1**  **< 1 < 1**  **< 1 < 1** 

**< 1 CFU** V. cholerae

**/ 100 mL**  **Discharge < 1**  **< 1 < 1**  **< 1 < 1** 

**Enterococci (MPN/100 mL)**  **N/A Post**  **Treatment Intake**  **< 1 < 1**  **< 1 < 1**  **< 1**

**< 100 CFU/ 100 mL**  **Discharge < 1**  **3 < 1**  **2 1** 

**Total Heterotrophic**  **Bacteria (MPN/mL)**  **N/A Post**  **Treatment Intake**  **<2 <2**  **<2 4**  **5**

**N/A Discharge**  **220,000 680,000**  **280,000 423,333**  **463,333**



### 3.3. Physical/Chemical Water Conditions

Total residual oxidant (TRO) and total residual chl orine (TRC) levels in the pre- and posttreatment intake water, filter backwash, and contro l intake water are shown in table 9. As expected, levels of TRC and TRO in intake pre-treat ment and control intake water were below regulatory concern. Also, as expected, in immediat e post-treatment intake water samples the
TRO and TRC were well above the allowable levels fo r discharge into receiving waters. Filter backwash, however, which was affected by low level electrolytic chlorination of the intake stream, was consistently below detection limits of TRC due to low initial levels, and rapid degradation of chlorine by high organic loads.GSI/LB/F/A/1 March 15, 2010 Page 36 of 58

Table 9. Average Total Residual Chlorine (TRC) and Total Residual Oxidant (TRO) Concentration (mg/L, ± std. dev.) from Trials A-E at Intake.

Sample Location Trial A Trial B Trial C Trial D Trial E

TRC TRO TRC TRO TRC TRO TRC TRO TRC TRO

**Control Intake (Tub 1)**  **<0.008 0.031**  **<0.008 0.027**  **<0.008 0.032**  **<0.008 0.027**  **<0.008 0.030** 

**Pre-Treatment Intake (Tub 4)**  **<0.008 0.035**  **<0.008 0.030**  **<0.008 <0.027**  **<0.008 0.033**  **<0.008 0.030** 

**Immediate Post Treatment**  **(Siemens Sample Port)**  **4.28 ±**

**0.21 4.20** 

**± 0.36**  **3.37 ±**

**0.03 3.36 ±** 

**0.07 3.06 ±** 

**0.21 3.08 ±** 

**0.14 3.33 ±** 

**0.28 3.31 ±** 

**0.11 3.64 ±** 

**0.07 3.49 ±** 

**0.07**

**Immediate Post Treatment**  **(GSI Sample Port)**  **5.23 ±**

**1.44 5.08** 

**± 0.75**  **4.19 ±**

**0.34 4.04 ±** 

**0.45 3.67 ±** 

**0.35 4.02 ±** 

**0.72 3.89 ±** 

**0.58 4.04 ±** 

**0.50 4.58 ±** 

**0.52 4.53 ±** 

**0.50**

**Immediate Post Treatment**  **(Tub 6) 1.99**  **2.29 1.66**  **1.75 1.31**  **1.45 1.85**  **1.87 1.96**  **1.84**

**Filter Backwash (Sample Port)**  **<0.008 -**  **<0.008 -**  **<0.008 -**  **<0.008 -**  **<0.008 -** 



### After five days retention in the 200 m 3 

### tanks, treated discharge samples had significantly higher levels than control discharge samples, but TRC leve ls in treated discharge was always 0.070 mg/L or less (table 10).

Table 10. Average Total Residual Chlorine (TRC) an d Total Residual Oxidant (TRO) Concentration (mg/L, std. dev.) from Trials A-E on Discharge.

Sample Location Trial A Trial B Trial C Trial D Trial E

TRC TRO TRC TRO TRC TRO TRC TRO TRC TRO

**Treatment Retention**  **Tank (Day 5, Prior to**  **Discharge) 0.058**  **0.084 0.064**  **0.065 0.055**  **0.062 0.070**  **0.070 0.069**  **0.064**

**Control Discharge**  **(Tub 1) <0.008**  **<0.027 <0.008**  **<0.027 <0.008**  **0.028 <0.008**  **<0.027 <0.008**  **<0.027**

**Treatment Discharge**  **(Tubs 4, 5, and 6)**  **0.063 ±**

**0.003 0.086 ±** 

**0.002 0.069 ±** 

**0.002 0.071 ±** 

**0.001 0.058 ±** 

**0.002 0.065 ±** 

**0.003 0.071 ±** 

**0.001 0.078 ±** 

**0.004 0.064** 

**± 0.006**  **0.083 ±**

**0.008**GSI/LB/F/A/1 March 15, 2010 Page 37 of 58



### 3.4. Residual Toxicity

GSI conducted Cold and Standard WET assays for each trial in the five valid trials series. The results showed fairly consistent effects with respe ct to residual TRC and TRO concentrations
across trials. There was no evidence of acute toxi c effects even in the 100 percent effluent samples. Outcomes of chronic biological toxicity as says varied somewhat across trials and species within each assay in the undiluted effluent samples. However, results consistently showed no toxicity in the more diluted samples.

3.4.1. TRC and TRO Concentrations in WET Tests

Across assays, in 100 percent effluent, TRC concent rations were higher in the Cold WET solutions, than Standard WET solutions. For the P. promelas and C. dubia assays, the mean TRC concentration was 0.032 - 0.040 mg/L in the Standar d WET assays, and 0.088-0.118 mg/L, in the Cold WET assays (table 11). In the S. capricornutum tests, the initial TRC concentrations ranged from 0.025 - 0.044 mg/L in the Standard WET assays, and from 0.097 - 0.134 mg/L in
the Cold WET tests. In all tests TRC concentrations were below the limit of quantification (LOQ) in dilutions of whole effluent (tables 11 and 12).

The range of TRO concentrations also differed betwe en Standard WET and Cold WET solutions.
In 100 % effluent across trials mean TRO concentrat ions for the P. promelas and C. dubia ranged from 0.060 - 0.070 mg/L in Standard WET solu tions, and 0.110 – 0.138 mg/L in Cold WET solutions (table 13). The initial TRO concentr ations across trials for the S. capricornutum bioassays ranged from 0.045 - 0.060 mg/L (table 13) . In Cold WET solutions they were 0.108- 0.127 mg/L.

Table 11. Mean (± Std. Err.) Total Residual Chlori ne (TRC) Concentration (mg/L) of Renewal Water

for Each Trial of P. promelas and C. dubia

WET Bioassays.

