GSI/LB/F/A/3 May 13, 2011

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# Final Report of the Land

‐ Based,

Freshwater Testing of the Lye (NaOH) Ballast Water Treatment System

May 13, 2011

Principal Investigator:

Allegra Cangelosi, NEMWI

Research Team:

Lisa Allinger, NRRI, UMD Mary Balcer, PhD, LSRI, UWS Lana Fanberg, LSRI, UWS Steve Hagedorn, LSRI, UWS

Tom Markee, LSRI, UWS

Nicole Mays, NEMWI Christine Polkinghorne, LSRI, UWS Kelsey Prihoda, LSRI, UWS Euan Reavie, PhD, NRRI, UMD Deanna Regan, LSRI, UWS

Donald Reid, Consultant Elaine Ruzycki, NRRI, UMD

Heidi Saillard, LSRI, UWS Tyler Schwerdt, AMI Engineering Heidi Schaefer, LSRI, UWS Matthew TenEyck, LSRI, UWSGSI/LB/F/A/3 May 13, 2011

Page 2 of 54



# Final Report of the Land

‐ Based,

Freshwater Testing of the Lye (NaOH)

Ballast Water Treatment System

May 13, 2011

Allegra Cangelosi Principal Investigator and Director Great Ships Initiative

Northeast-Midwest Institute 50 F St. NW, Suite 950 Washington, DC 20001

Phone: 202-464-4014

Email: acangelo@nemw.org

Approved for Release:

___________________________

PI Signature

May 13, 2011.GSI/LB/F/A/3 May 13, 2011

Page 3 of 54



## Table of Contents

Table of Contents

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

SUMMARY OF TABLES

............................................................................................................................... .... 5

EXECUTIVE SUMMARY

............................................................................................................................... ... 7

ACKNOWLEDGMENTS

............................................................................................................................... .... 8

1.0. INTRODUCTION

............................................................................................................................... .. 9

1.1. The Great Ships Initiative (GSI)

..................................................................................................... 9

1.2. The NaOH (Sodium Hydroxide, Lye) Ballast Water Treatment System

...................................... 10

1.3. Relationship of GSI Testing to the IMO Convention’s G8 and G9 Guidelines, and the USEPA

Environmental Technology Verification Program’s Protocol

..................................................... 10

2.0. METHODS

............................................................................................................................... ......... 12

2.1. Experimental Design and Set

‐ up

................................................................................................. 13

2.1.1. Experimental Infrastructure: The GSI Land

‐ Based Research, Development, Testing, and

Evaluation (RDTE) Facility

................................................................................................... 13

2.1.2. Challenge Conditions and Organism Injection Procedures

.................................................. 20

2.1.3. Sodium Hydroxide (NaOH) and Carbon Dioxide (CO 2 ) Dosing and Operational Parameter 21

2.1.4. Preventing Cross Contamination

......................................................................................... 23

2.2. Water Quality Analysis

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

2.2.1. pH

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

2.2.2. Total Suspended Solids (TSS), Including Mineral Matter (MM)

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

2.2.3. Non

‐ Purgeable Organic Carbon (NPOC) and Dissolved Organic Carbon (DOC), and

Determination of Particulate Organic Carbon (POC) Concentrations

................................ 25

2.2.4. Percent (%) Transmittance

.................................................................................................. 26

2.2.5. Water Quality Measurements using YSI Multiparameter Water Quality Sondes

............... 26

2.3. Viable Organism Analysis

............................................................................................................ 26

2.3.1. Organisms ≥ 50 μm in Minimum Dimension

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

2.3.2. Organisms ≥ 10 and  50 μm in Minimum Dimension

....................................................... 28

2.3.3. Organisms  10  m in Minimum Dimension

...................................................................... 30

2.4. Whole Effluent Toxicity (WET) Testing

....................................................................................... 31

2.5. Data Management

...................................................................................................................... 33GSI/LB/F/A/3 May 13, 2011

Page 4 of 54

2.5.1. Data Recording

......................................................................................................................... 33

2.5.2. Data Processing and Storage

.................................................................................................. 33

3.0. RESULTS........................................................................................................................

................... 34

3.1. Intake and Discharge Challenge Conditions

................................................................................ 34

3.1.1. Operational Conditions

....................................................................................................... 34

3.1.2. Physical, Chemical and Biological Challenge Conditions

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

3.1.3. In

‐ Tank Water Quality

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

3.2. Ballast Water Treatment System Biological Efficacy

.................................................................. 40

3.2.1. Organisms ≥ 50 μm in Minimum Dimension

....................................................................... 40

3.2.3. Organisms  10  m in Minimum Dimension

...................................................................... 44

3.2.4. Percent Reduction of Live Organisms in the Treatment Discharge

..................................... 45

3.3. Whole Effluent Toxicity (WET) Testing

....................................................................................... 46

4.0. QUALITY MANAGEMENT

................................................................................................................. 48

4.1. Quality Management Plan (QMP)

............................................................................................... 48

4.2. Quality Assurance Project Plan (QAPP)

....................................................................................... 48

4.3. Standard Operating Procedures (SOPs )

...................................................................................... 49

5.0. DISCUSSION OF RESULTS

................................................................................................................ 49

6.0. CONCLUSIONS

............................................................................................................................... .. 50

REFERENCES

............................................................................................................................... ................. 51

APPENDIX 1.

............................................................................................................................... ................. 52

List of GSI SOPs Relevant to the Land

‐ Based Evaluation of the NaOH Ballast Water Treatment System

.

............................................................................................................................... ..................... 52

APPENDIX 2

. ............................................................................................................................... ................. 54

Trimmed, Raw Microbial Analysis Data from Testing of Lye (NaOH) Ballast Water Treatment System 54GSI/LB/F/A/3 May 13, 2011

Page 5 of 54



## SUMMARY OF TABLES

Table 1. Comparison of USEPA ETV and IMO G8 Recommended Challenge Conditions to Ranges of

Various Physical, Chemical, and Biological Parameters in Ambient a Water from the Duluth

‐ Superior

Harbor of Lake Superior (June

– October).

................................................................................................. 12

Table 2. Timing of Intake and Discharge Operations during the NaOH Ballast Water Treatment System

Research and Development Trials at the GSI Land

‐ Based RDTE Facility.

................................................... 13

Table 3. Intake and Discharge Sample Points (SPs) and their Corresponding Sample Port Pitots and

Sample Collection Tubs.

.............................................................................................................................. 18

Table 4. Challenge Water Conditions for GSI Test Trials of the NaOH BWTS.

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

Table 5. Standard Operating Procedures Relative to Whole Effluent Toxicity (WET) Testing.

.................. 32

Table 6. Average Operational Parameters Measured During Ballasting Simulation of the Four Trials of

the NaOH Ballast Water Treatment System.

.............................................................................................. 34

Table 7. Operational Parameters Measured During the Deballasting Simulation for the Four Trials of the

NaOH Ballast Water Treatment System.

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

Table 8. Average (± Std. Dev.) Challenge Water Quality during Four Trials of the

.................................... 36

Table 9. Live Plankton Densities in Intake and Control Discharge During the Four NaOH Trials. Note: The

Live Intake Densities are an Average of the Control Intake and the Pre

‐ treatment Intake Samples.

........ 37

Table 10. Viable Microbial Densities (Average ± Standard Error of the Mean) in Intake and Control

Discharge During the Four NaOH Trials.

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

Table 11. Control and Treatment Retention Tank Water Quality (Average ± Std. Dev.) During the Three

‐ day Holding Period for Trials C and D of the NaOH BWTS Test.

................................................................. 39

Table 12. Live Treatment Discharge Densities and Treatment Discharge Sample Volume Analyzed Within

the ≥ 50  m Size Class During Four Trials of the NaOH Ballast Water Treatment System.

........................ 41

Table 13. Live Zooplankton Densities Across Taxa in Treatment Discharge from Four Trials of the NaOH

Ballast Water Treatment System.

............................................................................................................... 42

Table 14. Result of Statistical Comparison of Live Zooplankton Density in Control Discharge to

Treatment Discharge......................................................................................................................

............. 43

Table 15. Live Treatment Discharge Density and Treatment Discharge Sample Volume Analyzed Within

the ≥ 10  m and < 50  m Size Class during Four Trials of the NaOH Ballast Water Treatment System.

.... 43

Table 16. Result of Statistical Comparison of Live Phytoplankton Density in Control Discharge to

Treatment Discharge......................................................................................................................

............. 43

Table 17. Live Density (Average ± Standard Error of the Mean) of Regulated Microbes in the Treatment

Discharge from the Four Trials of the NaOH Ballast Water Treatment System.

........................................ 44

Table 18. Results of Statistical Comparisons of Live Microbe Density in Control Discharge and Treatment

Discharge in the <10

‐  m Size Class.

............................................................................................................ 45

Table 19. Percent Reduction of Live Organism Density in Treatment Discharge as Compared to Control

Discharge. Trial A had a Retention Time of Two Days; Trials B ‐ D had a Retention Time of Three Days. 46

Table 20. Average (± Standard Error of the Mean) Survival and Reproduction of C. dubia Exposed to

Whole Effluent Collected from NaOH Treatment Discharge During Trial D. Note: Statistical comparisons

were made within one trial only, i.e., no comparisons were made between trials.

.................................. 47GSI/LB/F/A/3 May 13, 2011

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Table 21. Average (± Standard Error of the Mean) Final Density of S. capricornutum Exposed to Whole

Effluent Collected from NaOH Treatment Discharge During Trial D.

......................................................... 47

Table 22. Average (± Standard Error of the Mean) P. promelas Survival and Dry Weight per Surviving

Minnow Exposed to Whole Effluent Collected from NaOH Treatment Discharge During Trials B and D.

. 48GSI/LB/F/A/3 May 13, 2011

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## EXECUTIVE SUMMARY

The Great Ships Initiative (GS I) provides independent, no-cost pe rformance verification testing services to developers of ballast water treatm ent systems (BWTSs) and processes at a purposebuilt, land-based ballast treatment test facility located in the Duluth-Superior Harbor of Lake Superior (Superior, WI). The GS I is capable of performing testing fully consistent with the requirements of the International Maritime Orga nization’s (IMO’s) International Convention for the Control and Management of Ships Ballast Water and Sediments (IMO, 2004) and the United States Environmental Protection Agency’s (U SEPA’s) Environmental Technology Verification Program (ETV; NSF Internationa l, 2010). GSI procedures, methods , materials and findings are also publicly accessible on the GSI website ( www.greatshipsinitiative.org ).

In July 2010, GSI conducted a land-based perfor mance evaluation test of a proposed BWTS developed by researchers from the U.S. Geol ogical Survey’s Leetown Science Center in Kearneysville, West Virginia. The proposed system involved a pplication of sodium hydroxide (NaOH, in the same formulation used for lye or caustic soda) to ballast water to raise pH, followed by application of carbon dioxide (CO 2 

### ) as a neutralization step prior to discharge of the ballast water to the receiving sy stem. The purpose of the land- based test of this system, consisting of four trials, was st atus testing for research and development. As such, the testing was based on, though not strictly consistent wit h, the IMO’s G8 Guidelines for Approval of Ballast Water Management Systems (IMO, 2008a), the IMO’s G9 Guidelines for Approval of Ballast Water Management Systems that make use of Active Substances (IMO, 2008b), and the USEPA’s ETV Program Generic Protocol for the Verification of Ballast Water Treatment Technology , v.5.1 (NSF International, 2010).

During the test, the NaOH BWTS was evaluated for its ability to: (a) successfully treat ballast water without interruption, (b) succ essfully neutralize tr eated ballast water to achieve Wisconsin Department of Natural Resources (WIDNR) permitting levels for har bor discharge (i.e., pH 6-9), (c) meet discharge target values for water chem istry/quality and biology that are approximately

consistent with the IMO Convention’s Annex D-2 discharge standards, and (d) discharge water after two- or three-day retention periods that is environmen tally benign (i.e., no residual toxicity) pursuant to USEPA water quality criteria.

The NaOH BWTS performed very well operationa lly and well enough biol ogically to warrant additional testing at the bench, land and ship-based scales. The system successfully treated ballast water without interrupti on, and successfully neutralized tr eated ballast water to achieve WIDNR permitting levels for harbor discharge (i.e., pH 6-9). The BWTS also significantly reduced live organism densities in treated discharge relative to control discharge in all size classes of organisms. Finally, in these tests, the BWTS performance met discharge target values that were approximately consistent with the IMO Convention’s Annex D- 2 discharge standards, though precision in this estimate was not possibl e given the research and development testing parameters. The only possible proble m that this testing revealed was that the water discharged after two- or three-day retenti on periods was not entirely environmentally benign (i.e., with no residual toxicity at the 100 % effluent dilution), though the leve l of residual toxicity in 100 %

effluent evident from these tests ma y not be of regulatory concern.GSI/LB/F/A/3 May 13, 2011

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## ACKNOWLEDGMENTS

The authors would like to express our sincere gr atitude to the Great Ships Initiative (GSI) Advisory Committee which provides invaluable i nput and encouragement to the GSI project.

We also wish to thank the ten United States and Canadian Great Lakes Ports which launched the GSI, and the Great Lakes Protection Fund whic h supported the initial scoping exercise. We sincerely thank the United States Department of Transportation, Mariti me Administration, and National Oceanic and Atmospheric Administration for their substantial financial and in-kind support for the construction of the state-of-the-art GSI land-based testing facility. We thank the United States and Canadian St. Lawrence Seaway organizations, the Legislative Citizens Commission on Minnesota Resources, the Universi ty of Wisconsin-Superior, and the City of Superior, WI, for their active financial a nd/or in-kind support for GSI operations.GSI/LB/F/A/3 May 13, 2011

Page 9 of 54



## 1.0. INTRODUCTION

In July 2010, the Great Ships Initiative (GSI) conducted a land- based evaluation of a proposed ballast water treatment system (BWTS) devel oped by researchers from the U.S. Geological Survey’s Leetown Science Center (Kearneysvi lle, WV). The BWTS involved application of sodium hydroxide (NaOH, in the same formulation used for lye or caustic soda) to ballast water on intake to increase the pH, followe d by application of carbon dioxide (CO 2 

### ) as a neutralization step prior to discharge of the ballast water to th e receiving system. The purpose of the evaluation was status testing for research and developm ent of the proposed NaOH BWTS. The objectives of the evaluation were to determine the ability of the NaOH BWTS to: (a) successfully treat ballast water without interruption, (b) successfu lly neutralize treated ballast water to achieve Wisconsin Department of Natural Resources (WIDNR) permitting levels for harbor discharge (i.e., pH 6-9), (c) meet discharg e target values for water chemistry/quality and biology that are approximately consistent with the IMO Conven tion’s Annex D-2 discharge standards, and (d) discharge water after two- or three-day retentio n periods that was environmentally benign (i.e., no residual toxicity) pursuant to USEPA water qua lity criteria. The te sting was based on, though not strictly consistent with, the IMO’s G8 Guid elines for Approval of Ballast Water Management Systems (IMO, 2008a), the IMO’s G9 Guidelines for Approval of Ballast Water Management Systems that make use of Active Subs tances (IMO, 2008b), and the USEPA ETV Generic Protocol for the Veri fication of Ballast Water Treatment Technology , v.5.1 (NSF International, 2010).



