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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, UWS 

   
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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. 
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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 (CO2) 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 ...................................................................................................................... 33
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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 54
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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 Ambienta 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. ..................................47
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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. .48
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EXECUTIVE SUMMARY

The Great Ships Initiative (GSI) provides independent, no-cost performance verification testing 

services to developers of ballast water treatment 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 GSI is capable of performing testing fully consistent with the 

requirements of the International Maritime Organization’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 (USEPA’s) Environmental Technology Verification 

Program (ETV; NSF International, 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 performance evaluation test of a proposed BWTS 

developed by researchers from the U.S. Geological Survey’s Leetown Science Center in 

Kearneysville, West Virginia. The proposed system involved application 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 (CO2) as a neutralization step prior to discharge of the 

ballast water to the receiving system. The purpose of the land-based test of this system, 

consisting of four trials, was status testing for research and development. As such, the testing 

was based on, though not strictly consistent with, 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) successfully neutralize treated ballast water to achieve Wisconsin 

Department of Natural Resources (WIDNR) permitting levels for harbor discharge (i.e., pH 6-9), 

(c) meet discharge target values for water chemistry/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 environmentally benign (i.e., no residual toxicity) 

pursuant to USEPA water quality criteria. 

The NaOH BWTS performed very well operationally and well enough biologically to warrant 

additional testing at the bench, land and ship-based scales. The system successfully treated 

ballast water without interruption, and successfully neutralized treated 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 possible given the research and development testing 

parameters. The only possible problem that this testing revealed was that the water discharged 

after two- or three-day retention periods was not entirely environmentally benign (i.e., with no 

residual toxicity at the 100 % effluent dilution), though the level of residual toxicity in 100 % 

effluent evident from these tests may not be of regulatory concern. 
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ACKNOWLEDGMENTS

The authors would like to express our sincere gratitude to the Great Ships Initiative (GSI) 

Advisory Committee which provides invaluable input 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 which supported the initial scoping exercise. We 

sincerely thank the United States Department of Transportation, Maritime 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 University of Wisconsin-Superior, and the City of 

Superior, WI, for their active financial and/or in-kind support for GSI operations. 
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1.0. INTRODUCTION

In July 2010, the Great Ships Initiative (GSI) conducted a land-based evaluation of a proposed 

ballast water treatment system (BWTS) developed by researchers from the U.S. Geological 

Survey’s Leetown Science Center (Kearneysville, 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, followed by application of carbon dioxide (CO2) as a neutralization 

step prior to discharge of the ballast water to the receiving system. The purpose of the evaluation 

was status testing for research and development 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) successfully neutralize treated ballast water to achieve 

Wisconsin Department of Natural Resources (WIDNR) permitting levels for harbor discharge 

(i.e., pH 6-9), (c) meet discharge target values for water chemistry/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 was environmentally benign (i.e., 

no residual toxicity) pursuant to USEPA water quality criteria. The testing was based on, though 

not strictly consistent with, 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 Generic 

Protocol for the Verification 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 globally. In support of that 

goal, GSI has established superlative 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, with the goal of facilitating the rapid progression 

of meritorious BWTSs through the research and development and approval processes to a 

market-ready condition. 

GSI has no involvement, intellectual or financial, in the mechanics, design or market success of 

the actual treatment systems it tests. To ensure that GSI tests are uncompromised by any real or 

perceived individual or team bias relative to test outcomes, GSI test activities are subject to 

rigorous quality assurance and quality control (QAQC) procedures 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 the performance of BWTSs 

with IMO guidelines, USEPA ETV Protocol, and other test facilities. GSI test protocols are as 

consistent as possible with the requirements of the IMO Convention for the Control and 
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Management of Ships’ Ballast Water and Sediments (IMO, 2004) and United States federal 

requirements (NSF International, 2010). In particular, 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 for the Verification 

of Ballast Water Treatment Technology, v.5.1 (NSF International, 2010). GSI procedures, 

methods, materials and findings are 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 Leetown 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 use as a BWTS. In 2008, GSI conducted bench-scale 

testing on the proposed NaOH BWTS and determined 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 utilized a version of the 

system that first increases the pH of ballast water on intake to pH 12, and then reduces the pH of 

the discharge water to less than 8.5 (but above 6.5) using carbon dioxide (CO2). 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 conduct independent, scientifically-sound, rigorous, and 

quality assured evaluations of BWTSs. At the same time, GSI seeks immediate relevance of its 

freshwater, land-based testing to regulatory processes such as those outlined in the IMO 