Exposure
Standard Whole Effluent Toxicity Bioassay Cold WET

Trial PPW CDW 0% 6.25% 12.5% 25% 50% 100% 100%

**A <0.008**  **(NA) <0.008** 

**(NA) <0.008** 

**(NA) <0.008** 

**(NA) <0.008** 

**(NA) <0.008** 

**(NA) <0.008** 

**(NA) 0.040** 

**(0.001) 0.102** 

**(0.003)**

**B <0.008**  **(NA) <0.008** 

**(NA) <0.008** 

**(NA) <0.008** 

**(NA) <0.008** 

**(NA) <0.008** 

**(NA) <0.008** 

**(NA) 0.032** 

**(0.002) 0.094** 

**(0.005)**

**C <0.008**  **(NA) <0.008** 

**(NA) <0.008** 

**(NA) <0.008** 

**(NA) <0.008** 

**(NA) <0.008** 

**(NA) <0.008** 

**(NA) 0.034** 

**(0.002) 0.088** 

**(0.005)**

**D <0.008**  **(NA) <0.008** 

**(NA) <0.008** 

**(NA) <0.008** 

**(NA) <0.008** 

**(NA) <0.008** 

**(NA) <0.008** 

**(NA) 0.039** 

**(0.002) 0.108** 

**(0.005)**

**E <0.008**  **(NA) <0.008** 

**(NA) <0.008** 

**(NA) <0.008** 

**(NA) <0.008** 

**(NA) <0.008** 

**(NA) <0.008** 

**(NA) 0.039** 

**(0.001) 0.118** 

**(0.006) NA-Not appropriate. If one or more value was less**  **than 0.008 mg/L this made calculating a meaningful a**  **standard error inappropriate. PPW – Fathead minnow reference control. CDW –**  C. dubia **reference control.** GSI/LB/F/A/1 March 15, 2010 Page 38 of 58

Table 12. Total Residual Chlorine (TRC) Concentrat ion (mg/L) of the Initial Exposure Solution for Each Trial of S. capricornutum WET Bioassays.

Exposure

Standard Whole Effluent Toxicity Bioassay Cold
WET

Trial SCW 0% 6.25% 12.50% 25% 50% 100% 100%

**A <0.008**  **<0.008 <0.008**  **<0.008 <0.008**  **<0.008 0.044**  **0.097**

**B <0.008**  **<0.008 <0.008**  **<0.008 <0.008**  **<0.008 0.025**  **0.105**

**C <0.008**  **<0.008 <0.008**  **<0.008 <0.008**  **<0.008 0.037**  **0.101**

**D <0.008**  **<0.008 <0.008**  **<0.008 <0.008**  **<0.008 0.039**  **0.134**

**E <0.008**  **<0.008 <0.008**  **<0.008 <0.008**  **<0.008 0.036**  **0.117 SCW –**  S. capricornutum **(green algae) reference control.**

Table 13. Mean (± Std. Err.) Total Residual Oxida nt (TRO) Concentration (mg/L) of Renewal Water

for Each Trial of P. promelas and C. dubia

WET Bioassays.

Exposure

Standard Whole Effluent Toxicity Bioassay Cold WET

Trial PPW CDW 0% 6.25% 12.5% 25% 50% 100% 100%

**A <0.027**  **(NA) <0.027** 

**(NA) <0.027** 

**(NA) <0.027** 

**(NA) <0.027** 

**(NA) <0.027** 

**(NA) 0.038** 

**(0.003) 0.060** 

**(0.006) 0.118** 

**(0.008)**

**B <0.027**  **(NA) <0.027** 

**(NA) <0.027** 

**(NA) <0.027** 

**(NA) <0.027** 

**(NA) 0.030** 

**(NA) 0.039** 

**(0.002) 0.062** 

**(0.005) 0.110** 

**(0.013)**

**C <0.027**  **(NA) <0.027** 

**(NA) <0.027** 

**(NA) <0.027** 

**(NA) <0.027** 

**(NA) 0.027** 

**(NA) 0.040** 

**(0.002) 0.062** 

**(0.002) 0.109** 

**(0.011)**

**D <0.027**  **(NA) <0.027** 

**(NA) <0.027** 

**(NA) <0.027** 

**(NA) <0.027** 

**(NA) 0.029** 

**(NA) 0.038** 

**(0.002) 0.067** 

**(0.003) 0.121** 

**(0.012)**

**E <0.027**  **(NA) <0.027** 

**(NA) <0.027** 

**(NA) <0.027** 

**(NA) <0.027** 

**(NA) <0.027** 

**(NA) 0.045** 

**(0.002) 0.070** 

**(0.004) 0.138** 

**(0.011) NA-Not appropriate. If one or more value was less**  **than 0.027 mg/L this made calculating a meaningful a standard error inappropriate. PPW – F**  **athead minnow reference control. CDW –** C. dubia **reference control.**GSI/LB/F/A/1 March 15, 2010 Page 39 of 58

Table 1 4 . Total R esidual O xidant (TRO) C oncentration (mg/L) of the I nitial Exposure Solution for Each Trial of S. capricornutum Bioassays.

Exposure

Standard Whole Effluent Toxicity Bioassay Cold
WET

Trial SCW 0% 6.25% 12.5% 25% 50% 100% 100%

**A <0.027**  **<0.027 <0.027**  **<0.027 0.034**  **0.037 0.060**  **0.114**

**B 0.036**  **<0.027 <0.027**  **<0.027 0.029**  **0.032 0.045**  **0.127**

**C 0.036**  **<0.027 <0.027**  **<0.027 0.030**  **0.033 0.060**  **0.124**

**D 0.033**  **<0.027 <0.027**  **<0.027 <0.027**  **0.042 0.057**  **0.123**

**E <0.027**  **<0.027 <0.027**  **<0.027 0.030**  **0.040 0.054**  **0.108 SCW –**  S. capricornutum **(green algae) reference control.**



### 3.4.2. Whole Effluent Toxic Effects

3.4.2.1. Standard WET Results

In the Standard WET tests, no statistically signifi cant acute toxic effect (i.e., reduction in survival) was detected across trials and test speci es (tables 15, 17, and 18). With respect to chronic toxicity in these tests, no effects were de tected at any effluent dilution relative to exposed fish P. promelas (table 15), or any other test species at 50 % effl uent dilution or below. Phytoplankton ( Selenastrum ) assays for two trials (Trials A and E) met data q uality criteria. 3 

### In Trial A, an estimated 77 % effluent dilution appear ed sufficient to reduce algal population growth by 25 %, while in Trial E no reduction in ce ll growth was observed even in 100 % effluent (table 16). Chronic (i.e., reproductive) effects in the zooplankton species C. dubia were observed in 100 % effluent in Standard WET tests in one out of two trials (tables 17 and 19; Trial A; p < 0.05). An estimated 43 % effluent dilution in Tr ial A, would reduce the mean number of young produced per female by 25 % (table 17). No chronic toxicity was observed in the other valid trial (Trial B).