### 1.1. The Great Ships Initiative (GSI)

Great Ships Initiative (GSI) is a regional effort devoted to ending the problem of ship-mediated invasive species in the Great Lakes-St. Lawrence Seaway System and globall y. In support of that goal, GSI has established superla tive freshwater ballast treatment evaluation capabilities at three scales—bench, land-based, and on board ship.

GSI awards independent status-testing services at no-cost to developers of BWTSs and processes determined to be promising. GSI status-testing is performed at the scale appropriate to the state of development of the target treatment system, w ith the goal of facilitating the rapid progression of meritorious BWTSs through the research a nd 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 that GSI tests are uncompromised by any real or perceived individual or team bias relative to te st outcomes, GSI test activities are subject to rigorous quality assurance and quality control (QAQC) proce dures and documentation (GSI, 2010a; GSI, 2010b). This attention to QAQC also assures high quality and credible evaluation of findings.

GSI has worked to standardize and calibrate its protocols to evaluate th e performance of BWTSs with IMO guidelines, USEPA ETV Protocol, and othe r test facilities. GSI test protocols are as consistent as possible with the requirement s of the IMO Convention for the Control andGSI/LB/F/A/3 May 13, 2011 Page 10 of 54



### Management of Ships’ Ballast Water and Se diments (IMO, 2004) and United States federal requirements (NSF International, 2010). In part icular, GSI testing directly supports the IMO’s G8 Guidelines for Approval of Ballast Water Management Systems (IMO, 2008a), the IMO’s G9 Guidelines for Approval of Ballast Water Management Systems that make use of Active Substances (IMO, 2008b), and the USEPA ETV Program’s Generic Protocol fo r the Verification of Ballast Water Treatment Technology , v.5.1 (NSF International, 2010). GSI procedures, methods, materials and findings ar e also not proprietary, and are publicly accessible on the GSI’s public website: www.greatshipsinitiative.org

.



### 1.2. The NaOH (Sodium Hydroxide, Lye) Ballast Water Treatment System

Researchers from the U.S. Geological Survey’s L eetown Science Center in Kearneysville, West Virginia developed the proposed system using sodium hydroxide (NaOH), in the formulation used for lye or caustic soda, for routine us e as a BWTS. In 2008, GSI conducted bench-scale testing on the proposed NaOH BWTS and determin ed that pH levels of 11.5, 12.0, and 12.5 were effective at killing the broad range of aquatic organisms tested; especially adult rotifers ( Brachionus calyciflorus ), the cladoceran Daphnia magna , and Eucyclops copepods (GSI, 2009). These bench-scale findings were encouraging and land-based testing of a scaled-up model of the NaOH BWTS was proposed and awarded by GSI. Land-based tests utili zed a version of the system that first increases the pH of ballast wate r on intake to pH 12, and then reduces the pH of the discharge water to le ss than 8.5 (but above 6.5) using carbon dioxide (CO 2 

### ). During retention, the ballast water remains at pH 12 and the pH is lowered just prior to discharge by recirculation between a Speece Cone-type carbonator and the ballast tank.



### 1.3. Relationship of GSI Testing to the IMO Convention’s G8 and G9 Guidelines, and the USEPA Environmental Technology Verification Program’s Protocol

The fundamental approach of GSI is to conduc t independent, scientifically-sound, rigorous, and quality assured evaluations of BWTSs. At the same time, GS I seeks immediate relevance of its freshwater, land-based testing to regulatory processes such as those outlined in the IMO

Convention and those under develo pment domestically in the United States and Canada. To that end, GSI protocols are rooted in the essential fe atures of the IMO’s G8 Guidelines for Approval of Ballast Water Management Systems (IMO, 2008a ), the IMO’s G9 Guidelines for Approval of Ballast Water Management Systems that make use of Active Substances (IMO, 2008b), and the USEPA ETV Program’s Generic Protocol for the Verifica tion of Ballast Water Treatment Technology, v.5.1 (NSF International, 2010). All aspects of the GSI land-based facility testing infrastructure (e.g. flow rate, re tention tank size, sample size, sample collection and analysis equipment and data logging) are directly consiste nt with these requirements. GSI also formally partners with the Maritime Environmental Res ource Center (MERC; Solo mons, MD), and other land-based test facilities, to ensure that GSI fr eshwater, land-based testin g can be complemented by comparable brackish/s alt water testing.GSI/LB/F/A/3 May 13, 2011 Page 11 of 54



### With respect to physical/chemical and biological charac teristics of the intake stream, GSI is fortunate in that its feed water source (i.e., the Duluth-Superior Harbor of Lake Superior) naturally meets many of the IMO G8 and the USEPA ETV requirements for intake organism densities and physical/chemical conditions during th e testing season (June to October, see Table 1). For those parameters that often do not naturally meet the IMO G8 and USEPA ETV requirements (e.g., total suspended solids, mine ral matter, particulate organic carbon, and phytoplankton), GSI has the ability to augment in take water to achieve recommended IMO/ETV parameter levels (Table 1). Other parameters may occasionally fall below the challenge water requirements (i.e., zooplankton and heterotrophic bacteria, see Table 1), but GSI conducts IMOand USEPA ETV-consistent tests only when they are sufficiently high. Though IMO and ETV protocols do not provide for them, GSI and the treatment system developer may also make a determination to set upper limits on certain water quality parameters, such as DOC concentrations, such that tests will be not be run when concentrations are exceedingly high, and these upper limits are reported in the test re port. GSI conducts and documents frequent monitoring of water chemistry a nd biology to predict valid run conditions for GSI, IMO G8, and USEPA ETV performance evaluati on/certification test trials.GSI/LB/F/A/3 May 13, 2011 Page 12 of 54

Table 1. Comparison of USEPA ETV and IMO G8 Recommended Challeng e Conditions to Ranges of Various Physical, Chemical, and Biological Parameters in Ambient a Water from the DuluthSuperior Harbor of Lake Superior (June – October).

Parameter US EPA ETV 1 Recommended

IMO G8 2 Duluth/Superior Harbor Ambient Ranges a

Temperature (°C) 4 – 35 No Requirement 4 - 30

Salinity (ppt) < 1 Two salinities, >10 ppt difference 0 – 1

Total Suspended Solids (mg/L) Min. 24 > 50 < 1 – 40

Mineral Matter (mg/L) Min. 20 No Requirement <1- 40

Particulate Organic Carbon (mg/L) Min. 4 > 5 < 0.1 – 3

Dissolved Organic Carbon (mg/L) Min. 6 > 5 6 – 30

Transmittance at 254 nm (%) b No Requirement No Requirement 14.0 – 68.5

Zooplankton ( ≥ 50  m/m 3 ) Min. 100,000 > 100,000 100,000 - 1,100,000

Phytoplankton ( ≥ 10 and  50  m/mL) Min. 1000 > 1,000 25 – 4,500

Heterotrophic Bacteria (MPN c /mL) Min. 1000 > 10,000 100 - 10,000

a Duluth-Superior Harbor ambient ranges were obtained from GSI monitoring data and records from June to October

2007 to 2010

b Measured on filtered Duluth-Superior Harbor water samples (May 2009 to October 2010)

c MPN = Most Probable Number



## 2.0. METHODS

Four NaOH BWTS trials took place at the GSI la nd-based test facility from July 6, 2010 to July 22, 2010. The experimental methods including pr ocedures for sampling and analysis of each physical, chemical and biological parameter and vari able are described below. All SOPs relevant to the NaOH BWTS tests are listed by analysis category in Appendix 1. Additional details on GSI’s standard operating procedures (SOPs) can be found at www.greatshipsinitiative.org

.

1 US Environmental Protection Agency, Environmental Technology Ve rification Program. Generic Prot ocol for the Verification of Ballast Water Treatment Technologies. Version 5.1. September, 2010. 2 IMO MEPC 57, Annex 3: Revised Guide lines for Approval of Ballast Water Management Systems (G8). April 4, 2008.GSI/LB/F/A/3 May 13, 2011 Page 13 of 54



### 2.1. Experimental Design and Set

‐ up

The NaOH BWTS test involved physical, chemi cal, and biological characterization of water samples upon uptake/intake of wate r, as well as, enumeration, sizi ng, and live/dead analysis of organisms in control and treated discharge water af ter a two- or three-day, in-tank holding time. The objective of the performance evaluation tria ls was to compare control (untreated) and treatment discharge in order to estimate the ef fects of the NaOH BWTS for its ability to: (a) successfully treat ballast water without interrup tion, (b) successfully neut ralize treated ballast water to achieve WIDNR permitting levels for harbor discharge (i.e., pH 6-9), (c) meet discharge target values for water chemistry/quality and bi ology that are approximatel y consistent with the IMO Convention’s Annex D-2 discharge standards, and (d) discharge water after two- or threeday retention periods th at is environmentally benign (i.e., no residual toxicity) pursuant to USEPA water quality criteria.

Table 2 shows the schedule of th e four trials, including the sequence of intake operations (simultaneous control and treatment) and discha rge operations (sequential, treatment then control).

Table 2. Timing of Intake and Discharge Operations during the NaOH Ballast Water Treatment System Research and Development Trials at the GSI Land-Based RDTE Facility.

Trial Treatment Timing of Operation

Intake Discharge

A Treatment 06 July 2010 11:08 – 12:08 8 July 2010 10:47-11:40

Control 13:01-13:55

B Treatment 09 July 2010 9:02 – 10:01 12 July 2010 10:53 – 11:45

Control 13:18 – 14:12

C Treatment 13 July 2010 9:02 – 10:02 16 July 2010 10:03 – 10:57

Control 12:51 – 13:45

D Treatment 19 July 2010 9:41 – 10:41 22 July 2010 10:12 – 11:07

Control 12:33 – 13:27

2.1.1. Experimental Infrastructure: The GSI Land

‐ Based Research, Development, Testing, and Evaluation (RDTE) Facility



### The test reported here evaluated the performa nce of the NaOH BWTS at GSI’s purpose-built, Land-Based Research, Developmen t, Testing and Evaluation (RDTE) Ballast Treatment Test Facility located in Superior, WI in the Duluth-Supe rior Harbor of Lake S uperior (Figures 1-3). Key features of the facility include:

 Four x 200 m 3 

### matched retention tanks with internal agitation for experimental water;GSI/LB/F/A/3 May 13, 2011 Page 14 of 54



###  Matched control and treatmen t intake flows up to 341 m 3 

### /hour;

 Highly automated flow and pressure control, monito ring and data logging;

 A freshwater estuary with plentiful a quatic life as a water intake source;

 Capacity to amend intake water to intensify challenge conditions;

 Semi-automated and validated faci lity sanitation between trials;

 High quality in-line or in-ta nk sampling and/or spiking;

 On-site laboratory space for live analysis of organisms in the ≥ 10 μ m and  50 μ m and ≥ 50 μ m size classes;

 Capacity to test treatment systems that operate on intake, discharge, in-tank, or combinations thereof;

 Off-site whole effluent toxicity (WET) testing; and

 Easy plug-in connections for treatment systems.

Figure 1. Location of GSI's Land-Based RDTE Facility in Superior, Wisconsin. Facility LocationGSI/LB/F/A/3 May 13, 2011 Page 15 of 54

Figure 2. Computer-Generat ed Rendering of the GSI Land-Based RDTE Facility.

Figure 3. Photo of the GSI Land-Based RDTE Facility.GSI/LB/F/A/3 May 13, 2011 Page 16 of 54



### GSI’s Land-Based RDTE Facility draws raw intake water from the Duluth-Superior Harbor at

400 m 3 

### /hr to 680 m 3 

### /hr. This main flow of intake wate r can be augmented with solids and/or organisms just prior to being split into control a nd treatment tracks (see injection points A and B; Figure 4).

A Y-split in the intake piping, just after a static mixer, simultaneously channels one half of the well-mixed 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 (Figure 4). The treatment track directs water through the experimental BWTS and into a 200 m 3 

### , cylindrical retent ion tank (Figure 4). The control track by-passes the treatment system and channels water directly into a matched control retention tank (Figure 4).

After a retention period, water is discharged sequentially from the treatment and control retention

tanks at 200 m 3 

### /hr to 340 m 3 

### /hr. The water is directed either back to the Duluth-Superior harbor,

to a 260-m 3 

### wastewater storage tank for subsequent di scharge to the City of Superior sewer, neutralization, or circulated to a second set of facility retention tanks (Figure 4).GSI/LB/F/A/3May 13, 2011Page 17 of 54Figure 4. Simplified Schematic of the GSI Land-Based RDTE Facility Showing Location of Sample Points, Sample Collection Tubs,Injection Points,Retention Tanks, and Treatment and Control Tracks. Note: Main intake and discharge lines are coded black.GSI/LB/F/A/3 May 13, 2011 Page 18 of 54



### Water is sampled continuously throughout ballas ting functions (i.e., intake, recirculation or discharge) through in-line sample points (SPs). Intake sampling takes place at paired intake sample points (SP#2 and SP#3) on the control and treatment tracks, respectively, and immediate post-treatment sampling occurs at SP#15 (Figure 4) . Typically, discharge biological sampling is conducted at SP#9, with samples for water qualit y analysis collected at SP#10 (Figure 4). All

these SPs consist of three identical sample ports spaced at regular in tervals in a length of straight pipe consistent with IMO guidelines, with the exception of SP#15, which has only one sample port. Each port is fitted with a cen ter-located, elbow-shaped pitot tube (90 o 

### ) which samples the water (Figure 5). This pitot design is based on one developed and validated analytically by the U.S. Naval Research Laboratory in Key West, Fl orida. The performance of the three identical sample ports at SP #2, 3, 9 and 10 was also validated empirically at GSI, and shown to produce equivalent, representative and unbi ased samples of water flow.

Figure 5. Simplified Schematic of a Sample Point (SP), Showing the Three Sample Ports.



### Sample water drawn by sample ports is transf erred simultaneously and continuously throughout ballasting operations (intake, reci rculation or discharge) from the sample ports to replicate 3.8 m 3


### sample collection tubs via clean 3.8 cm (internal diameter) flexible hoses and automated flowcontrolled pneumatic diaphragm valves. The samp le collection tubs, pictured in Figure 4, connect to the sample ports in the arrangement detailed Table 3. Though the same tubs serve as collection mechanisms for sample flow from more th an one pitot, only one such pitot is used at a time during any given sample collection event. Th e naming convention for an individual pitot is: “SP number” plus “sample port letter”. Sample collection tubs are labeled numerically 1-6.