Convention and those under development domestically in the United States and Canada. To that 

end, GSI protocols are rooted in the essential features 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 Verification 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, retention tank size, sample size, sample collection and analysis 

equipment and data logging) are directly consistent with these requirements. GSI also formally 

partners with the Maritime Environmental Resource Center (MERC; Solomons, MD), and other 

land-based test facilities, to ensure that GSI freshwater, land-based testing can be complemented 

by comparable brackish/salt water testing. 
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With respect to physical/chemical and biological characteristics 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 the 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, mineral matter, particulate organic carbon, and 

phytoplankton), GSI has the ability to augment intake 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 report. GSI conducts and documents frequent 

monitoring of water chemistry and biology to predict valid run conditions for GSI, IMO G8, and 

USEPA ETV performance evaluation/certification test trials. 

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

of Various Physical, Chemical, and Biological Parameters in Ambienta Water from the DuluthSuperior Harbor of Lake Superior (June – October). 

Parameter US EPA ETV1 Recommended 

IMO G82

Duluth/Superior Harbor 

Ambient Rangesa

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/m3) 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 

(MPNc/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 land-based test facility from July 6, 2010 to July 

22, 2010. The experimental methods including procedures for sampling and analysis of each 

physical, chemical and biological parameter and variable 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 Verification Program. Generic Protocol for the Verification of 

Ballast Water Treatment Technologies. Version 5.1. September, 2010. 

2 IMO MEPC 57, Annex 3: Revised Guidelines for Approval of Ballast Water Management Systems (G8). April 4, 2008.
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2.1. Experimental Design and Set‐up

The NaOH BWTS test involved physical, chemical, and biological characterization of water 

samples upon uptake/intake of water, as well as, enumeration, sizing, and live/dead analysis of 

organisms in control and treated discharge water after a two- or three-day, in-tank holding time. 

The objective of the performance evaluation trials was to compare control (untreated) and 

treatment discharge in order to estimate the effects of the NaOH BWTS for its ability to: (a) 

successfully treat ballast water without interruption, (b) successfully neutralize 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 biology that are approximately consistent with the 

IMO Convention’s Annex D-2 discharge standards, and (d) discharge water after two- or threeday retention periods that is environmentally benign (i.e., no residual toxicity) pursuant to 

USEPA water quality criteria. 

Table 2 shows the schedule of the four trials, including the sequence of intake operations 

(simultaneous control and treatment) and discharge 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 performance of the NaOH BWTS at GSI’s purpose-built, 

Land-Based Research, Development, Testing and Evaluation (RDTE) Ballast Treatment Test 

Facility located in Superior, WI in the Duluth-Superior Harbor of Lake Superior (Figures 1-3). 

Key features of the facility include: 

 Four x 200 m3

 matched retention tanks with internal agitation for experimental water; 
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 Matched control and treatment intake flows up to 341 m3/hour; 

 Highly automated flow and pressure control, monitoring and data logging; 

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

 Capacity to amend intake water to intensify challenge conditions; 

 Semi-automated and validated facility sanitation between trials; 

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

 On-site laboratory space for live analysis of organisms in the ≥ 10 μm and  50 μm 

and ≥ 50 μm 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 Location 
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Figure 2. Computer-Generated Rendering of the GSI Land-Based RDTE Facility. 

Figure 3. Photo of the GSI Land-Based RDTE Facility. 
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GSI’s Land-Based RDTE Facility draws raw intake water from the Duluth-Superior Harbor at 

400 m3/hr to 680 m3/hr. This main flow of intake water can be augmented with solids and/or 

organisms just prior to being split into control and 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 m3/hr to 340 m3/hr) to a treatment track and the other half (also 200 m3/hr 

to 340 m3/hr) to a matched control track (Figure 4). The treatment track directs water through the 

experimental BWTS and into a 200 m3, cylindrical retention 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 m3/hr to 340 m3/hr. The water is directed either back to the Duluth-Superior harbor, 

to a 260-m3

 wastewater storage tank for subsequent discharge to the City of Superior sewer, 

neutralization, or circulated to a second set of facility retention tanks (Figure 4). 
Figure 4. Simplified Schematic of the GSI Land-Based RDTE Facility Showing Location of Sample Points, Sample Colle