3.4.2.2. Cold WET Results

The Cold WET assays involved only 100 percent efflu ent solutions, and measured TRC and TRO values were significantly higher than in the St andard WET assays. Fathead minnow was unaffected across five trials. Acute toxicity (incr eased mortality) of C

. dubia was measured in one out of two valid trials (table 18; Trial A; p <0.05). There was chronic toxicity (reduced reproduction) detected in C. dubia in the same trial (table 19; Trial A; p <0.05). No such effects

on C

. dubia were detected in the other valid trial (tables 18 and 19; Trial B). In addition, 100 % effluent samples had no chronic toxicity effect on the algal species Selenastrum (data not shown).

3 Starting densities exceeded GSI standard operating procedure (SOP is based on US EPA methods).GSI/LB/F/A/1 March 15, 2010 Page 40 of 58

Table 15. Effective Concentration in 25 % of Test Organisms (EC 25 ), No-Observed-EffectConcentration (NOEC) and Lowest-Observed-Effect-Con centration (LOEC) for Pimephales promelas

Measured in terms of Reduced Survival or Growth of the Test Individuals when Exposed

to Effluent during Standard WET Tests.

Acute Toxicity (%Effluent) Chronic Toxicity (%Effluent)

Trial LC 50 (95% CI) NOEC LOEC EC 25 (95% CI) NOEC LOEC

**A >100**  **100 >100**  **>100 100**  **>100**

**B >100**  **100 >100**  **>100 100**  **>100**

**C >100**  **100 >100**  **>100 100**  **>100**

**D >100**  **100 >100**  **>100 100**  **>100**

**E >100**  **100 >100**  **>100 100**  **>100**

Table 16. Effective Concentration in 25 % of Test Organisms (EC 25 ), No-Observed-EffectConcentration (NOEC) and Lowest-Observed-Effect-Con centration (LOEC) for Selenastrum capricornutum Measured in terms of Reduced Cell Growth when Expos ed to Effluent during Standard WET Tests.

**a Inadequate survival of or too much variation in con**  **trol samples.** Chronic Toxicity (%Effluent)

Trial EC 25 (95% CI) NOEC LOEC

**A 77 (N/A)**  **50 100** 

**B a** 
**- -**  **-**

**C a** 
**- -**  **-**

**D a** 
**- -**  **-**

**E >100**  **100 >100** GSI/LB/F/A/1 March 15, 2010 Page 41 of 58

Table 17. Effective Concentration in 25 % of Test Organisms (EC 25 ), No-Observed-EffectConcentration (NOEC) and Lowest-Observed-Effect-Con centration (LOEC) for Ceriodaphnia dubia Measured in terms of Reduced Survival or the Mean N umber of Young Produced per Female when Exposed to Effluent during Standard WET Tests.

Acute Toxicity (%Effluent) Chronic Toxicity (%Effluent)

Trial LC 50 (95% CI) NOEC LOEC EC 25 (95% CI) NOEC LOEC

**A >100**  **100 >100**  **43 (32-70) 50**  **100**

**B >100**  **100 >100**  **>100 100**  **>100**

**C a** 
**- -**  **- -**  **- -** 

**D c** 
**- -**  **- -**  **- -** 

**E c** 
**- -**  **- -**  **- -** 

**a Inadequate survival in the control treatment group** 

**. c**  **Inadequate reproduction rate per female in control s.** 

Table 18. Average (n=10) Percent Mortality (Standa rd Error) of Ceriodaphnia dubia.

**a Inadequate survival in the control treatment group** 

**. c**  **Inadequate reproduction rate per female in control s.**  *** Statistically significant (** p **<0.05)** Trial Control 6.25 12.5 25 50 100 Cold WET 100%

**A 0 (0)**  **0 (0) 0 (0)**  **0 (0) 10 (6)**  **20 (8) 50 (16)** *

**B 10 (6)**  **10 (6) 10 (6)**  **0 (0) 0 (0)**  **0 (0) 0 (0)** 

**C a** 
**- -**  **- -**  **- -**  **-**

**D c** 
**- -**  **- -**  **- -**  **-**

**E c** 
**- -**  **- -**  **- -**  **-**GSI/LB/F/A/1 March 15, 2010 Page 42 of 58

Table 19. Average (n=10) Number of Young per Fe male (standard error) of Ceriodaphnia dubia

.

**a Inadequate survival in the control treatment group** 

**. c**  **Inadequate reproduction rate per female in control s.**  *** Statistically significant when compared to contro l (**  p **<0.05)**



### 3.4.3. Disinfection By-Products (DBPs)

Levels of all analyzed compounds were below the met hod reporting limits (see table 20 for MRLs) in all control water samples collected during Test Trials A-E. During Test Trials A-E, the only measureable concentrations of DBPs found i n the backwash samples were for several samples containing sodium chlorate ranging in conce ntration from 12 to 13 μ g/L (table 20). Measureable levels of many of the THMs and HAAs wer e found in the treated water samples collected immediately post treatment and from sampl es collected after the five day retention time. Total THM concentrations were higher in the samples collected after the five-day retention time than found in the samples collected immediatel y post treatment in all cases. Total THM concentrations ranged from 101 to 192 μ g/L in post-treatment sample water and 229 to 265 μ g/L after five-day retention (table 20). Total HAAs ra nged from 98 to 180 μ g/L in post-treatment and treated discharge sample water and displayed no definite pattern with time (table 20). Dichloroacetonitrile was the only haloacetonitrile compound out of four that were analyzed that had measureable concentrations in any of the water samples collected. Dichloroacetonitrile was measured only in water samples collected immediatel y post treatment, and concentrations ranged
from 2.9 to 4.1 μ



### g/L (table 20), but this compound was below the met hod reporting limit in treated water after a five-day retention. Sodium c hlorate concentrations in treated water collected immediately post treatment and on dischar ge, ranged from 370 to 450 μ g/L (table 20). Sodium bromate was not detected in any of the sampl es collected during intake or discharge. Trial Control 6.25 12.5 25 50 100 Cold WET 100%

**A 21 (1.5)**  **22 (0.9) 21 (1.2)**  **23 (1.0) 15 (1.5)**  **12 (0.9)*** **5 (1.4)***

**B 21 (1.0)**  **18 (1.7) 17 (1.4)**  **23 (0.8) 24 (0.6)**  **17 (0.8) 20.9 (1.8)** 

**C a** 
**- -**  **- -**  **- -**  **-**

**D c** 
**- -**  **- -**  **- -**  **-**

**E c** 
**- -**  **- -**  **- -**  **-**GSI/LB/F/A/1 March 15, 2010 Page 43 of 58

Table 20. Results from Analysis of Disinfection By-Products M easured in Samples Collected

during Siemens SiCURETM Performance Evaluations. Note: Te method reporting limits (MRLs) for each cl ass of chemicals is the summation of all the individual reporting limits for each chemical, and are as follows: trihalomethanes MRL = 2.0 μ g/L,
haloacetic acids MRL = 11.0 μ g/L, dichloroacetonitrile MRL = 0.5 μ g/L, and sodium chlorate MRL =
10.0 μ g/L.