Table 3. Intake and Discharge Sample Points (SPs) and their Corresponding Sample Port Pitots and Sample Collection Tubs.

INTAKE DISCHARGE

SP#2 SP#3 SP#15 SP#9 SP#10

Sample

Port Pitot a b c a b c a a b c a b c

Sample

Collection

Tub 1 2 3 4 5 6 6 3 & 6 2 & 5 1 & 4 3 & 6 2 & 5 1 & 4GSI/LB/F/A/3 May 13, 2011 Page 19 of 54



### An on-site mobile field laborator y (Figure 6) and stationary labor atory (Figure 7) provide space to support time-sensitive analyses associated with the GSI land-based tests, including live analysis of phytoplankton and zooplankton. The la boratories are climate- controlled, and have enough bench space to allow for simultaneous an alysis of samples by multiple personnel. All other analyses are conducted in laboratories of the Lake Superior Research Institute (LSRI) of the University of Wisconsin-Superior; appr oximately three miles from the facility.

Figure 6. The GSI Mobile Field Laboratory.

Figure 7. The GSI Stationary Laboratory.GSI/LB/F/A/3 May 13, 2011 Page 20 of 54

2.1.2. Challenge Conditions and Organism Injection Procedures



### The expected ranges of physical, chemical a nd biological challenge conditions for the NaOH BWTS test trials appear in Table 4. Ambien t Duluth-Superior Harbor water conditions were employed as the physical and chemical challenge conditions during all f our trials. Biological challenge conditions were ambient during Trials A- C. During Trial D, organism densities in the smaller of the two plankton size classes (i.e., ≥ 10 and  50 μ m) were augmented to achieve greater than 1000 cells/mL on intake and th ereby intensify challenge conditions. The phytoplankton injection pro cedure is detailed in GSI/SOP/LB/G/O/5

– Procedure for Injecting Organisms and Solids into the GSI Land-Based RDTE Facility . One to two days prior to the test trial, phytoplankton from the Duluth-Superior Har bor was collected and concentrated using 50-

to 80 μ m plankton nets towed from an outboard-power ed boat. The concentrated phytoplankton

was stored at the GSI Land-Ba sed RDTE Facility in holdi ng ponds equipped with aeration systems for less than 48 hours. Prior to inj ection, holding pond water c ontaining concentrated phytoplankton was mixed, sampled, and analyzed for live cell density. In addition, a sample of Duluth-Superior Harbor water was collected to determine the ambient live cell density. Based on the density of cells in the holding ponds and ambient intake water, the volume of phytoplankton concentrate that was needed to achie ve the desired density in intake water was calculated. This volume was added to the Organism Pressure Injection System (OPIS) vessel. The OPIS vessel was pressurized to 25 psi great er than the target system pressure. The phytoplankton concentrate was added at a constant rate to the intake wa ter via the pressure

differential for the entire duration of the intake procedure via Injection Point B (Figure 4). A static mixer installed in the main intake line ju st downstream of the two injection systems (SIS and OPIS) and prior to the main system “Y split ” (Figure 4) ensured that the concentration of added phytoplankton was equivalent in the control and treatment tracks of the facility. Gentle agitators installed in the contro l and treatment retention tanks ensured that live organisms, especially less motile organisms that may settle to the bottom of the tank during the retention period, were accounted for to the greatest extent possible in the discharge water analysis (see

GSI/SOP/LB/G/O/7 – Procedur e for Maintaining Solids Susp ension in the GSI Land-Based RDTE Facility’s Retention Tanks ).

.GSI/LB/F/A/3 May 13, 2011 Page 21 of 54

Table 4. Challenge Water Conditions for GSI Test Trials of the NaOH BWTS.

Parameter Expected Ranges for GSI NaOH BWTS Challenge

Water

Temperature (°C) Ambient (4 – 30*)

Salinity (ppt) Ambient (0 – 1*)

Total Suspended Solids, TSS (mg/L) Ambient ( ≥ 1 – 40*)

Particulate Organic Carbon, POC (mg/L) Ambient (< 0.1 – 3*)

Dissolved Organic Carbon, DOC (mg/L) Ambient (6 – 30*)

Mineral Matter, MM (mg/L) Ambient ( ≥ 1 – 40*)

Zooplankton ( ≥ 50  m/m 3 ) Ambient (100,000 – 1,100,000*)

Phytoplankton ( ≥ 10 and  50  m/mL) Trial A-C - Ambient (> 25*) Trial D – Concentrated Ambient( ≥ 1,000)

Heterotrophic Bacteria (MPN/mL) Ambient (100 – 10,000 MPN/mL*)

* Duluth-Superior Harbor ambient ranges were obt ained from GSI monitoring data and records from

June to October 2007 to 2010.

2.1.3. Sodium Hydroxide (NaOH) and Carbon Dioxide (CO 2 ) Dosing and Operational

Parameters



### A 50 % by weight sodium hydroxide (NaOH) so lution with a specific gravity of 1.53 was

metered into the intake water in the treatment track (Figure 4) to achieve a pH of 12. The pH of

the treatment water stream was monitored ever y ten seconds during the entire intake operation (i.e., before, during, and after Na OH injection) using an inline Signet pH sensor with built-in automatic temperature compensation (Georg Fi scher Signet LLC; El Monte, CA) that was located downstream of the dosing equipment. The inline pH sensor was calibrated prior to each trial’s intake operation according to the manufacturer’s instructions , using a two-point calibration with pH 7 and 10 buffers. The NaOH dosing pr ocedure began by partially closing the NaOH flow control valve (valve #9, Fi gure 8) in the main line of Treatment Lab #2 and opening the valves leading to the dosing pum ps (Figure 8). This valve c onfiguration created a pressure differential upon flow commencement that primed the two dosing pumps located in a side stream to the main line. Once the pressure diffe rential reached 8-10 pounds per square inch, both centrifugal dosing pumps were star ted to provide a combined flow of 30-40 gallons per minuteGSI/LB/F/A/3 May 13, 2011 Page 22 of 54



### (GPM). The supply valve from the NaOH tank was then opened and a flow of approximately 0.9

GPM was added into the 30-40 GPM from the dosing pumps by us ing a Venturi injector. A

technician monitored the flow rate of NaOH and maintained it at 0.9 GPM during the dosing procedure. The dosing proceeded until 450 pounds (or approximately 35.2 gallons) of the 50 % by weight NaOH solution had been injected as measured from a Salter/Brecknell model SBI100 electronic scale, then the valve leading from the NaOH tank was closed and a bypass line was opened to flush the injection lines with Duluth-S uperior Harbor water. The flushing of the NaOH dosing system continued for the remainder of the intake operation.

Figure 8. Photo of the NaOH Dosing Procedure Setup in Treatment Lab #2 of the GSI Land-Based Facility’s Stationary Laboratory.



### Following a two- or three-day retention time, the pH of the treated water was neutralized with carbon dioxide (CO 2 

### ) gas. An inline pH meter and Walchem pH sensor (Walchem; Holliston, MA) with built-in automatic temperature compen sation were located on the inlet pipe of a Speece Cone; the pH probe was calibrated using fresh pH 7 and 10 buffers as per the calibration procedure outlined in the operator’s manual. Water was removed from the treatment retention tank through an outlet centered in the base of the tank and plum bed from the existing 8” steel piping to a 6” diesel pump by flexible 4” PVC piping. The diesel pump drove the pH 12 water through a Speece Cone where the CO 2 

### gas was injected. The CO 2 

### was fed into the top of the Speece Cone so the inline rotameter read 15 st andard cubic feet per minute (SCFM) which equated to a gas flow of 34 SCFM (by applying a pressure correction factor of 2.268). The pressure in the apex of the cone was kept at 18 pounds per square inch gauge (PSIG) and the flow through the cone was maintained be tween 560 and 600 GPM usi ng a combination of NaOH Valve #9

Dosing Pump Suction Line

Dosing Pump Discharge LineGSI/LB/F/A/3 May 13, 2011 Page 23 of 54



### adjusting the discharge valve on th e cone and the pump speed. The CO 2 

### -enriched water was discharged subsurface back into the treatment retention tank using a PVC T-Joint that reduced the 6” feed to two, 3” jets orie nted in opposite di rections. The CO 2 

### flow continued at a rate of 15 SCFM until the inline pH probe indicated a pH of 8.5. Following, the flow of CO 2 

### was cut off,

and the neutralized water continue d to recirculate for an additi onal 10 minutes to ensure the water was well mixed. Prior to the discharge ope ration, a 1 L sample was collected at mid-depth (i.e., using a Kemmerer Sampler) from the trea tment retention tank, and measured using an Orion 3 Star pH Meter and pH Combination El ectrode (Thermo Scientific) according to §2.2.1 below, to confirm the pH was 6.5-8.5.

Flow control valves and system logic assured that sample fl ow rates were equivalent and proportional to intake and discharge flow rate s throughout each intake and discharge operation.

Flow rates were recorded conti nuously every five seconds by au tomated, in-line sensors located on the control track, treatment track, and on the di scharge line. Pressure readings were also recorded continuously at multiple points throughout the facility. These data, as well as, other operational and maintenance parameters (e.g., re tention tank volume and volume sampled) were measured and recorded continuously using a Hu man Machine Interface (H MI) installed at the GSI Land-Based RDTE Facility. The HMI has a 15” color touch display and is capable of detailing valve positions, pressure from the pre ssure meters, fill level of the ballast retention tanks, and flow rates in the cont rol and treatment lines, etc. The HMI console then saved the information to a specific file. An external comput er, connected to the HMI, was used to store the

data files.

2.1.4. Preventing Cross Contamination



### To minimize potential cross contamination of the treatment discharge water between trials, prior

to the first trial and after each te st trial, the interior of the re tention tanks were cleaned according

to GSI/SOP/LB/G/O/3

– Procedure for Cleaning and Verifyi ng Cleanliness of the Retention Tanks and Piping at the GSI Land-Based RDTE Facility . After each intake and discharge operation, the sampling equipment (i.e., sample collection tubs, drain spout hose and nozzle, plankton nets, etc.) was cleaned according to GSI/SOP/LB/G/O/4 – Procedure for Cleaning Sampling Equipment at the GS I Land-Based RDTE Facility . The GSI facility recirculation lines were flushed with potable water fro m an on-site potable water tank that had been verified to be free of living organisms. The flushing was unde rtaken after each intake and prior to each discharge operation. The thoroughn ess of the cleaning process was checked by partially filling a randomly selected treatment samp le collection tub with potable water, draining that water through a 35  m plankton net, and examining the filtrate for evidence of living organisms. The facility was deemed clean only if the filtrate wa ter was completely free of live Duluth-Superior Harbor zooplankton visible with a compound microscope at a magni fication of 40X to 100X (see

GSI/SOP/LB/G/O/3 ). Nets and other sample collection equipment were likewise validated for cleanliness prior to each sample operation (see GSI/SOP/LB/G/O/4 ).GSI/LB/F/A/3 May 13, 2011 Page 24 of 54



### 2.2. Water Quality Analysis

2.2.1. pH



### Samples for pH analysis were co llected during intake as follows:

 Three 1 L whole water samples were collect ed from the pre-treatment line (SP #3; Figure 4) at approximately 10, 30, and 50 minutes after the start of the intake procedure (which lasted approximately 57 minutes), and

 One 1 L whole water sample was collected from the treatment retention tank (Figure 4) after it was filled (i.e., using a Kemm erer Sampler) to confirm the pH was approximately 12.

The following samples were collected during discharge:

 Two 1 L whole water samples were collected from the treatment retention tank using a Kemmerer Sampler (Figure 4); one prior to neutralization of the treatment water via

CO 2 

### injection and one after the neutra lization procedure was completed,

 One 1 L whole water sample was collected from the treatment line approximately 30 minutes after the start of the discharge pro cedure via SP #10 (Figure 4, Trials A and B) and SP #15 (Figure 4, Trials C and D), and

 One 1 L whole water sample was collected from the control line approximately 30

minutes after the start of the control tank discharge proced ure (SP #10, Figure 4).

Sample analysis was conducted according to GSI/SOP/BS/RA/C/9

– Procedure for pH Meter Calibration and pH Measurement . Measurements were made usi ng an Orion 3 Star pH Meter and pH Combination Electrode (Thermo Scientific). The pH electrode was calibrated daily prior to use with certified pH buffers 4, 7, and 10. In addition, a pH “Check Buffer” (i.e., pH 12.45 buffer) was used to verify the accuracy of the pH electrode following ca libration at pH values greater than the most basic pH calibration buffer (i.e., pH  10). Prior to Trial A, the Automatic Temperature Compensation (ATC) probe was calibrated; the disp lay temperature was checked weekly during the testing and the ATC probe was recalibrated if needed.

Quality control measures consisted of collecting and analyzing one of the samples from all pH samples collected during th e four trials in duplicate.

2.2.2. Total Suspended Solids (TSS), Including Mineral Matter (MM)



### Samples for TSS analysis were collected during intake and discharge as follows:

 On intake, three 1 L whole water samples we re collected from the pre-treatment line (SP #3; Figure 4) approximately 10, 30, and 50 minutes after the start of the intake procedure.

 On discharge, one or three 1 L whole water samples were collected from the treatment line approximately 30 minutes (i.e., one sample) or 10, 30, and 50 minutesGSI/LB/F/A/3 May 13, 2011 Page 25 of 54



### (i.e., three samples) after the start of the discharge procedure. Samples were collected using SP #10 (Figure 4) for Trials A and B a nd SP #15 (Figure 4) for Trials C and D. In addition, one or three 1 L whole water samples were coll ected from the control line approximately 30 minutes (i.e., one samp le) or 10, 30, and 50 minutes (i.e., three samples) after the start of the control tank discharge procedure (SP #10, Figure 4).

Samples were collected in-line rather than from the sample collection tubs to avoid settling of suspended solids. This approach assured a mo re accurate measurement of solids and organic

carbon in the intake water.

Sample analysis was conducted according to GSI/SOP/BS/RA/C/8

– Procedure for Analyzing Total Suspended Solids (TSS) . The samples were vacuum f iltered through pre-washed, dried, and pre-weighed Whatman 934-AH glass fiber filter s. After the sample was filtered, it was dried in an oven and brought to constant weight. TSS values were dete rmined based on the weight of particulates on the filter and the volume of water filtered.

Quality control measures consisted of collecting and analyzing one of the samples from all TSS samples collected during the four trials in duplicate.

Mineral matter is defined as the difference between TSS and particulate organic matter (measured as POC). Therefore, MM concentrati ons were determined in each sample collected during these trials on intake following analysis of TSS, and the determination of POC as

calculated from the NPOC and DOC concentrations (see §2.2.3.).