Retention Tanks, and Treatment and Control Tracks. Note: Main intake and discharge lines are cod
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Water is sampled continuously throughout ballasting 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 quality analysis collected at SP#10 (Figure 4). All 

these SPs consist of three identical sample ports spaced at regular intervals 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 center-located, elbow-shaped pitot tube (90o) 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, Florida. 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 unbiased 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 transferred simultaneously and continuously throughout 

ballasting operations (intake, recirculation or discharge) from the sample ports to replicate 3.8 m3

sample collection tubs via clean 3.8 cm (internal diameter) flexible hoses and automated flowcontrolled pneumatic diaphragm valves. The sample 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 than one pitot, only one such pitot is used at a 

time during any given sample collection event. The 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 & 4 
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An on-site mobile field laboratory (Figure 6) and stationary laboratory (Figure 7) provide space 

to support time-sensitive analyses associated with the GSI land-based tests, including live 

analysis of phytoplankton and zooplankton. The laboratories are climate-controlled, and have 

enough bench space to allow for simultaneous analysis 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; approximately three miles from the facility. 

Figure 6. The GSI Mobile Field Laboratory. 

Figure 7. The GSI Stationary Laboratory. 
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2.1.2.		Challenge Conditions and Organism Injection Procedures

 The expected ranges of physical, chemical and biological challenge conditions for the NaOH 

BWTS test trials appear in Table 4. Ambient Duluth-Superior Harbor water conditions were 

employed as the physical and chemical challenge conditions during all four 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 μm) were augmented to achieve 

greater than 1000 cells/mL on intake and thereby intensify challenge conditions. The 

phytoplankton injection procedure 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 Harbor was collected and concentrated using 50- 

to 80 μm plankton nets towed from an outboard-powered boat. The concentrated phytoplankton 

was stored at the GSI Land-Based RDTE Facility in holding ponds equipped with aeration 

systems for less than 48 hours. Prior to injection, holding pond water containing 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 achieve 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 greater than the target system pressure. The 

phytoplankton concentrate was added at a constant rate to the intake water 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 just 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 control 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 – Procedure for Maintaining Solids Suspension in the GSI Land-Based 

RDTE Facility’s Retention Tanks).

. 
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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/m3) 

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 obtained from GSI monitoring data and records from 

June to October 2007 to 2010. 

2.1.3.			Sodium Hydroxide (NaOH) and Carbon Dioxide (CO2) Dosing and Operational

Parameters

A 50 % by weight sodium hydroxide (NaOH) solution 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 every ten seconds during the entire intake operation 

(i.e., before, during, and after NaOH injection) using an inline Signet pH sensor with built-in 

automatic temperature compensation (Georg Fischer 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 procedure began by partially closing the NaOH 

flow control valve (valve #9, Figure 8) in the main line of Treatment Lab #2 and opening the 

valves leading to the dosing pumps (Figure 8). This valve configuration 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 differential reached 8-10 pounds per square inch, both 

centrifugal dosing pumps were started to provide a combined flow of 30-40 gallons per minute 
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(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 using 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-Superior 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 (CO2) gas. An inline pH meter and Walchem pH sensor (Walchem; Holliston, 

MA) with built-in automatic temperature compensation 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 plumbed 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 CO2 gas was injected. The CO2 was fed into the top of the 

Speece Cone so the inline rotameter read 15 standard 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 between 560 and 600 GPM using a combination of 

NaOH Valve #9 

Dosing Pump Suction Line 

Dosing Pump Discharge Line 
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adjusting the discharge valve on the cone and the pump speed. The CO2-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 oriented in opposite directions. The CO2 flow continued at a rate of 15 

SCFM until the inline pH probe indicated a pH of 8.5. Following, the flow of CO2was cut off, 

and the neutralized water continued to recirculate for an additional 10 minutes to ensure the 

water was well mixed. Prior to the discharge operation, a 1 L sample was collected at mid-depth 

(i.e., using a Kemmerer Sampler) from the treatment retention tank, and measured using an 

Orion 3 Star pH Meter and pH Combination Electrode (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 flow rates were equivalent and 

proportional to intake and discharge flow rates throughout each intake and discharge operation. 

Flow rates were recorded continuously every five seconds by automated, in-line sensors located 

on the control track, treatment track, and on the discharge 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., retention tank volume and volume sampled) were 

measured and recorded continuously using a Human Machine Interface (HMI) 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 pressure meters, fill level of the ballast retention 

tanks, and flow rates in the control and treatment lines, etc. The HMI console then saved the 

information to a specific file. An external computer, 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 test trial, the interior of the retention tanks were cleaned according 

to GSI/SOP/LB/G/O/3 – Procedure for Cleaning and Verifying 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 GSI Land-Based RDTE Facility. The GSI facility recirculation lines 

were flushed with potable water from an on-site potable water tank that had been verified to be 

free of living organisms. The flushing was undertaken after each intake and prior to each 

discharge operation. The thoroughness of the cleaning process was checked by partially filling a 

randomly selected treatment sample 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 water was completely free of live Duluth-Superior 