Parameter Sample Trial A Trial B Trial C Trial D Trial E

**Trihalomethanes (**  μ **g/L) Intake - Filter** 

**Backwash Line** < **2.0** < **2.0** < **2.0** < **2.0** < **2.0**

**Post-Treatment (Tub 6)**  **100.5 123.8**  **121.4 191.7**  **161.6**

**Control Discharge (Tub 1)**  < **2.0** < **2.0** < **2.0** < **2.0** < **2.0**

**Treatment Discharge (Tub 6)**  **229.4 260.8**  **264.7 233.6**  **264.2**

**Haloacetic Acids (**  μ **g/L) Intake - Filter** 

**Backwash Line** < **11.0 b**  < **4.0** < **11.0** < **11.0** < **11.0**

**Post-Treatment (Tub 6)**  **127.2 a**  **Not Measured**  **123.8 158.1**  **156.4**

**Control Discharge (Tub 1)**  < **11.0 a**  **Not Measured**  < **11.0** < **11.0** < **11.0**

**Treatment Discharge (Tub 6)**  **180.4 101.5**  **97.8 117.2**  **104.1**

**Sodium chlorate (**  μ **g/L) Intake - Filter** 

**Backwash Line** < **10.0** < **10.0 13**  **12 13** 

**Post-Treatment (Tub 6)**  **420 380**  **370 450**  **400**

**Control Discharge (Tub 1)**  < **10.0** < **10.0** < **10.0** < **10.0** < **10.0**

**Treatment Discharge (Tub 6)**  **420 380**  **380 440**  **410**

**Dichloroacetonitrile (**  μ **g/L) Intake - Filter** 

**Backwash Line** < **0.5** < **0.5** < **0.5** < **0.5** < **0.5**

**Post-Treatment (Tub 6)**  **4.1 3.7**  **3.2 3.2**  **2.9**

**Control Discharge (Tub 1)**  < **0.5** < **0.5** < **0.5** < **0.5** < **0.5**

**Treatment Discharge (Tub 6)**  < **0.5** < **0.5** < **0.5** < **0.5** < **0.5**

**a Sample bottles broke in transit to the laboratory.** 

**b Zeros (not MRL values) were reported for all haloa**  **cetic acids analyzed, with the exception of**
**tribromoacetic acid for which the MRL of** < **4.0** μ **g/L was reported.**GSI/LB/F/A/1 March 15, 2010 Page 44 of 58



## 4.0. DISCUSSION OF RESULTS

The GSI tests on the SiCURE TM 

### Ballast Water Management System were comprehensive and informative of the treatment system’s potential per formance relative to IMO G8 guidelines in fresh water systems like the Great Lakes. The SiCU RE TM 

### Ballast Water Management System operated without interruption during five consecuti ve valid trials, and biological performance outcomes were remarkably consistent across trials. Treated discharge water from these valid trials contained densities of organisms within the regulated size classes that were below the IMO D-2 standards, as determined using methods document ed in this report. Though data on V.
cholera concentration are still pending, other microbial a nalyses showed system performance in keeping with IMO requirements for bacteria. Chemis try data generated across trials indicated the post-retention discharge to have well less than 0.1 mg/L total residual chlorine under ambient conditions. Ambient water collected immediately aft er treatment and held in a cold environment had TRC and TRO levels which slightly exceeded this level. However, in a real world application, the intake water would also be cold, a nd developers claim that the test system is designed to respond to this circumstance (reflected in oxidation-reduction potential, or ORP) with a reduction in chlorine generated and injected into the intake stream.

Disinfection by-products were detectable in post-tr eatment and post-retention water. Little aquatic chronic toxicity data are available for the DBPs tested. A comparison to United States Federal Drinking Water Maximum Contaminant Levels ( MCLs) show the total THM values in the treated water above the limits for total THMs ( 80 μ g/L) and HAA (60 μ g/L) in all of the treated water samples. However, this comparison is very conservative as it pertains to drinking water not subject to dilution effects.

There were two to five valid replicates of Standard and Cold WET assays for all organisms across trials. There were no statistically signific ant ( p < 0.05) acute toxic effects of treated discharge on any test species across assays and tri als. There were no statistically significant ( p < 0.05) detectable chronic toxicity effects across or ganisms and trials in 50 percent or lower effluent dilutions. Chronic toxicity effects (redu ced reproduction) in 100 percent effluent were detected in Trail A for test species of zooplankton and possibly phytoplankton in both the Standard and Cold WET assay, but not in other trial s or dilution.GSI/LB/F/A/1 March 15, 2010 Page 45 of 58



## 5.0. CONCLUSIONS

The Siemens SiCURE TM 

### Ballast Water Management System functioned properly during five consecutive trials, and was highly effective at red ucing live organism densities in the fresh water ambient conditions of Duluth-Superior Harbor, as am ended in these tests to achieve IMOconsistent challenge conditions. Live organisms in the regulated size classes were discharged in
densities below the IMO D-2 standard. Further data and testing are needed to fully characterize residual toxicity after a five day retention period , but we detected no consistent or pronounced
effect in these trials.



## REFERENCES

GSI (2009). Quality Assurance Project Plan for Grea t Ships Initiative (GSI) Bench-Scale and Land-Based Biological Tests (2009).

Hasan JA, Huq A, Tamplin ML, Siebeling RJ and Colwe ll RR (1994). A Novel Kit for Rapid
Detection of Vibrio cholerae O1. Clinical Microbiology; 32(1): 249-252

International Maritime Organization (IMO), 2004. In ternational Convention for the Control and Management of Ships Ballast Water and Sediments. As adopted by consensus at a Diplomatic Conference at IMO, London, England, February 13 200 4.

U.S. EPA, Office of Water (2002). Short-term Metho ds for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organi sms, 4 th 

### Edition. Accessed from: http://www.epa.gov/waterscience/methods/wet/disk3/i ndex.html

.



## APPENDICES

1. Design of the GSI Land-Based RDTE Facility
2. GSI SOPs Relevant to SiCURE TM 

### Tests
3. Average Density of Live Zooplankton in Treatment Di scharge
4. Data Quality Objectives, Criteria and Results
5. Operational Data SummaryGSI/LB/F/A/1 March 15, 2010 Page 46 of 58



## APPENDIX 1.

GSI LAND-BASED RDTE FACILITY: TECHNICAL INFORMATIO N

The GSI’s Land-Based Research, Development and Tech nology Evaluation (RDTE) Facility in Superior, Wisconsin is used to conduct full-scale b iological evaluations of prospective ballast treatments suitable to Seaway-sized vessels. The fa cility draws raw intake water and entrained organisms from Duluth-Superior Harbor at up to 680 m 3 

### /hr. After initial transport through 16 inch HDPE line to the facility, a carefully designe d “Y-split” in the intake piping simultaneously channels one half of the flow (up to 340 m 3 

### /hr) to a treatment track and one half (up to 340

m 3 

### /hr) to a matched control track (figure 1). Water i n the treatment track passes through the experimental ballast treatment system and into one of the 200 m 3 

### cylindrical treatment retention tanks (test tank #1 or #2; figure 1). Water in the control track by-passes the treatment system and is channeled directly into a matched control retent ion tank (control tank #1 or #2; figure 1). After storage (duration dependent on test requirements), the water is discharged sequentially from the treatment and control retention tanks at up to 340 m 3 

### /hr. Depending on the test scenario, the water is either discharged to the harbor or transfe rred to an alternate retention tank for subsequent transport to a wastewater facility, or t hrough the treatment system again for discharge
or retention.