2.2.3. Non

‐ Purgeable Organic Carbon (NPOC) and Dissolved Organic Carbon
(DOC), and Determination of Particulate Organic Carbon (POC)

Concentrations



### Samples for NPOC, DOC, and POC analysis we re collected immediately after TSS sample collection during intake only as follows:

 Three 125 mL whole water samples were co llected from the pre-treatment line (SP #3, Figure 4) approximately 10, 30, and 50 minu tes after the start of the operation.

In these tests, NPOC was measured as a surroga te for total organic carbon (TOC), though it may be a slight underestimate of TOC. The analytic al instrument used to measure NPOC purges the sample with air to remove inorganic carbon before measuring organi c carbon levels in the sample. Thus, the NPOC analysis does not in corporate any volatile or ganic carbon which may be present in the sample.

Sample analysis was conducted according to GSI/SOP/BS/RA/C/3– Procedures for Measuring Organic Carbon in Aqueous Samples . Upon arrival at LSRI, an aliquot of each 125 mL sample was filtered through a Whatman GF/F filter and acidi fied with hydrochloric acid for analysis of DOC. The remaining portion of the sample was aci dified with hydrochloric acid and analyzed for NPOC. A Shimadzu Total Organic Ca rbon Analyzer (Model TOC-5050A; ShimadzuGSI/LB/F/A/3 May 13, 2011 Page 26 of 54



### Scientific Instruments, Inc.; Columbia, MD) wa s employed for analysis of both NPOC and DOC.

Concentrations of NPOC and DOC were determ ined based on a calibration curve developed on the instrument using organic carbon standards prepared from potassium hydrogen phthalate. Reported particulate organic carbon (POC) concen trations were determined as the difference between the NPOC and DOC values for a given sample.

Quality control measures consisted of collecti ng and analyzing two of the samples from all organic carbon samples collected during the four trials in duplicate. A TOC reference standard (NSI Solutions Inc., Raleigh, NC QCI-062, Lot #051210-09) was analyzed once during testing to confirm the accuracy of the data being generated.

2.2.4. Percent (%) Transmittance



### An aliquot of the filtered porti on of each sample collected for TSS analysis was analyzed to determine percent transmittance. Sample analysis was conducted according to GSI/SOP/BS/RA/C/4 – Procedure for Determini ng Percent Transmittance (%T) of Light in

Water at 254 nm . A spectrophotometer set at 254 nm wa s used to measure %T of the filtered samples. Deionized water was used as a refere nce to adjust the spec trophotometer to 100 %T, and each filtered sample was measured in a pre-rinsed sample cuvette.

2.2.5. Water Quality Measurements using YSI Multiparameter Water Quality

Sondes



### Water quality was measured during each tria l using calibrated YSI Multiparameter Water Quality Sondes (YSI 6600 V2-4 Sondes; YSI Incorporated; Yellow Springs, OH, USA). The Sondes were calibrated prior to each trial following GSI/SOP/LB/G/C/4

- Procedure for Calibration, Deployment, and Storage of YSI Multiparameter Water Quality Sondes . The YSI Sondes have multiple probes that are able to measure the following parameters: dissolved oxygen, specific conductivity, salinity, temperature, pH, turbidity, and total chlorophyll. Water quality parameters were measured from approxima tely 1 L samples of water from each sample collection tub sampled on intake and discharge. Samples were taken immediately following collection of phytoplankton and microbial sample s, and each measurement was recorded on preprinted datasheets. In addition, water quality parameters in the control and treatment retention tanks were measured at mid-depth every 15 minutes during the two- or three-day holding time. Prior to discharge of the resp ective tanks, the Sondes were re moved and taken to the mobile laboratory where the data were later downloaded as test file s to a laptop computer using EcoWatch® for Windows® Software (v.3.18, 14 Apr il 2006; YSI Incorporat ed); the files were then translated to MS Excel files, which were stored on a laptop computer in the mobile laboratory and later uploaded to the GS I SharePoint intranet website.



### 2.3. Viable Organism Analysis

During these trials sample water for analysis of viable organisms was simultaneously and continuously collected from replicat e sample ports into identical 3.8 m 3 

### sample collection tubs during each intake, treatment discharge, and control discharge operation (retention tank discharge was sequential, treatment then control). Volumes retained were always greater thanGSI/LB/F/A/3 May 13, 2011 Page 27 of 54



### volumes recommended in the IMO Convention’ s G8 guidelines. The water in each sample collection tub constituted an independent, time -integrated, replicate sample of the 200 m 3


### experimental water mass.

2.3.1. Organisms ≥ 50 μm in Minimum Dimension

2.3.1.1. Sample Collection



### During the intake operation, i.e. the f illing of the treatment and control 200 m 3 

### retention tanks, the following time-integrated sample volumes we re collected and analyzed (additional samples were collected but were not analyzed) by continu ous flow from the intake lines simultaneously:

 One 2 m 3 

### sample from the pre-treatment intake line, and

 One 2 m 3 

### sample from the control intake line.

The pre-treatment and control samples served as replicate intake sub-samples for each trial.

During discharge the following time-integrated sa mple volumes were collected and analyzed (additional samples were collected but were not analyzed):

 Two time-integrated samples of 2 m 3 

### each (total volume 4 m 3 

### ) were continuously

collected from the treatm ent discharge line, and

 One 2 m 3 

### time-integrated sample was conti nuously collected from the control discharge line.

Flow control valves and system logic assured that sample fl ow rates were equivalent and proportional to intake and discha rge flow rates throughout each operation. Immediately after the sample collection tubs were filled, the phytopla nkton and microbial whole water samples were collected and Sonde readings recorded, followe d by the zooplankton samp le collection. The zooplankton samples were collected by dr aining the remaining volumes (i.e., 2 m 3 

### minus 5 L of rinse/Sonde water and the 1 L phytoplankton and mi crobial samples) from the sample collection tubs and concentrating through 35 μ m (50 μ m diagonal dimensions) pla nkton nets into 1 L codends for microscopic examination. See GSI/SOP/LB/RA/SC/6 - Procedure for Zooplankton Sample Collection . On intake, the zooplankton sample collection order alternated between collecting the pre-treatment or the control sample first. After the first sample was collected and analyzed, then the second sample was collected (either the control or the pre-treatment) and analyzed. On discharge, the treatment and contro l samples were also coll ected sequentially.

2.3.1.2. Live/Dead and Size Analysis



### All live/dead analysis was conducted according to GSI/SOP/LB/RA/SA/2 - Procedure for Zooplankton Sample Analysis , and took place within two hours of collecting and concentrating the individual samples. Micr ozooplankton (e.g., rotifers, copepod nauplii, and dreissenid veligers) and macrozooplankton (e.g., copepods, cla docerans, and insect larvae), all generally greater than or equal to 50 μ m in minimum dimension, were analyzed simultaneously byGSI/LB/F/A/3 May 13, 2011 Page 28 of 54



### separate taxonomists. Microz ooplankton subsamples were anal yzed in a Sedgewick-Rafter counting chamber by examination under a compound microscope at a magnification of 40X to 100X. Macrozooplankton were analyzed in a Wa rd’s Counting Wheel at a magnification of 20 to 30X using a dissecting microscope. Due to hi gh densities, quantification of zooplankton in the control intake, pre-treatment intake, and contro l discharge samples required analysis of subsamples and extrapolation to number live orga nisms per cubic meter. For these samples, a subsample was removed for analysis using a Hens on-Stempel pipette. The dead organisms (i.e., those organisms that did not move or respond to s timuli) were enumerated, then all organisms in the sample were killed by adding 50 % (v/v) a cetic acid solution (for microzooplankton) or Lugol’s solution (for m acrozooplankton) to the counting cham ber/wheel and the total number of organisms was enumerated. The number of live organisms was quantified by subtracting the number of dead organisms from the total numbe r of organisms in the counting chamber/wheel. The treatment discharge samples had lower densiti es allowing analysis of a greater proportion of the sample (see the “Results” section for the propor tion of sample volumes analyzed). Therefore, the treatment discharge samples were split in half using a Folsom Plankton Splitter. Half of the sample was analyzed for macrozooplankton and the other half was examined for microzooplankton. Only live organisms were enumer ated using standard movement and response to stimuli techniques.

Statistical analysis for the ≥ 50  m size class for the four trials was conducted using SigmaStat, version 3.5 (Systat Software, Inc.; Chicago, IL USA). A On e Way Analysis of Variance (ANOVA) was used to determine the differences in the mean values among the treatment groups if the data were normally distributed with e qual variance. If the data did not meet the assumptions of the One Way ANOVA, the data were transformed using either log (base 10), log normal, or square root transformation a nd a One Way ANOVA was used to compare the

transformed data. If transformation did not produce normally distributed data with equal variance, an appropriate non-parametr ic test was used. In all cases α =0.050.

Quality assurance measures during these trials in cluded live/dead analysis of four intake (i.e., one pre-treatment and three control samples) and one control discharge sample by two separate

taxonomists over the course of the four trials. The average percent similarity of taxonomic identification (live organisms only) and the averag e relative percent difference of the number of live organisms counted were calculated for all second analyses. In a ddition, all live organisms identified in the treatment discharge samples we re recorded and verified to be live by a second taxonomist, and the minimum visible dimension was measured using an eyepiece micrometer and recorded. Those organisms that were determined to be less than 50  m in minimum visible dimension were reported separately from the liv e zooplankton that did me et the size criterion detailed in Annex D-2 of the IMO Convention (IMO, 2004).

2.3.2. Organisms ≥ 10 and  50 μm in Minimum Dimension

2.3.2.1. Sample Collection



### For live analysis of organisms ≥ 10 and < 50 μ m in minimum dimension, during intake the following whole water samples were collected:GSI/LB/F/A/3 May 13, 2011 Page 29 of 54



###  One 1 L sample was collected immediately after filling from the pre-treatment sample collection tub (Tub #4, Figure 4), and

 One 1 L sample was collected from the control sample collection tub (Tub #1, Figure 4).

The pre-treatment and control samples served as replicate intake sub-samples for each trial.

During discharge:

 Three 1 L samples were collected from the th ree treatment sample collection tubs (Tubs #4-#6, Figure 4), and

 One 1 L sample was collected from the control tank via the sample collection tub (Tub #1, Figure 4).

The three, 1 L treatment discharge samples were composited for analysis. Analysis of all samples occurred on-site within 1.5 hours of samp le collection, with samples stored in coolers during the interim. Prior to analysis, samples were concentrated through 10 μ m mesh plankton netting and stored in a 25 mL sample container. See GSI/SOP/LB/RA/SC/3 - Procedure for Algae/Small Protozoa Sample Collection

.

2.3.2.2. Sample Analysis



### Sample analysis was conducted according to GSI/SOP/LB/RA/SA/1 - Procedure for Algae/Small Protozoan Sample Analysis. A 1.5 mL subsample of the concen trated sample was transferred to a 2 mL sample container, with 4 μ L of fluorescein diacetate (FDA) viability stain stock solution added. The subsample was then allowed to inc ubate in the dark for 5 minutes. The 1.5 mL incubated sample was mixed and 1.1 mL was imme diately transferred to a Sedgwick-Rafter cell, covered and placed on the stage of a microscope that was set for simultaneous observation using brightfield and epifluorescence. At least two horizont al transects were c ounted (an area known to reflect greater than 1 mL of original sample water), aiming for at least 100 entities (i.e., unicellular organism, colony or filament) counte d. If time permitted, additional transects were counted to increase statistical power. Single cell entities and cells comprising colonial and

filamentous entities were characterized as follows: alive = cells showing obvious green fluorescence from cell contents; dead = cells s howing no or very little evidence of green fluorescence from cell contents; and ambiguous = cells or entities that cannot be clearly identified as alive or dead (were uncommon). R ecords were kept of tran sect lengths and widths

so that the total counted area and volume analyzed could be calculated later.

Entities less than 10 μ m in all visible dimensions or greater than 50 μ m in minimum visible dimension were not counted. Counting and measur ement of all other 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 width or total filament length if cells could not be discerned). The re maining concentrated sample in the 25 mL bottle was archived using a preservative (formalin or Lugol’s) for long-term storage.GSI/LB/F/A/3 May 13, 2011 Page 30 of 54



### Statistical analysis for the ≥ 10- and < 50  m size class for the four tr ials was conducted using SigmaStat, version 3.5 (Systat Software, In c.; Chicago, IL USA). A One Way ANOVA was

used to determine the differences in the mean values among the treatment groups if the data were normally distributed with equal variance. If th e data did not meet the assumptions of the One Way ANOVA, the data were transformed using eith er log (base 10), log normal, or square root transformation and a One Way ANOVA was used to compare the transformed data. If transformation did not produce norm ally distributed data with e qual variance, an appropriate non-parametric test was used. In all cases α =0.050.

Quality assurance measures included analysis of one intake sample and three discharge samples (i.e., two treatment and one control discharge) by two separate taxonomists using a dual-headed microscope (i.e., both taxonomists analyzed the same sample at the same time) over the four trials of the NaOH BWTS. In addition, subs ample analysis was conducted on two treatment discharge samples (over the entire four-trial NaOH BWTS Test ) by a single taxonomist (i.e., one taxonomist analyzed two separate aliquots from one sample) to determine within sample precision. The average percent si milarity of taxonomic identific ation and the average relative percent difference of the number of live organi sms counted were calculated for all second analyses.

2.3.3. Organisms  10  m in Minimum Dimension



### Control and treatment samples for these trials were collected and analyzed for heterotrophic bacteria and three specific indicator organism s for waterborne pathogens: total coliform bacteria, Escherichia coli and Enterococcus spp.

2.3.3.1. Sample Collection



### Whole water samples were collected as follows:

 On intake, three 1 L samples were coll ected immediately after filling the pretreatment sample collection tub and collection of the ≥ 10 and  50  m size class sample (Tub #4, Figure 4).

 On discharge, three 1 L samples were co llected immediately after tank discharge from the treatment sample collection t ubs (Tubs #4-#6, Figure 4), and three 1 L samples were collected from the control retention tank via Tub #1 (Figure 4) after

collection of the ≥ 10 and  50  m size class sample.

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

2.3.3.2. Sample Analysis



### Viable heterotrophic bacteria we re enumerated according to GSI/SOP/BS/RA/MA/1 – Procedure for Quantifying Heterotrophic Pl ate Counts (HPCs) using IDEXX’s SimPlate® for HPC Method

.GSI/LB/F/A/3 May 13, 2011 Page 31 of 54



### This method utilizes the IDEXX SimPlate® for HPC Method (IDEXX Laboratories, Inc.; Westbrook, Maine), which is based on IDEXX La boratories’ patented multiple enzyme technology.