Harbor zooplankton visible with a compound microscope at a magnification 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). 
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2.2. Water Quality Analysis		

2.2.1. pH

Samples for pH analysis were collected during intake as follows: 

 Three 1 L whole water samples were collected 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 Kemmerer 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 

CO2 injection and one after the neutralization procedure was completed, 

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

minutes after the start of the discharge procedure 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 procedure (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 using 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 calibration 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 display 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 the 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 were 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 minutes 
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(i.e., three samples) after the start of the discharge procedure. Samples were collected 

using SP #10 (Figure 4) for Trials A and B and SP #15 (Figure 4) for Trials C and D. 

In addition, one or three 1 L whole water samples were collected from the control line 

approximately 30 minutes (i.e., one sample) 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 more 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 filtered through pre-washed, dried, 

and pre-weighed Whatman 934-AH glass fiber filters. After the sample was filtered, it was dried 

in an oven and brought to constant weight. TSS values were determined 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 concentrations 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 were collected immediately after TSS sample 

collection during intake only as follows: 

 Three 125 mL whole water samples were collected from the pre-treatment line (SP 

#3, Figure 4) approximately 10, 30, and 50 minutes after the start of the operation. 

In these tests, NPOC was measured as a surrogate for total organic carbon (TOC), though it may 

be a slight underestimate of TOC. The analytical instrument used to measure NPOC purges the 

sample with air to remove inorganic carbon before measuring organic carbon levels in the 

sample. Thus, the NPOC analysis does not incorporate any volatile organic 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 acidified with hydrochloric acid for analysis of 

DOC. The remaining portion of the sample was acidified with hydrochloric acid and analyzed 

for NPOC. A Shimadzu Total Organic Carbon Analyzer (Model TOC-5050A; Shimadzu 
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May 13, 2011 

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

Concentrations of NPOC and DOC were determined based on a calibration curve developed on 

the instrument using organic carbon standards prepared from potassium hydrogen phthalate. 

Reported particulate organic carbon (POC) concentrations were determined as the difference 

between the NPOC and DOC values for a given sample. 

Quality control measures consisted of collecting 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 portion 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 Determining Percent Transmittance (%T) of Light in 

Water at 254 nm. A spectrophotometer set at 254 nm was used to measure %T of the filtered 

samples. Deionized water was used as a reference to adjust the spectrophotometer 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 trial 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 approximately 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 samples, 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 respective tanks, the Sondes were removed and taken to the mobile 

laboratory where the data were later downloaded as test files to a laptop computer using 

EcoWatch® for Windows® Software (v.3.18, 14 April 2006; YSI Incorporated); 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 GSI SharePoint intranet website. 

2.3. Viable Organism Analysis

During these trials sample water for analysis of viable organisms was simultaneously and 

continuously collected from replicate sample ports into identical 3.8 m3

 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 than 
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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 m3 

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 filling of the treatment and control 200 m3

 retention tanks, 

the following time-integrated sample volumes were collected and analyzed (additional samples 

were collected but were not analyzed) by continuous flow from the intake lines simultaneously: 

 One 2 m3 sample from the pre-treatment intake line, and 

 One 2 m3

 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 sample volumes were collected and analyzed 

(additional samples were collected but were not analyzed): 

 Two time-integrated samples of 2 m3

 each (total volume 4 m3) were continuously 

collected from the treatment discharge line, and 

 One 2 m3 time-integrated sample was continuously collected from the control 

discharge line. 

Flow control valves and system logic assured that sample flow rates were equivalent and 

proportional to intake and discharge flow rates throughout each operation. Immediately after the 

sample collection tubs were filled, the phytoplankton and microbial whole water samples were 

collected and Sonde readings recorded, followed by the zooplankton sample collection. The 

zooplankton samples were collected by draining the remaining volumes (i.e., 2 m3 minus 5 L of 

rinse/Sonde water and the 1 L phytoplankton and microbial samples) from the sample collection 

tubs and concentrating through 35 μm (50 μm diagonal dimensions) plankton 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 control samples were also collected 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. Microzooplankton (e.g., rotifers, copepod nauplii, and dreissenid 

veligers) and macrozooplankton (e.g., copepods, cladocerans, and insect larvae), all generally 

greater than or equal to 50 μm in minimum dimension, were analyzed simultaneously by 
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separate taxonomists. Microzooplankton subsamples were analyzed in a Sedgewick-Rafter 

counting chamber by examination under a compound microscope at a magnification of 40X to 