Treatment and control intake and discharge water is sampled at pressure/flow controlled in-line sample points (SPs). Intake samples are collected c oncurrently on the control and treatment tracks respectively (using SP#2 and SP#3, figure 2) . Post-treatment samples are collected from SP#15 (figure 2). Discharge samples are collected f rom one of two discharge sample points (SP#9, or SP#10; figure 2), with sequential samplin g of control and treatment water. At each of these SPs there are three replicate sample ports wi th a center-located 3.8 cm internal diameter (ID) elbow-shaped pitot tube (figure 3) connected t o a 3.8 cm ID PVC transfer pipe that carries the sample water to one of six 3.8. m 3 

### collection tubs located at a centralized sampling station (figure 2). Injection Ports A and B (figure 1) can be used to meter fine grade Arizona Test Dust, and concentrated algae harvested from the Duluth-Su perior Harbor into the intake stream to assure adequate concentrations of Total Suspended S olids (TSS) and live phytoplankton in intake and control discharge. Other SPs shown in figure 2, with one port per SP, are used for calibration testing the facility itself and not typically used for sample collection during a treatment system
evaluation.

A mobile field laboratory and on-site stationary st ructure provides bench-scale facilities to support time-sensitive assays associated with tests conducted at the GSI Land-Based RDTE Facility. The mobile laboratory is located at the f acility during testing but may be moved to other sites in the Great Lakes-St. Lawrence Seaway System to support GSI shipboard tests when required. Both laboratories are climate-controlled with enough desk and counter space to allow for simultaneous microscopic and analytical analysi s of samples. In addition, laboratories of the University of Wisconsin-Superior’s Lake Superior Re search Institute (LSRI) and the University
of Minnesota-Duluth’s Natural Resources Research In stitute provide non-time sensitive analysis of samples from the land-based tests. Since both fa cilities are only a few miles from the facility, samples can be easily transported for rapid analysi s.GSI/LB/F/A/1 March 15, 2010 Page 47 of 58

Figure 1. Simplified Schematic of the GSI Land-Base d RDTE Facility.GSI/LB/F/A/1 March 15, 2010 Page 48 of 58

Figure 2. Schematic of the GSI Land-Based RDTE Faci lity Showing the Location of the Intake and Discharge Sample Points (i.e., SP #s 2, 3, 9, and 1 0), and Corresponding Sample Collection Tubs.

Figure 3. Schematic of a Sample Point (SP) Showing the Design of the Three Sample Port Pitots.GSI/LB/F/A/1 March 15, 2010 Page 49 of 58



## APPENDIX 2.

GSI SOPs Relevant to Siemens SiCURETM Performance Ev aluation

SOP/Analysis Category SOP ID Code SOP Title Date of Issue

**Health and Safety GSI/SOP/LB/G/S/1**  **Procedure for Ensuring Worker Health and Safety at the GSI Land-Based RDTE Facility**  **June 26, 2009**

**Facility Operation GSI/SOP/LB/G/O/1**  **Procedure for Operating the GSI Land-Based RDTE Facility**  **July 20, 2009**

**Facility Operation GSI/SOP/LB/G/O/3**  **Procedure for Cleaning the Retention Tanks and Othe r**  **Equipment at the GSI Land-Based RDTE Facility July 20, 2009** 

**Facility Operation GSI/SOP/LB/G/O/5**  **Procedure for Injecting Organisms and Solids into t he**  **GSI Land-Based RDTE Facility September 1, 2009** 

**Facility Operation GSI/SOP/LB/G/O/7**  **Procedure for Solids Resuspension After Retention a t**  **the GSI Land-Based RDTE Facility DRAFT** 

**Sample Handling and Custody**  **GSI/SOP/G/RA/SC/3 Procedure for Labeling Samples Collected at the GSI**  **Land-Based RDTE Facility July 15, 2009** 

**Water Chemistry GSI/SOP/BS/RA/C/2**  **Procedure for Determining Total Residual Oxidants (TRO) in Water**  **July 7, 2009**

**Water Chemistry GSI/SOP/BS/RA/C/6**  **Procedure for Analyzing Total Residual Chlorine Concentrations in Water**  **July 15, 2009**

**Water Quality GSI/SOP/BS/RA/C/3**  **Procedures for Measuring Organic Carbon in Aqueous Samples**  **May 28, 2009**

**Water Quality GSI/SOP/BS/RA/C/8**  **Procedure for Analyzing Total Suspended Solids (TSS )**  **June 30, 2009**

**Sample Collection GSI/SOP/LB/RA/SC/8**  **Procedure for Collecting Physical/Chemical Data and Samples at the GSI Land-Based RDTE Facility.**  **DRAFT**

**Sample Collection GSI/SOP/LB/RA/SC/1**  **Procedure for Collecting Biological Sample Water vi a**  **In-Line Sample Ports July 20, 2009** 

**Sample Collection GSI/SOP/LB/RA/SC/6**  **Procedure for Zooplankton Sample Collection July 20, 2009** 

**Zooplankton Sample Analysis**  **GSI/SOP/LB/RA/SA/2 Procedure for Zooplankton Sample Analysis**  **July 20, 2009**GSI/LB/F/A/1 March 15, 2010 Page 50 of 58

**Sample Collection GSI/SOP/LB/RA/SC/3**  **Procedure for Algae/Small Protozoa Sample Collectio n**  **June 26, 2009**

**Phytoplankton Sample Analysis**  **GSI/SOP/LB/RA/SA/1 Procedure for Algae/Small Protozoan Sample Analysis**  **June 26, 2009**

**Sample Collection GSI/SOP/LB/RA/SC/4**  **Procedure for Microbial Sample Collection July 16, 2009** 

**Microbial Sample Analysis**  **GSI/SOP/LB/RA/SA/3 Procedure for the Colony Blot Preparation for**  **Enumeration of Culturable** Vibrio chloreae **DRAFT**

**Microbial Sample Analysis**  **GSI/SOP/BS/RA/MA/1 Procedure For Quantifying Heterotrophic Plate Count**  **s (HPCs) Using IDEXX’s SimPlate® for HPC Method**  **July 17, 2009**