The most probable number (MPN) per 100 mL of total coliform bacteria, E. coli ( GSI/SOP/BS/RA/MA/4

- Procedure for the Detection and Enumeration of Total Coliforms and E. coli Using IDEXX's Colilert® ) and enterococci ( GSI/SOP/BS/RA/MA/3 - Procedure for the Detection and Enumeration of En terococcus using EnterolertTM) were determined using QuantiTray/2000® with Colilert® and EnterolertTM, respectively, which are both based on IDEXX’s patented Defined Substrate Technology (DST®; IDEXX Laboratories, Inc.; Westbrook, Maine).

Statistical analysis for all four types of bacteria in the <10  m size class (i.e., total coliform bacteria, E. coli

, Enterococcus spp ., and total heterotrophic bacteria) for the four trials was conducted using SigmaStat, version 3.5 (Systat So ftware, Inc.; Chicago, IL USA). A One Way

ANOVA was used to determine the differences in the mean values among the treatment groups if the data were normally distributed with equal vari ance. If the data did not meet the assumptions of the One Way ANOVA, the data were transformed using either log (base 10), log normal, or square root transformation and a One Way ANOVA was used to compare the transformed data. If transformation did not produce normally distribut ed data with equal va riance, an appropriate non-parametric test was used. In all cases α =0.050.

Quality control samples analyzed for each intake and discharge operation included a media blank and a positive control for E. coli /total coliforms and Enterococcus spp ., and a media blank for heterotrophic bacteria. Quality assurance measures included analysis of at least 10 % of the samples in duplicate from the total number of samples collected over the four trials. The average relative percent difference of all duplicates anal yzed during the trials was calculated separately

for E. coli

, Enterococcus spp ., and heterotrophic bacteria.



### 2.4. Whole Effluent Toxicity (WET) Testing

GSI’s whole effluent toxicity (WET) testi ng of the NaOH BWTS was conducted using three freshwater species as desc ribed in Table 5. The WET tests were conducted on P. promelas and

C. dubia using Trial B treatment discharge, and on P. promelas

, C. dubia , and S. capricornutum using Trial D treatment discharge.

The WET of treatment discharge water was de termined using standard USEPA procedures (USEPA, 2002) following a three-day retentio n period in the land-based facility’s 200 m 3


### treatment retention tank (Figure 4). Sample water (i.e., 19 L), collected from one of the treatment discharge sample collection tubs usi ng a 20 L, high-density, pol yethylene container, was immediately transported to LSRI and used upon arrival to set up the WET tests. Following set up of the tests, the remaining sample water was st ored at 4 °C in the dark to preserve as much of the initial water quality/chemistry properties as possible, and portions (i.e ., 2 to 3 L) of the discharge sample water was warmed to 25 °C each day to serve as renewal water for the bioassay. Filtered Duluth-Superior Harbor wa ter (i.e., filtered through a Whatman 934-AH
Glass Microfiber Filter, 1.5  m particle retention in liquid) served as the control, and treatmentsGSI/LB/F/A/3 May 13, 2011 Page 32 of 54



### consisted of 0 % treatment discharge water (i.e ., filtered harbor water control), 100 % treatment discharge water, and a pe rformance control (i.e., Ceriodaphnia dubia and Pimephales promelas culture water, and algae growth media for Selenastrum capricornutum ). All tests were conducted in temperature-controlled incubators or water baths, or at ambient room temperature following the SOPs listed in Table 5. Differences in mean percent survival (for C. dubia and P. promelas ), mean dry weight values (for P. promelas ), mean S. capricornutum cell density, and mean

number of C. dubia young per female between the 0 % an d 100 % treatment discharge groups were analyzed for statistical significance at α = 0.05 using a One-Way Analysis of Variance and a post hoc statistical comparison.

The WET tests were initiated with healthy, vigorous organisms. To determine the overall health of the test organisms, reference toxicant tests were performed with the cladoceran Ceriodaphnia

dubia and the minnow Pimephales promelas prior to the start of each definitive test or at least once per month. In addition, a performance control was used for all species tested. The performance control consists of the normal cu lturing conditions for each species, providing the test organisms with the optimal environment fo r survival, growth, and reproduction. Therefore, the performance control, along with the referen ce toxicant tests, provi des verification of the health of the test organisms. To determine the validity of the WET tests, percent survival of C.

dubia and P. promelas , dry weights of surviving P. promelas , mean S. capricornutum cell density, and mean number of young per female C. dubia in the controls were compared to the test acceptability criteria published in the USEPA’s Short-term Methods for Estimating the Chronic Toxicity of Effluents and R eceiving Waters to Freshwater Organisms (2002). Class I weights were used to verify the accuracy of the laboratory balance according to GSI/SOP/BS/RA/GL/1 – Procedure for Verification of Laboratory Balances . Daily or weekly

calibration of test meters ensu red optimal performance. The P. promelas drying process is verified by re-weighing a percentage of the fish after they have been dried for an additional length of time in the oven.

Table 5. Standard Operating Procedures Relative to Whole Effluent Toxicity (WET) Testing.

GSI SOP Code Test Type Test Species Test Endpoint

GSI/SOP/BS/RA/WET/1 Short-Term, Chronic Cladoceran ( Ceriodaphnia dubia) Survival and Reproduction

GSI/SOP/BS/RA/WET/2 Short-Term, Chronic Fathead Minnow ( Pimephales promelas) Survival and Growth (growth measured via dry weight)

GSI/SOP/BS/RA/WET/3 Short-Term, Chronic Green Alga ( Selenastrum capricornutum) Growth (measured via direct density counts)GSI/LB/F/A/3 May 13, 2011 Page 33 of 54



### 2.5. Data Management

2.5.1. Data Recording



### All biological and chemical data were recorded by hand (using inde lible ink) on pr e-printed data collection forms and/or in bound, uniquely-identifie d laboratory notebooks that were specific to

the NaOH BWTS test. The data that were record ed on pre-printed data collection forms were secured in uniquely-identified thr ee ring binders, specific to the type of data and to the treatment technology.

Biological and chemical data that were recorded by hand were entered into either a MS Access Database that was designed, developed, and is maintained by the GSI Database Manager (i.e., microbial, phytoplankton, and zooplankton data) or the data were entered into a MS Excel spreadsheet (i.e., water chemistry and WET test data; see GSI/SOP/G/RA/DM/1 - Procedure for Data Entry, Data Quality Control, and Database Management ).

In-tank water quality data (e.g., temperatur e, pH, dissolved oxygen, specific conductivity, salinity, turbidity, and total chlo rophyll) was measured continuously every fifteen minutes during each retention period and automatically recorded in a text file, which was la ter translated to a MS Excel spreadsheet. Facility data (e.g., flow rates and pressure measurements) were electronically recorded every five seconds during intake and di scharge. This data was exported to MS Excel for subsequent analysis, and is stored by AMI Engineers on a secure network and on GSI

SharePoint.

Following completion of the NaOH BWTS trials, a thorough review of all data sheets and laboratory notebooks was undertaken to ensure co mpliance with the documentation procedures

outlined in all relevant GSI SOPs and in the GSI Land-Based Quality Assurance Project Plan (GSI, 2010a). A percentage of data that was recorded by hand and entered into MS Access or MS Excel was verified against the original raw data. This process also included verification of formulas and calculations (i.e., hand-calculation of data). The percentage of verified raw data ranged from 10 % to 100 % of the original raw data, depending on th e data type. More detail on

the GSI’s data validation activities is additiona lly detailed in Section 7 of the GSI Quality Assurance Project Plan for Land-Based Tests (G SI, 2010a). This section also details the acceptable values, where appropriate, for the fo llowing quality objectives : accuracy, precision, completeness, comparability, representativeness, and sensitivity.

2.5.2. Data Processing and Storage



### After examination and quality assurance analysis, the data distribution files from the MS Access database were posted to the LSRI’s Local Area Network (LAN) in an or ganized hierarchical folder system. All electronic data files stored on the LSRI’s secured LAN can be accessed only by GSI personnel. The GSI Database Manager is the single point of c ontrol for access to the LSRI LAN. The LSRI LAN is automatically back ed up every 24 hours. A backup of the database was also made regularly to avoid any loss of data following computer /electronic glitches.GSI/LB/F/A/3 May 13, 2011 Page 34 of 54



### Electronic data files, including MS Excel files, are stored on the LSRI LAN as well as GSI’s internal SharePoint website, which acts as a secondary data backup/storage mechanism. All original raw data will be stored in a climate-cont rolled, secure archive room at the LSRI for five years after this repo rt is finalized.



## 3.0. RESULTS

Four trials (Trials A-D) of the NaOH BWTS’s biological effectiveness were completed, with WET tests incorporated into two trials (Trial B and D). During these trials, there were no significant deviations fr om the above methods.



### 3.1. Intake and Discharge Challenge Conditions

3.1.1. Operational Conditions



### Operational conditions, measured continuously duri ng intake, for all four trials were extremely

consistent with each other and between treat ment and control tracks of the GSI Land-Based Facility. Flow rate was slightly below 200 m 3 

### /hour and pressure in the facility lines was 30.4 – 31.6 psi (Table 6). On discharge, the flow dura tion, flow rate, and pre ssure was very similar

between the treatment and c ontrol tracks (Table 7).

Table 6. Average Operational Parameters Measured During Ballasting Simulation of the Four

Trials of the NaOH Ballast Water Treatment System.

Trial Flow Duration (min) Treatment

Flow Rate

(m 3 /h) Total Volume

of Water Treated (m 3 ) Control

Flow Rate

(m 3 /h) Pressure (psi)

A 56.9 198.6 188.3 199.1 30.4

B 56.5 198.9 187.3 198.9 31.0

C 56.5 198.2 186.6 199.3 31.3

D 56.5 198.4 186.8 199.5 31.6GSI/LB/F/A/3 May 13, 2011 Page 35 of 54

Table 7. Operational Parameters Measured During the Deballasting Simulation for the Four Trials of the NaOH Ballast Water Treatment System.

Treatment Control

Trial Flow

Duration

(min) Flow

Rate Pressure (psi) Flow

Duration

(min) Flow

Rate Pressure (psi) (m 3 /h) (m 3 /h)

A 53.3 200 30.2 52.6 198 28.9

B 52.3 197 31.2 54.1 195 31.1

C 54.2 196 30.9 54.7 195 31.4

D 54.5 195 31.5 54.0 192 31.4

3.1.2. Physical, Chemical and Biological Challenge Conditions



### A summary of the physical/chemical conditions of intake water are provided in Table 8. The ambient TSS was characteristically low, ranging from 1.3 mg/L to 2.4 mg/L, and averaging 2.0 mg/L for the four trials. The NPOC was entir ely DOC, as the average of both parameters was 13.4 mg/L throughout the four trials. The %T range d from 24.8 to 29.4 in th e first three trials, but in Trial D rose substantially (42.7 %T). The average % T acr oss the four trials was 31.0 %T. The challenge water pH ranged from 7.75 to 7.99 during all four trials.

The average pH of the post-treatment water collect ed immediately prior to the water entering the treatment retention tank was 12.02, achieving the ta rget pH of 12.00. Sample s collected from the treatment retention tank just prior to neutralization had an average pH was 12.04, showing that the pH of the treated water did not change significantly during the holding time. After the neutralization process, samples measured from the treatment tank and in-line from the treatment discharge averaged 8.17 and 8.14, respectively). The neutralized discharge pH met the targetvalue of less than 8.5, and was within the WIDNR permitting levels for discharge to the DuluthSuperior Harbor (i.e., pH 6 to 9).GSI/LB/F/A/3 May 13, 2011 Page 36 of 54

Table 8. Average (± Std. Dev.) Challenge Water Quality during Four Trials of the NaOH Ballast Water Treatment System.

Parameter Desired pH

Value Sample Trial A Trial B Trial C Trial D Summary ( n =4)

pH 1 7-9 (Ambient) Pre-Treatment

Intake 7.76 ± 0.01 7.83 ± 0.04 7.99 ± 0.04 7.75 ± 0.02 7.83 ± 0.11

12.00 Post-Treatment

Intake 12.00 12.04 12.04 12.00 12.02 ± 0.02

12.00 In-Tank Before

Neutralization 12.06 12.07 11.99 12.02 12.04 ± 0.04

≤ 8.5 In-Tank After

Neutralization 8.27 8.21 7.81 8.39 8.17 ± 0.25

7-9 Treatment Discharge NA 8.22 7.81 8.40 8.14 ± 0.30

TSS (mg/L) N/A Intake 1.3 ± 0.2 2.0 ± 0.1 2.4 ± 0.1 2.1 ± 0.1 2.0 ± 0.5

NPOC (mg/L) N/A Intake 13.5 ± 0.5 15.3 ± 0.3 14.4 ± 0.2 10.3 ± 0.2 13.4 ± 2.2

DOC (mg/L) N/A Intake 13.7 ± 0.3 15.3 ± 0.1 14.3 ± 0.3 10.3 ± 0.2 13.4 ± 2.2

POC (mg/L) N/A Intake -0.2 ± 0.7 -0.1 ± 0.2 0.1 ± 0.3 0.0 ± 0.4 -0.1 ± 0.1

%T (254 nm) N/A Intake 24.8 ± 0.5 26.9 ± 0.1 29.4 ± 0.2 42.7 ± 0.8 31.0 ± 8.1

1 The sample temperature was measured simultaneously with sample pH measurements. The average sample temperature was 20.9 ± 1.8 °C in pre-treatment intake, 23.1 ± 1.9°C in post-treatment in take, 22.6 ± 1.5 °C in the

treatment retention tank before and after neutraliz ation, and 21.0 ± 1.0°C in treatment discharge.



### The live plankton densities in intake and control di scharge samples, for the ≥ 10  m and < 50



m size class and for the larger plankton size class (i.e., ≥ 50 μ m), are summarized in Table 9. During all four trials, average intake densit ies of live zooplankton (i.e., those organisms ≥ 50

μ m) ranged from 28,331/m 3 

### (Trial A) to 648,158/m 3 

### (Trial C), for an average of 291,229/m 3



### . The zooplankton community was comprised mainly of dreissenid mussel veligers; the rotifers Keratella, Polyarthra, and Synchaeta ; calanoid and cyclopoid cope pods; and the cladoceran

Bosmina. The target intake density of ≥ 100,000/m 3 

### was achieved during a ll but the first trial (Trial A). The density of live organisms in the control discharge samples after the two- or threeday holding period ranged from 22,047/m 3 

### (Trial A) to 725,980/m 3 

### (Trial C), which was 78 % to 112 % of the starting densities. Cont rol discharge densities in Trials B and C were slightly higher than their corresponding intake de nsities (1% to 12% respectively), likely due to reproduction of the rotifers ( Keratella spp. and Polyarthra spp. ) and the cladoceran Bosmina

.