100X. Macrozooplankton were analyzed in a Ward’s Counting Wheel at a magnification of 20 

to 30X using a dissecting microscope. Due to high densities, quantification of zooplankton in the 

control intake, pre-treatment intake, and control discharge samples required analysis of subsamples and extrapolation to number live organisms per cubic meter. For these samples, a 

subsample was removed for analysis using a Henson-Stempel pipette. The dead organisms (i.e., 

those organisms that did not move or respond to stimuli) were enumerated, then all organisms in 

the sample were killed by adding 50 % (v/v) acetic acid solution (for microzooplankton) or 

Lugol’s solution (for macrozooplankton) to the counting chamber/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 number of organisms in the counting chamber/wheel. 

The treatment discharge samples had lower densities allowing analysis of a greater proportion of 

the sample (see the “Results” section for the proportion 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 enumerated 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 One 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 equal 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 and 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-parametric test was used. In all cases α=0.050. 

Quality assurance measures during these trials included 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 average relative percent difference of the number of 

live organisms counted were calculated for all second analyses. In addition, all live organisms 

identified in the treatment discharge samples were 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 live zooplankton that did meet 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 μm in minimum dimension, during intake the 

following whole water samples were collected: 
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May 13, 2011 

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 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 three 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 sample collection, with samples stored in coolers 

during the interim. Prior to analysis, samples were concentrated through 10 μm 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 concentrated sample was transferred to 

a 2 mL sample container, with 4 μL of fluorescein diacetate (FDA) viability stain stock solution 

added. The subsample was then allowed to incubate in the dark for 5 minutes. The 1.5 mL 

incubated sample was mixed and 1.1 mL was immediately transferred to a Sedgwick-Rafter cell, 

covered and placed on the stage of a microscope that was set for simultaneous observation using 

brightfield and epifluorescence. At least two horizontal transects were counted (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) counted. 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 showing 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). Records were kept of transect lengths and widths 

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

Entities less than 10 μm in all visible dimensions or greater than 50 μm in minimum visible 

dimension were not counted. Counting and measurement 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 remaining concentrated sample in the 25 mL bottle was archived 

using a preservative (formalin or Lugol’s) for long-term storage. 
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Statistical analysis for the ≥ 10- and < 50 m size class for the four trials was conducted using 

SigmaStat, version 3.5 (Systat Software, 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 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 and 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-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, subsample 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 similarity of taxonomic identification and the average relative 

percent difference of the number of live organisms 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 organisms 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 collected 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 collected immediately after tank discharge 

from the treatment sample collection tubs (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 were enumerated according to GSI/SOP/BS/RA/MA/1 – Procedure 

for Quantifying Heterotrophic Plate Counts (HPCs) using IDEXX’s SimPlate® for HPC Method. 
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This method utilizes the IDEXX SimPlate® for HPC Method (IDEXX Laboratories, Inc.; 

Westbrook, Maine), which is based on IDEXX Laboratories’ 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 Enterococcus 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 Software, 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 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 and 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-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 analyzed 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) testing of the NaOH BWTS was conducted using three 

freshwater species as described 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 determined using standard USEPA procedures 

(USEPA, 2002) following a three-day retention period in the land-based facility’s 200 m3

treatment retention tank (Figure 4). Sample water (i.e., 19 L), collected from one of the 

treatment discharge sample collection tubs using a 20 L, high-density, polyethylene 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 stored 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 water (i.e., filtered through a Whatman 934-AH 

Glass Microfiber Filter, 1.5 m particle retention in liquid) served as the control, and treatments 
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May 13, 2011 

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consisted of 0 % treatment discharge water (i.e., filtered harbor water control), 100 % treatment 

discharge water, and a performance 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 % and 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 culturing conditions for each species, providing the 

test organisms with the optimal environment for survival, growth, and reproduction. Therefore, 

the performance control, along with the reference toxicant tests, provides 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 Receiving 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 ensured 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) 
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2.5. Data Management

2.5.1.		Data Recording

All biological and chemical data were recorded by hand (using indelible ink) on pre-printed data 

collection forms and/or in bound, uniquely-identified laboratory notebooks that were specific to 

the NaOH BWTS test. The data that were recorded on pre-printed data collection forms were 

secured in uniquely-identified three 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., temperature, pH, dissolved oxygen, specific conductivity, 

salinity, turbidity, and total chlorophyll) was measured continuously every fifteen minutes during 

each retention period and automatically recorded in a text file, which was later translated to a MS 

Excel spreadsheet. Facility data (e.g., flow rates and pressure measurements) were electronically 

recorded every five seconds during intake and discharge. 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 compliance 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 the data type. More detail on 

the GSI’s data validation activities is additionally detailed in Section 7 of the GSI Quality 

Assurance Project Plan for Land-Based Tests (GSI, 2010a). This section also details the 

acceptable values, where appropriate, for the following 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 organized 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 control for access to the 

LSRI LAN. The LSRI LAN is automatically backed 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 

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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-controlled, secure archive room at the LSRI for five 

years after this report 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 from the above methods.