**Microbial Sample Analysis**  **GSI/SOP/BS/RA/MA/3 Procedure for the Detection and Enumeration of**  Enterococcus

**using EnterolertTM July 17, 2009** 

**Microbial Sample Analysis**  **GSI/SOP/BS/RA/MA/4 Procedure for the Detection and Enumeration of Tota**  **l Coliforms and**  E. coli

**Using IDEXX's Colilert® July 17, 2009** 

**Whole Effluent Toxicity Testing**  **GSI/SOP/BS/RA/WET/1 Procedure for Assessing Chronic Residual Toxicity o**  **f a Ballast Treatment System to**  Ceriodaphia dubia **July 16, 2009**

**Whole Effluent Toxicity Testing**  **GSI/SOP/BS/RA/WET/2 Procedure for Assessing Chronic Residual Toxicity o**  **f a Ballast Treatment System to the Fathead Minnow** 

**(** Pimephales promelas **) July 16, 2009** 

**Whole Effluent Toxicity Testing**  **GSI/SOP/BS/RA/WET/3 Procedure for Assessing Chronic Residual Toxicity o**  **f a Ballast Treatment System to the Green Alga**  **(** Selenastrum capricornutum **) DRAFT** 

**Data Management GSI/SOP/G/RA/DM/1**  **Procedure for Data Entry, Data Quality Control and Database Management**  **Available on request (Internal document)** GSI/LB/F/A/1 March 15, 2010 Page 51 of 58



## APPENDIX 3.

Average Density (per m 3 

## ) of Live Zooplankton in Treatment Discharge during

the Trials of the SiCURETM System.

Test Trials: Trial A Trial B Trial C Trial D Trial E

Total Vol. Treatment Discharge Analyzed, m 3 : 3.84 3.96 6.11 5.97 6.05

Greater than 50 μ

m (min. dimension)

Taxa Group Species Avg. Density (per m 3 ) Avg.

Density (per m 3 ) Avg.

Density (per m 3 ) Avg.

Density (per m 3 ) Avg.

Density (per m 3 )
**Arachnida Mite 0.17**

**Cladocerans Bosmina 0.17**  **Daphnia 0.17 Copepods Copepod 0.79 0.17**  **Diptera Chironomid 2.08 4.03 2.79 2.36 0.66**
**Nematoda Nematode 0.33 Ostracoda Ostracod 0.17**  **Dreissena Dreissenid (Zebra Mussel) 0.50 Nauplii Copepod Nauplii 1.56 2.52 1.32 0.34 0.66**  **Tardigrade Tardigrade 0.49**

**Rotifers Bdelloid 0.52 0.33** 
**Keratella 0.33**

Greater than 50 μ

m (min. dimension) Total: 4.95 7.54 4.44 4.04 1.49

Less than 50 μ

m (min. dimension)

Taxa Group Species Avg. Density (per m 3 ) Avg.

Density (per m 3 ) Avg.

Density (per m 3 ) Avg.

Density (per m 3 ) Avg.

Density (per m 3 )

**Rotifers Lecanidae 5.21 12.57 7.52 3.02 0.99**

Less than 50 μ

m (min. dimension) Total: 5.21 12.57 7.52 3.02 0.99GSI/LB/F/A/1 March 15, 2010 Page 52 of 58



## APPENDIX 4. DATA QUALITY OBJECTIVES, CRITERIA

AND RESULTS

Table 1. Water Quality Analyses.

Data Quality
Indicator Evaluation Process/

Performance Measurement Data Quality Objective Performance Measurement Result

Precision Samples (10%) are collected

and analyzed in duplicate – with performance measured by average relative percent difference (RPD) of all duplicate analyses performed during test

trials. <20% average

(± SD) RPD. **Percentage of Water**

**Quality Samples**
**Collected and Analyzed in Duplicate:** 

17% Total Suspended Solids: 4.5%

(±6.7%) RPD , n=6

Dissolved Organic Carbon: 2.4% (±1.8) RPD , n=6

^Particulate Organic Carbon: 111.4% (±100.6%) RPD , n=6

Accuracy Performance is measured by

average percent difference (%D) between all measured and
nominal reference standard
values from test trials. <20% average (± SD) D. **Percentage of Analysis**

**Days Containing a Reference Standard:** 

100% Total Suspended Solids: 4.3% (±2.7%) D , n=5

Dissolved Organic Carbon: 1.0% (±1.0) D , n=5

Representativeness Pre-treatment/control and post-treatment/treatment samples are handled and analyzed in the same manner. Not Applicable – Qualitative. All pre-treatment and post-treatment water quality

intake samples, as well as all control and treatmen t discharge samples, were collected, handled, and analyzed in the same manner (using the appropriate GSI SOPs).

Comparability Routine procedures are conducted according to appropriate SOPs to ensure consistency between tests. Not Applicable – Qualitative. The following GSI SOPs were used for all water qual ity analyses conducted during the test trials: - GSI/SOP/BS/RA/C/3 – Procedures for Measuring Organic Carbon in Aqueous Samples - GSI/SOP/BS/RA/C/8 – Procedure for Analyzing Total

Suspended Solids (TSS)

Completeness Number of valid data obtained from the performance
measurement system vs. number performance measurement samples analyzed. Performance is measured by percent completeness (%C). >90% C. Total Suspended Solids: 10/10 performance
measurements met DQO = 100% C

Dissolved Organic Carbon: 10/10 performance
measurements met DQO = 100% C

^Particulate Organic Carbon: 2/5 performance
measurements met DQO = 40% C

^Water quality parameter having performance measure ment result that did not meet data quality objectiv e specified in GSI QAPP (2009).GSI/LB/F/A/1 March 15, 2010 Page 53 of 58

Table 2. Chemistry Analyses.

Data Quality
Indicator Evaluation Process/

Performance Measurement Data Quality Objective Performance Measurement Result

Precision Samples (10%) are collected

and analyzed in duplicate – with performance measured by average relative percent difference (RPD) of all duplicate analyses performed during test trials. <20% average (± SD) RPD. **Percentage of**

**Chemistry Samples Collected and Analyzed**  **in Duplicate:** 20% Total Residual Chlorine: 4.8% (±5.3%) RPD , n=11

Total Residual Oxidants: 8.2% (±7.0%) RPD , n=11

Bias Performance is measured by

average percent spikerecovery (%SPR) of all analyses performed during
test trials. 75%-110% average (± SD)

SPR. **Percentage of**

**Chemistry Samples Collected, Spiked, and**  **Analyzed:** 18% (TRC)
and 15% (TRO) Total Residual Chlorine: 98.6% (±15.5%) RPD , n=10

Total Residual Oxidants: 95.7% (±3.8%) RPD , n=8

Accuracy Performance is measured by

average percent difference (%D) between all measured
and nominal reference
standard values from test

trials. <20% average (± SD) D. **Percentage of Analysis**

**Days Containing a Reference Standard:** 

100% Total Residual Chlorine: 4.8% (±3.0%) D , n=10

Total Residual Oxidants: 7.2% (±1.1%) D , n=10

Representativeness Pre-treatment/control and post-treatment/treatment samples are handled and analyzed in the same manner. Not Applicable – Qualitative. All pre-treatment and post-treatment water quality intake

samples, as well as all control and treatment disch arge samples, were collected, handled, and analyzed in t he same manner (using the appropriate GSI SOPs).