The live organism density for the ≥ 10 and  50  m organism size class on intake, consisting mainly of phytoplankton, ranged from 67.56 cells/m L (Trial A) to 661.91 cells/mL (Trial D, in

which the intake stream was amended with concen trated harbor algae; Table 9). The community of protists comprised, in d ecreasing relative abundance, chai n-forming diatoms (largely

Aulacoseira ), coccoid green algae (largely Gonium ), filamentous blue-green algae ( Oscillatoria ), miscellaneous microflagellates, and fr ee-living centric di atoms, such as Cyclotella . The densityGSI/LB/F/A/3 May 13, 2011 Page 37 of 54



### of live phytoplankton in the cont rol discharge sample s ranged from 16.00 cells/mL (Trial C) to 275.95 cells/mL (Trial D), for an average of 93.43 cel ls/mL (Table 9). This represented 4 % to 42 % of the intake densities (Tab le 9). Although organism densit ies were lower in the control discharge samples, the relative abundance of taxa were similar to that observe d in intake samples.

Table 9. Live Plankton Densities in Intake and Control Discharge During the Four NaOH Trials. Note: The Live Intake Densities are an Average of the Control Intake and the Pre-treatment Intake Samples.

Organism Size Category Sample Trial A Trial B Trial C Trial D Average ± Std.

Dev. ( n =4)

≥ 50 μ m (#/m 3 ) Intake 28,331 384,216 648,158 104,210 291,229 ± 141,462

Control Discharge 22,047 389,885 725,980 102,478 310,098 ± 159,531

≥ 10 and < 50 μ m (#cells/mL) Intake 67.56 276.63 417.01 661.91 355.78 ± 124.77

Control Discharge 27.44 54.31 16.00 275.95 93.43 ± 61.37



### Concentrations of organisms in the < 10  m size class in the intake and control discharge samples during the four NaOH Trials are provided in Table 10. Overall intake densities within this size class were highest duri ng Trials C and D. Total colif orm bacteria ranged from 204

MPN/100 mL in Trial A to 552 MPN/100 mL in Trial D. E. coli ranged from 41 MPN/100 mL (Trial A), to 107 MPN/100 mL (Trial C). Enterococci ranged from 35 MPN/100 mL (Trial A) to 164 MPN/100 mL (Trial D). Finally, total hetero trophic bacteria range d from 400 MPN/mL in

Trial A to 1240 MPN/mL in Trial B (Table 10). In the control di scharge, indicator organisms for waterborne pathogens were more sparse, which is to be expected as the retention tank is not a favorable environment to support growth of these organisms. The total coliform bacteria ranged from 19 MPN/100 mL (Trial B) to 129 MPN/100 mL (Trial D), an overall decline of 78 % relative to intake. The overall average E. coli density was 6 MPN/100 mL. Enterococci ranged

from 4 MPN/100 mL in Trial A to 143 MPN/100 mL in Trial B. Total heterotrophic bacteria ranged from 424 MPN/mL (Trial A) to 1833 MPN/mL (Trial D).GSI/LB/F/A/3 May 13, 2011 Page 38 of 54

Table 10. Viable Microbial Densities (Average ± Standard Error of the Mean) in Intake and Control Discharge During the Four NaOH Trials.

< 10  m Size Class Group Sample Trial A Trial B Trial C Trial D Summary ( n =4)

Total Coliform

Bacteria (MPN/100 mL) Intake 239 ± 53 204 ± 20 536 ± 40 552 ± 46 383 ± 93

Control Discharge 123 ± 32 19 ± 4 51 ± 2 129 ± 7 81 ± 27

E. Coli (MPN/100 mL) Intake 41 ± 2 55 ± 4 107 ± 5 73 ± 5 69 ± 14

Control Discharge 11 ± 2 1 ± 0.5 a 3 ± 1 9 ± 1 6 ± 2

Enterococci (MPN/100 mL) Intake 35 ± 7 40 ± 14 139 ± 26 164 ± 31 95 ± 33

Control Discharge 4 ± 0.3 143 ± 88 68 ± 8 76 ± 3 73 ± 28

Total Heterotrophic

Bacteria (MPN/mL) Intake 400 ± 200 1240 ± 30 1033 ± 491 1117 ± 164 948 ± 187

Control Discharge 424 ± 63 1225 ± 200 900 ± 321 1833 ± 67 1096 ± 296

a One or more values were below the limit of detection (LOD). Half the value of the LO D was used for calculations. See Appendix 2 for raw data.

3.1.3. In

‐ Tank Water Quality



### Control and treatment retention tank water quality data are presented in Table 11. Two values are reported for the treatment tank: the values m easured prior to the start of the neutralization period, and the values measured after neutralizati on and just prior to the start of the discharge operation. Only data from Trials C and D are re ported; the YSI Sondes were not calibrated prior to Trial A or Trial B; therefore, the accuracy of the water quality data from these trials cannot be

assured.

Temperature and dissolved oxygen were similar in the control and treatment tanks throughout the entire holding period, includi ng after neutralization of the treatment tank. However, several parameters were different as a result of the NaOH injection into the treatment track. As expected, the pH of the treatment tank water was significantly higher than the control tank water. In trials C and D, the average pH of water in the tr eatment tank prior to neutralization was 11.80 and 11.85, respectively. In contrast, the average pH of water in the control tank was 7.72 for Trial C and 7.45 for Trial D. The pH of water held in the treatment tank following neutralization with

CO 2 

### was 7.57 and 8.18 for the two trials respectiv ely, thereby meeting Wisconsin DNR permit requirements for discharge to the harbor.

In both trials reported in Table 11 (Trials C and D) the specific conductivity in the treatment tank prior to neutralization was on average 14.5 to 17.4 times higher, than in the control tank during the three-day holding period. Th e neutralization pro cess decreased the specific conductivity by approximately half, but on average the post-neut ralization conductiv ity just prior to treatmentGSI/LB/F/A/3 May 13, 2011 Page 39 of 54



### discharge was still 6.6 to 7.6 times higher than in the control tank. In addition, the treatment tank salinity (as calculated by the YSI Sonde based on specific conductivity) increa sed as a result of the NaOH injection. On average, the salinity in the treatment tank prior to neutralization was 15.5 to 20.4 times higher than in the control tank. The neutralizati on process reduced the salinity by about half from the average salinity measured prior to CO 2 

### injection but the levels in the treatment tank just prior to discharge were still an average of eight times hi gher than the levels in the control tank just prior to di scharge in both Trial C and D.

The turbidity of water held in the control and treatment tanks during Trial D was similar; however, during Trial C the turbid ity reading for the control tank water as substantially higher than all other readings for cont rol and as compared to the trea tment tank water (i.e., average 12.7

NTU in the control tank and 1.8 NTU in the trea tment tank), possibly indicating a problem with the Sonde probe post calibration. In both trials, the turb idity of the treatment tank did not change after the neutralization process.

Table 11. Control and Treatment Retention Tank Water Quality (Average ± Std. Dev.) During the Three-day Holding Period for Trials C and D of the NaOH BWTS Test.

Parameter Retention

Tank Sample

Period Trial C Trial D

Temperature (°C) Control Entire

Retention 21.95 ± 0.24 ( n =288) 21.33 ± 0.23 ( n =283)

Treatment Before

Neutralization 21.98 ± 0.25 ( n =277) 21.47 ± 0.23 ( n =276)

Prior to Discharge 22.19 ( n =1) 21.99 ( n =1)

Specific Conductivity (mS/cm) Control Entire

Retention 0.224 ± 0.001 ( n =288) 0.179 ± 0.001 ( n =283)

Treatment Before

Neutralization 3.258 ± 0.016 ( n =277) 3.113 ± 0.021 ( n =276)

Prior to Discharge 1.475 ( n =1) 1.365 ( n =1)

Salinity (ppt) Control Entire

Retention 0.11 ± 0.00 ( n =288) 0.08 ± 0.00 ( n =283)

Treatment Before

Neutralization 1.71 ± 0.01 ( n =277) 1.63 ± 0.01 ( n =276)

Prior to Discharge 0.74 ( n =1) 0.68 ( n =1)

pH Control Entire

Retention 7.72 ± 0.06 ( n =288) 7.45 ± 0.02 ( n =283)

Treatment Before

Neutralization 11.80 ± 0.02 ( n =277) 11.85 ± 0.02 ( n =276)

Prior to Discharge 7.57 ( n =1) 8.18 ( n =1)

Turbidity (NTU) Control Entire

Retention 12.7 ± 0.6 ( n =288) 0.6 ± 0.3 ( n =283)

Treatment Before

Neutralization 1.8 ± 0.2 ( n =277) 0.8 ± 0.5 ( n =276)

Prior to Discharge 1.4 ( n =1) 0.7 ( n =1)GSI/LB/F/A/3 May 13, 2011 Page 40 of 54

Parameter Retention

Tank Sample

Period Trial C Trial D

Dissolved Oxygen

(% Saturation) Control Entire

Retention 89.2 ± 3.1 ( n =288) 83.1 ± 0.9 ( n =283)

Treatment Before

Neutralization 89.8 ± 2.2 ( n =277) 83.3 ± 0.9 ( n =276)

Prior to Discharge 86.8 ( n =1) 81.0 ( n =1)

Dissolved Oxygen (mg/L) Control Entire

Retention 7.80 ± 0.24 ( n =288) 7.36 ± 0.10 ( n =283)

Treatment Before

Neutralization 7.78 ± 0.16 ( n =277) 7.29 ± 0.10 ( n =276)

Prior to Discharge 7.53 ( n =1) 7.06 ( n =1)



### 3.2. Ballast Water Treatment System Biological Efficacy

Plankton densities and associated sample vol umes relevant to live organisms in the ≥ 50  m and

≥ 10  m to < 50  m size classes in control and treatment discharge samples from Trials A, B, C

and D are summarized in Tables 12 to 16. In ad dition, live densities of f our groups of bacteria (i.e., total coliform, E. coli

, Enterococcus spp., and total heterotr ophic) in the <10  m size class are reported in Tables 17 and 18. Percent reduction of live orga nism density in the treatment discharge as compared to the cont rol discharge of organisms in the ≥ 50  m, ≥ 10 and < 50  m,
and < 10  m size classes is summarized in Table 19.

3.2.1. Organisms ≥ 50 μm in Minimum Dimension



### Average live organism densities in treated discharge and total vol ume of treated discharge water analyzed during Trials A, B, C and D are reported in Table 12. Significant amounts of dead material in the treatment discharge samples limite d the sample volume that could be analyzed for live organisms ≥ 50 μ m in minimum dimension prior to maximum sample holding time (i.e., two hours). Sample volumes analyzed for live macrozooplankton ranged from 0.6 m 3 

### to 2.2 m 3 

### and sample volumes analyzed for live mi crozooplankton ranged from 0.1 to 0.3 m 3 

### (Tables 12 and 13). These low volumes resulted in low st atistical certainty of density estimates.GSI/LB/F/A/3 May 13, 2011 Page 41 of 54

Table 12. Live Treatment Discharge Densities and Treatment Discharge Sample Volume Analyzed

Within the ≥ 50  m Size Class During Four Trials of the NaOH Ballast Water Treatment System.

Treatment Discharge Density/ Vol. Analyzed Trial A Trial B Trial C Trial D Avg. ± SEM

Density (#/m 3 ) 0.5 0.0 19 0.0 4.9 ± 4.8

Total Vol. Analyzed (m 3 ) 2.2

MacroZP

0.2

MicroZP 1.3

MacroZP

0.3

MicoZP 0.8

MacroZP

0.1

MicroZP 0.6

MacroZP

0.1

MicroZP 1.2 ± 0.7 MacroZP

0.2 ± 0.1 MicroZP



### Overall, the average live organism densities ≥ 50 μ m in minimum dimension in treated discharge across all four trials was 4.9 live organisms/m 3 

### (Table 12). Table 13 provides the relative densities of live organisms across taxa in the treate d discharge from the four trials. In Trial A,

one live ostracod was observed in the treated disc harge sample resulting in a density estimate of 0.5 live organisms per m 3 

### (Tables 12 and 13). There we re no live organisms measuring ≥ 50  m in minimum dimension observed in trials B or D treatment discharge (Table 12), however, there was one chironomid larvae each (both measuring 40  m and in minimum dimension) observed in Trial B and Trial D treatment discharge resu lting in a density estimate of 0.80 and 1.67 per m 3


### respectively (Table 13). Trial C treated disc harge had a total of 19.2 live zooplankton per m 3 

### ≥

50  m (Table 12), including chironomid larvae, plan aria, copepods, and dreissenid larvae (Table 13).GSI/LB/F/A/3 May 13, 2011 Page 42 of 54

Table 13. Live Zooplankton Densities Across Taxa in Treatment Discharge from Four Trials of the NaOH Ballast Water Treatment System.

Test Trials: Trial A Trial B Trial C Trial D

Total Vol. Treatment Discharge Analyzed, m 3 : 2.21 (0.24 MicroZP) 1.28 (0.30 MicoZP) 0.82 (0.14 MicroZP) 0.60 (0.08 MicroZP)



### ≥ 50 μ m (min. dimension)

Taxa Group Avg. Density (per m 3 ) Avg. Density (per m 3 ) Avg. Density (per m 3 ) Avg. Density (per m 3 ) Copepod: Calenoid/Cyclopoid 2.43

Chironomid 4.86

Planaria 4.86

Ostracod 0.50
Dreissenid (Zebra Mussel) 7.01 Equal to or Greater than 50

μ

m (min. dimension) Total: 0.50 0.00 19.16 0.00



### < 50 μ m (min. dimension)

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

Chironomid 0.80 1.22 1.67

Less than 50 μ m (min. dimension) Total: 0.00 0.80 1.22 1.67

< 50  m (min. dimension); Observed but not Quantified

Taxa Group Observations/

Comments Observations/

Comments Observations/

Comments Observations/

Comments

Eggs/Cysts A few observed. Many observed (~85,000/m 3 live). Many observed.

Phytoplankton A few observed with chlorophyll.

Copepod Nauplii Four live

observed under dissecting scope.



### The results of the statis tical comparison between live organism density in the treatment discharge and in the control discharge are shown in Ta ble 14. The Kruskal-Wallis One Way ANOVA was used to compare the two groups, as the data were normally dist ributed but did not have equal

variance and transformation of th e data (e.g., log base 10, log norma l, and square root) did not successfully produce data that me t the assumptions of a One Way ANOVA. After four trials of the NaOH BWTS, overall live zoopl ankton density in the treated discharge was significantly ( p = 0.029) lower than that of the control discharge.GSI/LB/F/A/3 May 13, 2011 Page 43 of 54

Table 14. Result of Statistical Comparison of Live Zooplankton Density in Control Discharge to Treatment Discharge. The hypothesis tested was that the NaOH BWTS significant ly reduces the number of live organisms on discharge in comparison to untreated, control discharge.