3.1. Intake and Discharge Challenge Conditions

3.1.1. Operational Conditions

Operational conditions, measured continuously during intake, for all four trials were extremely 

consistent with each other and between treatment and control tracks of the GSI Land-Based 

Facility. Flow rate was slightly below 200 m3/hour and pressure in the facility lines was 30.4 – 

31.6 psi (Table 6). On discharge, the flow duration, flow rate, and pressure was very similar 

between the treatment and control 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 

(m3/h) 

Total Volume 

of Water 

Treated (m3) 

 Control 

Flow Rate 

(m3/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 31.6 
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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) (m3

/h) (m3/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 entirely DOC, as the average of both parameters was 

13.4 mg/L throughout the four trials. The %T ranged from 24.8 to 29.4 in the first three trials, 

but in Trial D rose substantially (42.7 %T). The average % T across 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 collected immediately prior to the water entering the 

treatment retention tank was 12.02, achieving the target pH of 12.00. Samples 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 target 

value of less than 8.5, and was within the WIDNR permitting levels for discharge to the DuluthSuperior Harbor (i.e., pH 6 to 9). 
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May 13, 2011 

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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) 

pH1

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 intake, 22.6 ± 1.5 °C in the 

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

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

m size class and for the larger plankton size class (i.e., ≥ 50 μm), are summarized in Table 9. 

During all four trials, average intake densities of live zooplankton (i.e., those organisms ≥ 50 

μm) ranged from 28,331/m3 (Trial A) to 648,158/m3 (Trial C), for an average of 291,229/m3. 

The zooplankton community was comprised mainly of dreissenid mussel veligers; the rotifers 

Keratella, Polyarthra, and Synchaeta; calanoid and cyclopoid copepods; and the cladoceran 

Bosmina. The target intake density of ≥ 100,000/m3

 was achieved during all 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/m3

 (Trial A) to 725,980/m3 (Trial C), which was 78 % to 

112 % of the starting densities. Control discharge densities in Trials B and C were slightly higher 

than their corresponding intake densities (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/mL (Trial A) to 661.91 cells/mL (Trial D, in 

which the intake stream was amended with concentrated harbor algae; Table 9). The community 

of protists comprised, in decreasing relative abundance, chain-forming diatoms (largely 

Aulacoseira), coccoid green algae (largely Gonium), filamentous blue-green algae (Oscillatoria), 

miscellaneous microflagellates, and free-living centric diatoms, such as Cyclotella. The density 
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May 13, 2011 

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of live phytoplankton in the control discharge samples ranged from 16.00 cells/mL (Trial C) to 

275.95 cells/mL (Trial D), for an average of 93.43 cells/mL (Table 9). This represented 4 % to 

42 % of the intake densities (Table 9). Although organism densities were lower in the control 

discharge samples, the relative abundance of taxa were similar to that observed 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 μm (#/m3) 

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 μm 

(#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 during Trials C and D. Total coliform 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 heterotrophic bacteria ranged from 400 MPN/mL in 

Trial A to 1240 MPN/mL in Trial B (Table 10). In the control discharge, 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). 
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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 ± 0.5a 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 LOD 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 measured prior to the start of the neutralization 

period, and the values measured after neutralization and just prior to the start of the discharge 

operation. Only data from Trials C and D are reported; 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, including 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 treatment 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 

CO2 was 7.57 and 8.18 for the two trials respectively, 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. The neutralization process decreased the specific conductivity by 

approximately half, but on average the post-neutralization conductivity just prior to treatment 
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May 13, 2011 

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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) increased 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 neutralization process reduced the salinity 

by about half from the average salinity measured prior to CO2 injection but the levels in the 

treatment tank just prior to discharge were still an average of eight times higher than the levels in 

the control tank just prior to discharge 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 turbidity reading for the control tank water as substantially higher 

than all other readings for control and as compared to the treatment tank water (i.e., average 12.7 

NTU in the control tank and 1.8 NTU in the treatment tank), possibly indicating a problem with 

the Sonde probe post calibration. In both trials, the turbidity 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)
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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 volumes 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 addition, live densities of four groups of bacteria 