Comparability Routine procedures are conducted according to appropriate SOPs to ensure consistency between tests. Not Applicable – Qualitative. The following GSI SOPs were used for all water qual ity analyses conducted during the test trials: -GSI/SOP/BS/RA/C/2 – Procedure for Determining Total Residual Oxidants (TRO) in Water -GSI/SOP/BS/RA/C/6 – Procedure for Analyzing Total Residual Chlorine (TRC) Concentrations in

Water

Completeness Number of valid data obtained from the performance
measurement system vs. number performance
measurements collected. Performance is measured by percent completeness (%C). >90% C. Total Residual Chlorine: 29/31 performance measurements
met DQO = 94% C

Total Residual Oxidants: 28/29 performance measurements

met DQO = 97% C

Sensitivity The limit of detection (LOD)

and quantification (LOQ) for the analytical method used is

reported. Dependent upon

the analyte and

instrumentation. Total Residual Chlorine: LOD = 3 μg/L ; LOQ = 8 μg/L

Total Residual Oxidants: LOD = 8 μg/L ; LOQ= 27 μg/LGSI/LB/F/A/1 March 15, 2010 Page 54 of 58

Table 3. Phytoplankton (Organisms <50 μm) Analyses.

Data Quality
Indicator Evaluation Process/

Performance Measurement Data Quality Objective Performance Measurement Result

Bias Samples (10%) are analyzed by

two separate taxonomists – with performance measured by average percent similarity (PS) of taxonomic identification (live entities) and average relative percent difference (RPD) of the
number of live entities counted for all second (QA) analyses performed during test trials. >60% average (± SD) PS and <20% average (± SD) RPD. **Percentage of**

**Phytoplankton Samples Analyzed by a** 
**Second Taxonomist:**

35% 97.5% (± 2.1%) PS , n=5

3.30% (± 3.27) RPD , n=5

Representativeness Pre-treatment/control and post-treatment/treatment samples are handled and analyzed in the same manner. Not Applicable – Qualitative. All pre-treatment and post-treatment phytoplankton

samples collected on intake, as well as all control and treatment discharge samples, were collected, handle d, and analyzed in the same manner (using the appropri ate

GSI SOPs).

Comparability Routine procedures are conducted according to appropriate SOPs to ensure consistency between tests. Not Applicable – Qualitative. The following GSI SOP was used for all phytoplankto n

sample analyses conducted during the test trials: - GSI/SOP/LB/RA/SA/1 – Procedure for Algae/Small

Protozoan Sample Analysis

Completeness Number of valid data obtained from the performance
measurement system vs. number performance measurement samples analyzed. Performance is measured by percent completeness (%C). >90% C. 9/10 performance measurements met DQO = 90% CGSI/LB/F/A/1 March 15, 2010 Page 55 of 58

Table 4. Zooplankton (Organisms >50 **μ** m) Analyses.

Data Quality
Indicator Evaluation Process/

Performance Measurement Data Quality Objective Performance Measurement Result

Bias Samples (10%) are analyzed by

two separate taxonomists – with performance measured by average percent similarity (PS) of taxonomic identification (live organisms only) and average relative percent difference (RPD) of the number of live organisms counted for all second (QA) analyses performed during test trials. >90% average (± SD) PS and <20% average (± SD) RPD. **Percentage of**

**Zooplankton Samples Analyzed by a Second** 

**Taxonomist:** 16% ^Microzooplankton: 80.0% (± 9.4%) PS , n=4 and 42.4% (± 32.2%) RPD, n=4

Macrozooplankton: 97.3% (± 2.5%) PS , n=5 and 4.0% (± 3.9%) RPD , n=5

Representativeness Pre-treatment/control and post-treatment/treatment samples are handled and analyzed in the same manner. Not Applicable – Qualitative. All pre-treatment and post-treatment zooplankton

samples collected on intake, as well as all control and treatment discharge samples, were collected, handle d, and analyzed in the same manner (using the appropri ate

GSI SOPs).

Comparability Routine procedures are conducted according to appropriate SOPs to ensure consistency between tests. Not Applicable – Qualitative. The following GSI SOP was used for all zooplankton

sample analyses conducted during the test trials: - GSI/SOP/LB/RA/SA/2 (DRAFT) – Procedure for Zooplankton Sample Analysis

Completeness Number of valid data obtained from the performance
measurement system vs. number performance measurement samples analyzed. Performance is measured by percent completeness (%C). >90% C. ^Microzooplankton: 2/8 performance measurements
met DQO = 25% C

Macrozooplankton: 9/10 performance measurements

met DQO = 90% C

^Zooplankton analysis parameter having performance measurement result that did not meet data quality objective specified in GSI QAPP (2009).GSI/LB/F/A/1 March 15, 2010 Page 56 of 58

Table 5. Microbial Analyses.

Data Quality
Indicator Evaluation Process/

Performance

Measurement Data

Quality Objective Performance Measurement Result

Precision Samples (10%) are

analyzed in duplicate – with performance measured by average relative percent difference (RPD) of all duplicate analyses performed during
test trials. <20%

average (± SEM) RPD. **Percentage of Microbial**

**Samples Analyzed in Duplicate:**  20% ^E. coli: 23.25% (±9.31%) RPD

, n=12

^Total Coliforms: 36.36% (±14.99) RPD

, n=12

^Enterococcus spp .: 26.96% (±10.27%) RPD

,

n=10

^Heterotrophic: 53.93% (±24.96%) RPD

, n=10

^Vibrio spp .: 31.08% (±8.41%) RPD

, n=16

Bias Samples (10%) are counted

by two separate analysts – with performance measured by average relative percent difference (RPD) for all second counts performed during test trials. <20% average (± SEM) RPD. **Percentage of Microbial**

**Samples Counted by a Second Analyst:**  40% (Total Coliforms); 30% ( **E. coli**

, **Enterococcus**

**spp** ., and Heterotrophic Bacteria); and 10% ( **Vibrio spp.)** E. coli: 0.00% (±0.00%) RPD

, n=24

Total Coliforms: 4.17% (±4.17) RPD

, n=32

Enterococcus spp .: 13.02% (±12.65%) RPD

, n=24

Heterotrophic: 0.84% (±0.58%) RPD

, n=24

^Vibrio spp .: 43.06% RPD

, n=15

Representativeness Pre-treatment/Control and

post-treatment/treatment samples are handled and analyzed in the same

manner. Not

Applicable

– Qualitative. All pre-treatment and post-treatment water quality intake samples, as well

as all control and treatment discharge samples, wer e collected, handled, and analyzed in the same manner (using the appropri ate GSI SOPs).