Treatment

Group Mean Density ( n =4) Std. Dev. SEM t p Probability of Trial Resulting in No Difference

Control Discharge 310,098 live/m 3 319,063 159,531

26.000 0.029 1 in 34 Treatment Discharge 5 live/m 3 9 5

3.2.2. Organisms ≥ 10 and  50 μm in Minimum Dimension



### In the ≥ 10 and < 50  m size class, live organism densities ranged from 0.2 cell/mL (Trial B) to 2.5 cells/mL (Trial D), for an average of 1.0 cel l/mL (Table 15). The volume of treatment discharge water analyzed was 5.5 mL to 8.9 mL (Table 15). While few in number, surviving organisms in treated water were taxonomically various including diatoms, green algae, bluegreen algae, and protozoans.

Table 15. Live Treatment Discharge Density and Treatment Discharge Sample Volume Analyzed

Within the ≥ 10  m and < 50  m Size Class during Four Trials of the NaOH Ballast Water Treatment System.

Treatment Discharge Density/ Vol. Analyzed Trial A Trial B Trial C Trial D Avg. ± SEM

#cells/mL 0.3 0.2 1.0 2.5 1.0 ± 0.5

Total Vol. Analyzed (mL) 8.9 5.5 8.2 7.1 7.4 ± 1.5



### The results of the One Way ANOVA are provide d in Table 16 below. The data were not normally distributed; therefore, the data were transformed usi ng log (base 10) transformation. Overall, the live phytoplankton density in the treatment discharge was significantly ( p = 0.002) lower as compared to the unt reated, contro l discharge.

Table 16. Result of Statistical Comparison of Live Phytoplankton Density in Control Discharge to Treatment Discharge. The hypothesis tested was that the NaOH BWTS significant ly reduces the number of live organisms on discharge in comparison to untreated, control discharge.

Treatment

Group Mean Density ( n =4) Std. Dev. SEM t p Probability of Trial Resulting in No Difference

Control Discharge 93 cells/mL 123 61

5.200 0.002 1 in 500 Treatment Discharge 1 cell/mL 1 0.5GSI/LB/F/A/3 May 13, 2011 Page 44 of 54

3.2.3. Organisms  10  m in Minimum Dimension



### The density of live organisms in the  10  m size class in treatment discharge from the four NaOH BWTS trials are presented in Table 17. Th e total coliform bacteria concentrations in treated discharge ranged from less than the limit of detection (i.e.,  1 MPN/100 mL) in Trials A, B, and D to a maximum of 2 MPN/100 mL in Trial C. For a ll four trials the E. coli density was

 1 MPN/100 mL (i.e., the limit of detection) in treatment discharge. The live density of

Enterococci ranged from 2 MPN/100 mL (Trials A a nd D) to 42 MPN/100 mL (Trial B) in treatment discharge. Total hete rotrophic bacteria densities in treatment discharge ranged from 116 MPN/mL (Trial B) to 363 MPN/mL (Trial A), which is an average of 80 % less heterotrophic bacteria as compar ed to the control discharge.

Table 17. Live Density (Average ± Standard Error of the Mean) of Regulated Microbes in the

Treatment Discharge from the Four Trials of the NaOH Ballast Water Treatment System.

 1 0  m Size Class Group Trial A Trial B Trial C Trial D Summary ( n =4)

Total Coliform Bacteria (MPN/100 mL)  1  1 2 ± 2 a  1  1

E. Coli (MPN/100 mL)  1  1  1  1  1

Enterococci (MPN/100 mL) 2 ± 1 a 42 ± 42 a 6 ± 5 2 ± 1 13 ± 10

Total Heterotrophic Bacteria (MPN/mL) 363 ± 0 116 ± 6 222 ± 24 174 ± 21 219 ± 53

a One or more values were below the limit of detection (LOD). Half the value of the LO D was used for calculations. See Appendix 2 for raw data.



### The results of the One Way ANOVA are provided in Table 18 below. There was a significant ( p < 0.05) reduction in live organism density in the treatment discharg e as compared to the control discharge for all groups analyzed except Enterococcus spp .; with reductions in total coliform bacteria being the most pronounced. The total coliform and E. coli data did not meet the assumptions of the One Way ANOVA. The total coliform data were transformed using log (base 10) transformation. Transformation (e.g., log (bas e 10), natural log, and square root) of the E.

coli data did not produce normally distributed data with equal variance; therefore, the nonparametric Kruskal-Wallis One Way ANOVA on Ranks was used to compare differences

between the treatment and control. The Enterococcus spp. data and the total heterotrophic data were analyzed using a One Way ANOVA.GSI/LB/F/A/3 May 13, 2011 Page 45 of 54

Table 18. Results of Statistical Comparisons of Live Microbe Density in Control Discharge and Treatment Discharge in the <10-  m Size Class. The hypothesis tested was that the NaOH BWTS significant ly reduces the number of live organisms on discharge in comparison to untreated, control discharge.

Type of

Bacteria Treatment Group Mean Density

( n =4) Std.

Dev. SEM t p Probability of Trial Resulting in No

Difference

Total

Coliform

Bacteria Control Discharge 80.50 MPN

/100 mL 54.19 27.10

7.881 0.0002 1 in 5000 Treatment Discharge 0.88 MPN

/100 mL 0.75 0.38

E. coli Control Discharge 6.00

MPN/100 mL 4.76 2.38

26.000 0.029 1 in 34 Treatment Discharge 0.50

MPN/100 mL 0.00 0.00

Enterococcus

spp. Control Discharge 72.75 56.85 28.42

1.989 0.094 1 in 11 Treatment Discharge 13.00 19.43 9.713

Total Heterotrophic

Bacteria Control Discharge 1095.50 591.55 295.78

2.918 0.027 1 in 37 Treatment Discharge 218.75 105.48 52.74

3.2.4. Percent Reduction of Live Organisms in the Treatment Discharge



### Percent reduction of live organism density in the treatment discharge as compared to the control discharge for all three regulat ed size classes was calculate d by the following equation: ൬ െ 1 	 ൤ൌ ݃݁ݎ݄ܿܽݏ݅ܦ	ݐ݉݁݊ݐ݁ܽݎܶ	݅݊	݊݋݅ݐܿݑܴ݁݀	ݐܿ݁݊ݎܲ݁ ݕݐ݅ݏ݁݊ܦ	݃݁ݎ݄ܿܽݏ݅ܦ	ݐ݉݁݊ݐ݁ܽݎܶ

ݕݐ݅ݏ݁݊ܦ	݃݁ݎ݄ܿܽݏ݅ܦ	݈݋ݎݐ݊݋ܥ 100% ൈ ൰൨



### Table 19 summarizes the percent reduc tion in live organism density in treatment discharge for all four trials of the NaOH BWTS. The largest per cent reduction of live organism density was seen

in the ≥ 50  m (zooplankton) and ≥ 10  m and < 50  m (phytoplankton) size classes. There was a reduction of 100 % live zooplankton in the treat ment discharge as compared to the control discharge during Trials B and D. Trials A a nd C saw an approximate reduction of 100 % of the live zooplankton density in the trea tment discharge, although 0.5 live/m 3 

### and 19 live/m 3 

### were counted in each trial, respectively. Trials B and D were also the most successful for phytoplankton with an approximate treatment disc harge reduction of 10 0 % (5.5 live cells/mL were counted) in Trial B and 99 % reduction in Tr ial D. There was a reduction of 99 % and 94 % in Trials A and C, respectively, for an overall average of 98 % reduction during all four trials. The NaOH BWTS was less effective for the organisms in the < 10  m size class. There was a greater than one-log reduction in live coliform bacteria density in treatment discharge as compared to control discharge (i.e., average of 98 % reduction in live density in treatment discharge). The live density of E. coli

, Enterococcus spp. , and heterotrophic ba cteria in treatment discharge was reduced less than one log as compared to the control discharge. There was anGSI/LB/F/A/3 May 13, 2011 Page 46 of 54



### overall average of 81 % reduction in live E. coli , 77 % reduction in live Enterococcus spp. , and

68 % reduction in live he terotrophic bacteria.

Table 19. Percent Reduction of Live Organism Density in Treatment Discharge as Compared to Control Discharge. Trial A had a Retention Time of Two Days; Trials B - D had a Retention Time of Three Days.

Organism Size Category Group Trial A Trial B Trial C Trial D Summary ( n =4)

≥ 50  m Zooplankton 100% a 100% 100% a 100% 100% ± 0%

≥ 10  m and <50  m Phytoplankton 99% 100% a 94% 99% 99% ± 3%

<10  m b Total Coliform

Bacteria 100% a,c 97% c 96% 99% c 98% ± 2%

E. coli 95% c 50% c 83% c 94% c 81% ± 21%

Enterococci 50% 71% 91% 97% 77% ± 21%

Total Heterotrophic

Bacteria 14% 91% 75% 91% 68% ± 36%

a Live organisms were found during this trial. The percent reduction was rounded up to 100% although complete elimination of live organisms in tr eatment discharge was not observed.

b Percent reduction of live bacteria d ensity in treatment discharge as comp ared to control discharge is based on the average ( n = 3 samples) live organism density per tr ial in treatment and control discharge.

c Average ( n =3) treatment discharge density values were below t he limit of detection (LOD), which is 1 MPN/100 mL. Half the value of the LOD, or 0.5 MPN/100 mL was used for per cent reduction calculations.



### 3.3. Whole Effluent Toxicity (WET) Testing

WET tests were conducted on P. promelas and C. dubia using Trial B treatme nt discharge, and

on P. promelas

, C. dubia , and S. capricornutum using Trial D treatment discharge. The performance controls (i .e., culture water for P. promelas and C. dubia , and algae media for S.
capricornutum ) met test acceptability criteria in all cases, with the exception of the C. dubia

survival criterion in Trial B. The untreated f iltered harbor water controls (0 % treatment discharge water) met the test acceptability criter ia for all species tested during both Trials B and

D.

The survival of C. dubia was only 70 % in the Trial B performance control (i.e., hard reconstituted culture water), and therefore these re sults were discarded and are not reported here.

The WET test conducted on C. dubia using Trial D whole effluent showed no lethal effect (Table 20). However, there was a significant ( p  0.05) reduction in the mean number of young produced per female in the 100 % Effluent group as compared to the 0 % Effl uent group (Table 20). In Trial D, each female in the 100 % Effluent produ ced an average of 20 young as compared to 36 young per female in the 0 % Effluent (Table 20). Th is result indicates a potential effect of 100 % treatment discharge on cladoceran reproduction.

In the WET test conducte d on the algal species S. capricornutum using treatment discharge from Trial D, the average cell density at test te rmination in the 100 % Effluent group (3,896,875 cells/mL) was significantly ( p  0.05) higher as compared to the 0 % Effluent group (2,875,000GSI/LB/F/A/3 May 13, 2011 Page 47 of 54



### cells/mL), suggesting a possible eff ect of 100 % Effluent might be enhanced algal growth (Table 21).

Exposure to 100 % BWTS treatment discharge water did not affect P. promelas survival or growth in either Trial B or Trial D (Table 22). However, these organisms did display behavioral differences from their counterparts exposed to C ontrol tank discharge. During both Trials B and D, organisms in treated discharge continuously swam in a circular pattern, an effect not observed

in the harbor water control.

Table 20. Average (± Standard Error of the Mean) Survival and Reproduction of C. dubia Exposed

to Whole Effluent Collected from NaOH Treatm ent Discharge During Trial D. Note: Statistical comparisons were made within one trial only, i.e., no comparisons were made between trials.

Treatment Group TRIAL D

Survival (%) No. Young per

Female

C. dubia Culture Water (Performance Control) 100 ± 0.0 33 ± 3.2

0% Effluent (Untreated Harbor Water) 100 ± 0.0 36 ± 4.0

100% Effluent 90 ± 10.0 20 ± 3.4 a

Test acceptability criteria: 80 % or greater survival and an average of 15 more young per female in the controls.

a The difference in average number of young per female is greater than would be expected by chance; the 100% Effluent group is statistically ( p  0.05) less than the 0% Effluent group.

Table 21. Average (± Standard Error of the Mean) Final Density of S. capricornutum Exposed to Whole Effluent Collected from NaOH Treatment Discharge During Trial D.

Treatment Group S. capricornutum Density (cells/mL) CV (%)

Algae Growth Media (Performance Control) 2,240,625 ± 218,146 19.5

0% Effluent (Untreated Harbor Water) 2,875,000 ± 112,384 7.8

100% Effluent 3,896,875 ± 162,850 a 8.4

Test acceptability criteria: Control flask must exceed 1 * 10 6 cells/mL and not vary more than 20 % among replicates.

a The difference in average cell density is greater than would be ex pected by chance; the 100% Effluent group is statistically ( p  0.05) greater than the 0% Effluent group.GSI/LB/F/A/3 May 13, 2011 Page 48 of 54

Table 22. Average (± Standard Error of the Mean) P. promelas Survival and Dry Weight per Surviving Minnow Exposed to Whole Effluent Collected from NaOH Treatment Discharge During

Trials B and D.

Treatment Group TRIAL B TRIAL D

Survival (%) Dry Weight per Survivor (mg) Survival (%) Dry Weight per Survivor (mg)
Laboratory Water (Performance Control) 98 ± 1.7 0.45 ± 0.02 100 ± 0.0 0.54 ± 0.03

0% Effluent (Untreated Harbor Water) 100 ± 0.0 0.47 ± 0.01 100 ± 0.0 0.58 ± 0.02

100% Effluent 95 ± 3.2 0.53 ± 0.02 100 ± 0.0 0.57 ± 0.02

Test acceptability criteria: 80 % or greater survival in the controls; average dry weight per surviving organism in the controls equal to or exceeding 0.25 mg.



## 4.0. QUALITY MANAGEMENT

GSI uses a wide variety of quality management documents and records to implement its quality management system. These include quality system documentation (i.e., the GSI Quality Management Plan), project-specific documentati on (i.e., Quality Assurance Project Plans), and routine procedures documentation (i.e ., Standard Operating Procedures).



### 4.1. Quality Management Plan (QMP)

Detailed information on the structure and organi zation of GSI’s quality system can be found in the GSI Quality Management Pl an (GSI, 2010b). Electronic c opies of this document are available upon request. The GSI QMP covers a ll aspects of GSI’s commitment to quality including policies and procedures; cr iteria for and areas of application; roles, responsibilities, and authorities; assessment and respons e; and quality improvement. It is the framework for planning, implementing, documenting, and assessing the GS I’s quality assurance and quality control (QAQC) activities.