(i.e., total coliform, E. coli, Enterococcus spp., and total heterotrophic) in the <10 m size class 

are reported in Tables 17 and 18. Percent reduction of live organism density in the treatment 

discharge as compared to the control 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 volume 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 limited the sample volume that could be analyzed for 

live organisms ≥ 50 μm in minimum dimension prior to maximum sample holding time (i.e., two 

hours). Sample volumes analyzed for live macrozooplankton ranged from 0.6 m3

 to 2.2 m3 and 

sample volumes analyzed for live microzooplankton ranged from 0.1 to 0.3 m3

 (Tables 12 and 

13). These low volumes resulted in low statistical certainty of density estimates. 
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May 13, 2011 

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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 (#/m3) 0.5 0.0 19 0.0 4.9 ± 4.8 

Total Vol. Analyzed (m3) 

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 μm in minimum dimension in treated discharge 

across all four trials was 4.9 live organisms/m3 (Table 12). Table 13 provides the relative 

densities of live organisms across taxa in the treated discharge from the four trials. In Trial A, 

one live ostracod was observed in the treated discharge sample resulting in a density estimate of 

0.5 live organisms per m3

 (Tables 12 and 13). There were 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 resulting in a density estimate of 0.80 and 1.67 per m3 

respectively (Table 13). Trial C treated discharge had a total of 19.2 live zooplankton per m3 ≥

50 m (Table 12), including chironomid larvae, planaria, copepods, and dreissenid larvae (Table 

13). 
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May 13, 2011 

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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, m3: 

2.21 

(0.24 MicroZP)

1.28 

(0.30 MicoZP) 

0.82 

(0.14 MicroZP) 

0.60 

(0.08 MicroZP) 

≥ 50 μm (min. dimension)

Taxa Group Avg. Density 

(per m3

) 

Avg. Density 

(per m3)

Avg. Density 

(per m3)

Avg. Density 

(per m3

)

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 

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

< 50 μm (min. dimension)

Taxa Group Avg. Density 

(per m3

)

Avg. Density 

(per m3)

Avg. Density 

(per m3)

Avg. Density 

(per m3

)

Chironomid 0.80 1.22 1.67 

Less than 50 μm (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/m3

 live). Many observed. 

Phytoplankton A few observed 

with chlorophyll. 

Copepod Nauplii 

Four live 

observed under 

dissecting scope. 

The results of the statistical comparison between live organism density in the treatment discharge 

and in the control discharge are shown in Table 14. The Kruskal-Wallis One Way ANOVA was 

used to compare the two groups, as the data were normally distributed but did not have equal 

variance and transformation of the data (e.g., log base 10, log normal, and square root) did not 

successfully produce data that met the assumptions of a One Way ANOVA. After four trials of 

the NaOH BWTS, overall live zooplankton density in the treated discharge was significantly (p = 

0.029) lower than that of the control discharge. 
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Table 14. Result of Statistical Comparison of Live Zooplankton Density in Control Discharge to 

Treatment Discharge. 

The hypothesis tested was that the NaOH BWTS significantly 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/m3 319,063 159,531 

26.000 0.029 1 in 34 Treatment 

Discharge 5 live/m3 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 cell/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 provided in Table 16 below. The data were not 

normally distributed; therefore, the data were transformed using log (base 10) transformation. 

Overall, the live phytoplankton density in the treatment discharge was significantly (p = 0.002) 

lower as compared to the untreated, control 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 significantly 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 0.5 
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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. The 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 all 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 and D) to 42 MPN/100 mL (Trial B) in 

treatment discharge. Total heterotrophic 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 compared 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 ± 2a 1 1 

E. Coli 

(MPN/100 mL) 1 1 1 1 1 

Enterococci (MPN/100 mL) 2 ± 1a 42 ± 42a 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 LOD 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 discharge 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 (base 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. 
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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 significantly 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 regulated size classes was calculated by the following equation: 