Comparability Routine procedures are

conducted according to appropriate SOPs to ensure consistency between tests. Not Applicable

– Qualitative. The following GSI SOPs were used for all microbial analyses conducted during the test trials: -GSI/SOP/BS/RA/MA/1 – Procedure for Quantifying Heterotrophic Plate Counts (HPCs) using IDEXX’s SimPlate® for HPC Method -GSI/SOP/BS/RA/MA/3 – Procedure for the Detection and Enumeration of

**Enterococcus** using Enterolert® -GSI/SOP/BS/RA/MA/4 – Procedure for the Detection and Enumeration of

Total Coliforms and **E. coli** using IDEXX’s Colilert® -GSI/SOP/RDTE/SA/M/3 – Procedure for Colony Blot Preparation for the

Enumeration of Culturable **Vibrio cholerae**

Completeness Number of valid data
obtained from the performance measurement system vs. number of performance
measurements collected.
Performance is measured by percent completeness

(%C). >90% C. ^E. coli : 31/36 performance measurements met DQO = 86% C

^ Total Coliforms: 36/44 performance measurements met DQO = 82% C

^Enterococcus spp .: 27/34 performance measurements met DQO = 79% C

^ Heterotrophic: 27/34 performance measurements met DQO = 79% C

^Vibrio spp .: 15/31 performance measurements met DQO = 48% C

Sensitivity The limit of detection (LOD)

for the analytical method used is reported. Dependent upon the analytical technique
used. E. coli LOD: <1 MPN/100 mL

Total Coliforms LOD: <1 MPN/100 mL
Enterococcus spp . LOD: <1 MPN/100 mL
Heterotrophic LOD: <2 MPN/1 mL
Vibrio spp . LOD: 0 CFU/100 mL ^Microbial analysis having performance measurement result that did not meet data quality objective spe cified in GSI QAPP (2009).GSI/LB/F/A/1 March 15, 2010 Page 57 of 58

Table 6. Whole Effluent Toxicity (WET) Test s

.

Data Quality
Indicator Evaluation Process/

Performance

Measurement Data Quality

Objective Performance Measurement Result

Bias Experiment Bias:

Monthly reference

toxicant tests are
conducted on test organisms. Performance is measured by sensitivity of the test organisms relative to
historical values

. LC 50 value
within 2
standard
deviations of
the historical

LC 50 average C. dubia : LC 50 values from reference toxicant tests performed dur ing WET

Testing were within 2 SD of the historical average (501.5 and 498.4 mg/L KCl), n=2.

P. promelas: LC 50 value from reference toxicant test performed 9/200 9 was within 2 SD of the historical average (6.45 g/L NaC l), n=1.

S. capricornutum: No data, reference toxicant test not conducted on this test organism.

Operator Bias: Experimental units (10%) are counted by two separate analysts – with performance measured by average relative percent difference (RPD)
of the number of live test organisms counted for all second analyses. <10% average RPD (±SD) C. dubia: 39% of

experimental units counted by a second analyst C. dubia: 0.1% (± 0.23%) RPD , n=1055

P. promelas: 35% of experimental units counted by a second analyst P. promelas: 0.1% (± 0.1%) RPD , n=442

S. capricornutum: 0% of experimental units counted by a second analyst. S. capricornutum: No data, RPD could not be
determined

Representativeness Control groups

(reference and dilution control) and treatment groups are handled and analyzed in the same

manner. Not

Applicable – Qualitative. All control groups (reference and dilution controls ) and all treatment groups

were set up, handled, and analyzed in the same mann er (using the

appropriate GSI SOPs).

Comparability Routine procedures are

conducted according to appropriate SOPs to ensure consistency
between tests. Not

Applicable – Qualitative. The following GSI SOPs were used for all WET Testin g conducted during the
test trials: -GSI/SOP/BS/RA/WET/1 – Procedure for Assessing Chronic Residual Toxicity of a Ballast Treatment System to **Ceriodaphnia dubia** -GSI/SOP/BS/RA/WET/2 – Procedure for Assessing Chronic Residual Toxicity of a Ballast Treatment System to the Fathe ad Minnow ( **Pimephales promelas**  ) -GSI/SOP/BS/RA/WET/3 (DRAFT) – Procedure for Assessing Chronic Residual Toxicity of a Ballast Treatment System to the Green Alga

( **Selenastrum capricornutum** )

Completeness Number of valid data

obtained from the performance
measurement system vs. number of performance
measurements collected.
Performance is measured by percent completeness (%C). >90% C. Ceriodaphnia dubia: 1075/1075 performance measurements met DQO =

100% C

Pimephales promelas: 443/443 performance measurements met DQO =

100% C

Selenastrum capricornutum: No data, %C could not be determined.GSI/LB/F/A/1 March 15, 2010 Page 58 of 58



## APPENDIX 5. OPERATIONAL DATA SUMMARY

Trial A Trial B Trial C Trial D Trial EMain SystemFill 

### Total Volume Control Retention Tank m^3 192 192 193 193 193

Treatment Retention Tank m^3 193 193 193 193 192

Avg Flow Control Track m^3/hr 199 200 199 199 200

Treatment Track m^3/hr 199 198 199 199 198
Avg System

Pressure Both Tracks kPa 132 130 132 129 130Discharge 

### Total Volume Control Retention Tank m^3 180 181 181 181 182

Treatment Retention Tank m^3 182 179 184 179 182

Avg Flow Control Track m^3/hr 200 196 196 199 199

Treatment Track m^3/hr 200 198 197 199 199

Avg System

Pressure Control Track kPa 135 130 128 125 123

Treatment Track kPa 125 124 128 127 130 Avg Values Calculated from the point of the by

- p ass valve shut until the pump is off.

Trial A Trial B Trial C Trial D Trial ESampling SystemFill 

### Control **Sample Tub 1** 

### m^3 2.09 2.09 2.09 2.08 2.09

**Sample Tub 2** 

### m^3 2.08 2.08 2.08 2.07 2.08

Treatment **Sample Tub 4** 

### m^3 2.11 2.09 2.09 2.08 2.11

**Sample Tub 5** 

### m^3 2.11 2.09 2.09 2.08 2.11

Post

Treatment **Sample Tub 6** 

### m^3 2.09 2.09 2.08 2.07 2.09Drain 

### Control **Sample Tub 1** 

### m^3 3.78 2.07 2.06 2.04 2.05

**Sample Tub 2** 

### m^3 3.77 2.07 2.06 2.03 2.03

**Sample Tub 3** 

### m^3 2.07 2.06 2.03 2.04

Treatment **Sample Tub 4** 

### m^3 3.88 2.02 2.06 2.02 2.04

**Sample Tub 5** 

### m^3 3.87 2.02 2.06 2.01 2.04
**Sample Tub 6** 

### m^3 2.01 2.06 2.01 2.04 Avg Values calculated from the measurable start of flow to the end of measurable flow