### 4.2. Quality Assurance Project Plan (QAPP)

Additional information and deta ils regarding the ac tivities undertaken by GSI to assure the quality and credibility of its research at th e Land-Based RDTE Facility can be found in GSI’s Land-Based Quality Assurance Project Plan (GSI, 2010a). This document is available electronically upon request. The QAPP covers all aspects of quality assurance/quality control (QAQC), including data quality indicators, eval uation processes, performance measures and acceptance criteria; instrument certification an d calibration; personnel training requirements; documents and records; data management; a nd QAQC assessments and response actions.GSI/LB/F/A/3 May 13, 2011 Page 49 of 54



### 4.3. Standard Operating Procedures (SOPs)

SOPs are used to implement all GSI test activiti es. This facilitates consistent conformance to technical and quality system requirements and in creases data quality. The SOPs include both programmatic and technical proce sses and procedures such as or ganism culturing; operation of the GSI Land-Based RDTE facility; sample co llection, labeling, analysis and custody; and safety. Appendix 1 provides a list of GSI SOPs relevant to land-based test activities.



## 5.0. DISCUSSION OF RESULTS

The NaOH BWTS operated effectively during the four trials conducted on the proposed

NaOH/CO 2 

### treatment process. In particular, the BW TS led to highly si gnificant reductions in live organism densities in all taxonomic categories evaluated in treated discharge relative to control discharge. It is not possible to assess pe rformance against a discharge standard using the results from these land-based trials; the testi ng conditions and quality assurances were not at IMO- or ETV-consistent levels given the resear ch and development objective of the test series.

In these tests, there was a nearly 100 % re duction in live orga nisms greater than 50  m in minimum dimension (zooplankton) in the treatment discharge as compared to the control discharge, with zero live organisms found in Trials B and D and very few live organisms found in Trials A and C. The overall percent reducti on of live phytoplankton density in the treatment discharge was greater than 97 % as compared to the control discharge density during the four

trials of the NaOH BWTS. In terms of micr obial organisms, any reductions caused by the treatment system in these tests were less than one log in magnitude. Coliform bacteria and E.

coli appeared most sensitive to the BWTS of the organisms tested, decreasing these microbial organisms by an average of 98% and 81%, respectiv ely across the four tria ls. The process had very little effect if any on total heterotrophic bacteria; though densities (i.e. MPN per mL) were on average lower in treated discharge than cont rol discharge. The overall percent reduction was less pronounced for the Enterococci and total heterotrophic bacteria, with greater than 77 % and 67 % reduction as compared to th e control, respectively.

Treatment effectiveness in this larger size class of organisms (greater than 50  m in minimum dimension) appeared roughly consistent with IMO D-2 standards. That is, in three trials, the density estimates were well below IMO D-2 standards (0.0/m 3 

### to 0.5/m 3 

### ; Trials A, B, and D). In one trial (Trial C), the estim ate was clearly above it (19/m 3 

### ), but it was impo ssible to discern a difference between any of these estimates (above or below) and the 10 live organisms per m 3


### benchmark from a statistical standpoint given the small sample volumes analyzed (Miller, et al

, 2011). With no filter system associated with this treatment process, the detritus in the samples subject to analysis was too great to allow analysis of sufficient sample volumes in the time period required to afford a precise estimate of di scharge densities. This problem will likely not go away when the treatment process is subjec ted to actual certification testing, setting up a situation in which many more trials or many more microscopists are needed to generate adequate statistical certainty.GSI/LB/F/A/3 May 13, 2011 Page 50 of 54



### The relatively high density value for live organisms from the ≥ 50  m size class in the Trial C treatment discharge is most likely an artifact resulting from the extremely low sample volumes subject to analysis. To illustrate the effect of this sample condition, consider that Dreissenids figure prominently in Trial C treatment discharge, contributing to nearly one-half of the live organism density. However, due to the sma ll amount of sample volume analyzed for microzooplankton (0.14 m 3 

### ), the discovery of just one live dreissenid during sample analysis accounted for that density estimate. It is interesting to note that the intake and control discharge density of organisms in the ≥ 50  m size class was significantly highe r in Trial C as compared to the other three trials, ranging from 2 - 23 time s higher on intake and 2 - 33 times higher in the control discharge (Table 9), but this difference does not itse lf account for th e disparity. Operational sources of error were likely not th e cause. Cross contamination was prevented in Trial C, as well as the other three trials, and sa mples were analyzed to ensure no live organisms were present in the potable water source used for cleaning, and treatment tubs prior to each discharge operation (see Section 2.1.3.). In ad dition, a thorough review of the raw data did not reveal any potential contamination from the pl ankton net or sampling equipment, nor did the procedural audits conducted du ring the NaOH Trials point out any potential contamination from those sources. The density es timates for organisms in the ≥ 10 and < 50  m size class in treated discharge were consistently low, but our ability to conclude performance within the IMO D-2

standard is nonetheless limited he re by sample size as well (Miller et al ., 2011).

WET test findings were indicative of chronic toxicity and behavi oral effects of 100 % treatment discharge effluent water. Undiluted discharge from the treatment process led to inhibition of cladoceran reproduction, accelerated algal growth, a nd circular swimming in fish. It should be

noted that 100 % effluent is not the condition that aquatic organisms will be subject to in reality at the point of ballast outfall since dilution occurs upon the ballast discharge making contact with the receiving system. Instead it is indicative of a potential fo r residual toxicity. More WET testing using additional dilution le vels should be undertaken in a ny follow-up land-based tests to corroborate the findings and determ ine the percent dilution at whic h toxicity is observed, and at what dilution it no longer is detectable.

In addition, follow-up bench tests at GSI will seek to better elucidate the cause for this effect. The effects may be due to the in creased conductivity or residual CO 2 

### from the neutralization process in the 100 % effluent water as compared to the harbor water control. Bench-scale tests

should seek to rule out attributes in the treated water that could cont ribute to an effect in order to better isolate the causal agent(s).



## 6.0. CONCLUSIONS

The NaOH (Sodium Hydroxide, Lye) BWTS pe rformed very well operationally and well enough biologically to warrant additional testing at the bench, land and ship-based scales. The system successfully treated ballast wa ter without interruption, and su ccessfully neutralized treated ballast water to achieve WIDNR permitting levels for harbor discharge (i.e., pH 6-9). The BWTS also significantly reduced live organism dens ities in treated discharge relative to control discharge in all size classes of organisms. Fi nally, in these tests, the BWTS performance metGSI/LB/F/A/3 May 13, 2011 Page 51 of 54



### discharge target values that we re approximately consistent w ith the IMO Convention’s Annex D2 discharge standards, though prec ision in this estimate was not possible given th e research and development testing parameters. This testing revealed that the water discharged after two- or three-day retention periods wa s not entirely environmentally benign (i.e., with no residual toxicity at the 100 percent effl uent dilution), though the level of residual toxicity in 100 %

effluent evident from these tests ma y not be of regulatory concern.



## REFERENCES

Great Ships Initiative (2009). Technical Repor t on the Sodium Hydroxide (NaOH) Bench-Scale Test Findings. Available at: http://www.nemw.org/GSI /GSI-BS-P-TR-NaOH.pdf

.

GSI (2010a). Quality Assurance Project Plan for Great Ships Initiative (GSI) Land-Based Tests (2010). Northeast-Midwest In stitute, Washington, DC.

GSI (2010b). Great Ships Initiative (GSI) Qual ity Management Plan (QMP), 2010. NortheastMidwest Institute, Washington, DC.

International Maritime Organi zation (IMO), 2004. International Convention for the Control and Management of Ships Ballast Water and Sediment s. As adopted by consensus at a Diplomatic Conference at IMO, Lon don, England, February 13 2004.

International Maritime Organi zation (2008a). Guidelines fo r Approval of Ballast Water Management Systems (G8), Resolution ME PC.174 (58). Adopted on 10 October 2008.

International Maritime Organization (2008b). Procedure for Approval of Ballast Water Management Systems that Make use of Active Substances (G9), Resolution MEPC.169 (57). Adopted on 04 April 2008.

NSF International (2010). Generi c Protocol for the Verification of Ballast Water Treatment Technology. Prepared for the U.S. Enviro nmental Protection Agency, Environmental Technology Verification Program in cooperation with the U.S. Coast Guard, Environmental Standards Division, Washington, DC, and the U.S. Naval Research Laboratory, Center for Corrosion Science and Engineering, Wash ington, DC. Version 5.1., September 2010.

USEPA, Office of Water (2002). Short-term Methods for Estima ting the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms, 4 th 

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

.GSI/LB/F/A/3 May 13, 2011 Page 52 of 54



## APPENDIX 1.

List of GSI SOPs Relevant to the Land-Ba sed Evaluation of the NaOH Ballast Water Treatment System.

SOP CODE SOP TITLE CATEGORY SUBCATEGORY

GSI/SOP/G/A/RK/1 Procedure for Record Keeping Administration Record Keeping

GSI/SOP/G/RA/DM/1 Procedure for Data Entry, Data Quality Control and Database Management Research

Activities Data Management

GSI/SOP/G/RA/SC/2 Procedure for Labeling Samples Collected at the GSI Land-Based RDTE Facility Research

Activities Sample Custody

GSI/SOP/BS/RA/GL/1 Procedure for Verification of Laboratory Balances Research

Activities General Laboratory

GSI/SOP/BS/RA/WET/1 Procedure for Assessing Chronic Residual Toxicity of a Ballast Treatment System to Ceriodaphnia dubia Research

Activities Residual Toxicity

GSI/SOP/BS/RA/WET/2 Procedure for Assessing Chronic Residual Toxicity of a Ballast Treatment System to the Fathead Minnow ( Pimephales promelas ) Research

Activities Residual Toxicity

GSI/SOP/BS/RA/WET/3 Procedure for Assessing Chronic Residual Toxicity of a Ballast Treatment System to the Green Alga ( Selenastrum capricornutum ) Research

Activities Residual Toxicity

GSI/SOP/BS/RA/MA/1 Procedure For Quantifying Heterotrophic Plate Counts (HPCs) Using IDEXX’s SimPlate® for HPC

Method Research

Activities Microbial Analysis

GSI/SOP/BS/RA/MA/3 Procedure for the Detection and Enumeration of

Enterococcus Using EnterolertTM Research

Activities Microbial Analysis

GSI/SOP/BS/RA/MA/4 Procedure for the Detection and Enumeration of

Total Coliforms and E. coli Using IDEXX's Colilert® Research

Activities Microbial Analysis

GSI/SOP/BS/RA/MP/1 General Microbiology Preparation Procedures Research

Activities Microbial Procedures

GSI/SOP/BS/RA/C/3 Procedures for Measuring Organic Carbon in Aqueous Samples Research

Activities Chemistry

GSI/SOP/BS/RA/C/4 Procedure for Determini ng Percent Transmittance (%T) of Light in Water at 254 nm Research

Activities ChemistryGSI/LB/F/A/3 May 13, 2011 Page 53 of 54

SOP CODE SOP TITLE CATEGORY SUBCATEGORY

GSI/SOP/BS/RA/C/8 Procedure for Analyzing Total Suspended Solids (TSS) Research

Activities Chemistry

GSI/SOP/BS/RA/C/9 Procedure for pH Meter Calibration and pH

Measurement Research

Activities Chemistry

GSI/SOP/LB/G/O/1 Procedure for Operating the GSI Land-Based RDTE Facility General Operation

GSI/SOP/LB/G/O/2 Procedure for Sampling and Analyzing Treated Water in the Retention Tanks Prior to Discharge General Operation

GSI/SOP/LB/G/O/3 Procedure for Cleaning and Verifying Cleanliness ofthe Retention Tanks and Piping at the GSI LandBased RDTE Facility General Operation

GSI/SOP/LB/G/O/4 Procedure for Cleaning Sampling Equipment at the GSI Land-Based RDTE Facility General Operation

GSI/SOP/LB/G/O/5 Procedure for Injecting Organisms and Solids into the GSI Land-Based RDTE Facility General Operation

GSI/SOP/LB/G/O/7 Procedure for Maintaining Solids Suspension in the GSI Land-Based RDTE Facility’s Retention Tanks General Operation

GSI/SOP/LB/G/C/4 Procedure for Calibration, Deployment, and Storage of YSI Multiparameter Water Quality Sondes General Calibration

GSI/SOP/LB/G/S/1 Procedure for Ensuring Worker Health and Safety at the GSI Land-Based RDTE Facility General Safety

GSI/SOP/LB/RA/SC/1 Procedure for Collecting Biological Sample Water via In-Line Sample Ports Research

Activities Sample Collection

GSI/SOP/LB/RA/SC/3 Procedure for Algae/Small Protozoa Sample

Collection Research

Activities Sample Collection

GSI/SOP/LB/RA/SC/4 Procedure for Microbial Sample Collection Research

Activities Sample Collection

GSI/SOP/LB/RA/SC/6 Procedure for Zooplankton Sample Collection Research

Activities Sample Collection

GSI/SOP/LB/RA/SA/1 Procedure for Algae/Small Protozoan Sample Analysis Research

Activities Sample Analysis

GSI/SOP/LB/RA/SA/2 Procedure for Zooplankton Sample Analysis Research

Activities Sample AnalysisGSI/LB/F/A/3 May 13, 2011 Page 54 of 54



## APPENDIX 2.

Trimmed, Raw Microbial Analysis Data fr om Testing of Lye (NaOH) Ballast Water Treatment System

Trial Sample Location Sample

Tub Rep. Total

Coliform

Bacteria E. coli Enterococcus

spp. Heterotrophic

Bacteria

MPN/100 mL MPN/100 mL MPN/100 mL MPN/1 mL

A PRE

‐ TREATMENT

INTAKE 4 1 179 42 21 200

2 192 36 45 200

3 345 44 40 800

CONTROL

DISCHARGE 1 1 186 12 4 514

2 79 8 5 454

3 105 12 4 303

TREATMENT

DISCHARGE 4

1 <1 <1 1 363

5 <1 <1 4 363

6 1 <1 1 363

B PRE

‐ TREATMENT

INTAKE 4 1 210 50 35 1210

2 166 52 19 1210

3 236 62 67 1301

CONTROL

DISCHARGE 1 1 17 2 313 1150

2 14 <1 101 923

3 26 <1 16 1603

TREATMENT

DISCHARGE 4

1 <1 <1 <1 104

5 <1 <1 126 124

6 <1 <1 <1 120

C PRE

‐ TREATMENT

INTAKE 4 1 548 113 173 1000

2 461 111 155 1900

3 598 96 88 200

CONTROL

DISCHARGE 1 1 55 4 84 800

2 48 2 65 400

3 51 3 56 1500

TREATMENT

DISCHARGE 4

1 6 <1 1 266

5 <1 <1 15 183

6 <1 <1 1 216

D PRE

‐ TREATMENT

INTAKE 4 1 614 67 186 800

2 461 69 204 1200

3 579 82 103 1350

CONTROL

DISCHARGE 1 1 118 6 80 1900

2 142 11 71 1700

3 127 10 78 1900

TREATMENT

DISCHARGE 4

1 <1 <1 1 216

5 <1 <1 2 155

6 <1 <1 4 151