ݕݐ݅ݏ݁݊ܦ	݃݁ݎ݄ܿܽݏ݅ܦ	ݐ݉݁݊ݐ݁ܽݎܶ൬ െ 1 	 ൤ൌ ݃݁ݎ݄ܿܽݏ݅ܦ	ݐ݉݁݊ݐ݁ܽݎܶ	݅݊	݊݋݅ݐܿݑܴ݁݀	ݐܿ݁݊ݎܲ݁

100% ൈ ൰൨ ݕݐ݅ݏ݁݊ܦ	݃݁ݎ݄ܿܽݏ݅ܦ	݈݋ݎݐ݊݋ܥ

Table 19 summarizes the percent reduction in live organism density in treatment discharge for all 

four trials of the NaOH BWTS. The largest percent 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 treatment discharge as compared to the control 

discharge during Trials B and D. Trials A and C saw an approximate reduction of 100 % of the 

live zooplankton density in the treatment discharge, although 0.5 live/m3

 and 19 live/m3 were 

counted in each trial, respectively. Trials B and D were also the most successful for 

phytoplankton with an approximate treatment discharge reduction of 100 % (5.5 live cells/mL 

were counted) in Trial B and 99 % reduction in Trial 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 bacteria in treatment 

discharge was reduced less than one log as compared to the control discharge. There was an 
GSI/LB/F/A/3 

May 13, 2011 

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

68 % reduction in live heterotrophic 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 mb

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 treatment discharge was not observed. b Percent reduction of live bacteria density in treatment discharge as compared to control discharge is based on the 

average (n = 3 samples) live organism density per trial in treatment and control discharge. c

 Average (n =3) treatment discharge density values were below the 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 percent reduction calculations. 

3.3. Whole Effluent Toxicity (WET) Testing

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 

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 filtered harbor water controls (0 % treatment 

discharge water) met the test acceptability criteria 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 results 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 % Effluent group (Table 20). In 

Trial D, each female in the 100 % Effluent produced an average of 20 young as compared to 36 

young per female in the 0 % Effluent (Table 20). This result indicates a potential effect of 100 % 

treatment discharge on cladoceran reproduction. 

In the WET test conducted on the algal species S. capricornutum using treatment discharge from 

Trial D, the average cell density at test termination in the 100 % Effluent group (3,896,875 

cells/mL) was significantly (p0.05) higher as compared to the 0 % Effluent group (2,875,000 
GSI/LB/F/A/3 

May 13, 2011 

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cells/mL), suggesting a possible effect 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 Control 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 Treatment 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 ± 3.4a

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 ± 162,850a 8.4 

Test acceptability criteria: Control flask must exceed 1 * 106 cells/mL and not vary more than 20 % among replicates. a The difference in average cell density is greater than would be expected by chance; the 100% Effluent group is statistically 

(p0.05) greater than the 0% Effluent group. 
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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 documentation (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 organization of GSI’s quality system can be found in 

the GSI Quality Management Plan (GSI, 2010b). Electronic copies of this document are 

available upon request. The GSI QMP covers all aspects of GSI’s commitment to quality 

including policies and procedures; criteria for and areas of application; roles, responsibilities, and 

authorities; assessment and response; and quality improvement. It is the framework for planning, 

implementing, documenting, and assessing the GSI’s quality assurance and quality control 

(QAQC) activities. 

4.2. Quality Assurance Project Plan (QAPP)

Additional information and details regarding the activities undertaken by GSI to assure the 

quality and credibility of its research at the 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, evaluation processes, performance measures and 

acceptance criteria; instrument certification and calibration; personnel training requirements; 

documents and records; data management; and QAQC assessments and response actions. 
GSI/LB/F/A/3 

May 13, 2011 

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4.3. Standard Operating Procedures (SOPs)

SOPs are used to implement all GSI test activities. This facilitates consistent conformance to 

technical and quality system requirements and increases data quality. The SOPs include both 

programmatic and technical processes and procedures such as organism culturing; operation of 

the GSI Land-Based RDTE facility; sample collection, 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/CO2 treatment process. In particular, the BWTS led to highly significant reductions in 

live organism densities in all taxonomic categories evaluated in treated discharge relative to 

control discharge. It is not possible to assess performance against a discharge standard using the 

results from these land-based trials; the testing conditions and quality assurances were not at 

IMO- or ETV-consistent levels given the research and development objective of the test series. 

In these tests, there was a nearly 100 % reduction in live organisms 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 reduction 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 microbial 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%, respectively across the four trials. 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 control 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 the 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/m3

 to 0.5/m3; Trials A, B, and D). In 

one trial (Trial C), the estimate was clearly above it (19/m3), but it was impossible to discern a 

difference between any of these estimates (above or below) and the 10 live organisms per m3

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 discharge densities. This problem will likely not 

go away when the treatment process is subjected to actual certification testing, setting up a 

situation in which many more trials or many more microscopists are needed to generate adequate 

statistical certainty. 
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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 small amount of sample volume analyzed for 

microzooplankton (0.14 m3), 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 higher in Trial C as compared to 

the other three trials, ranging from 2 - 23 times higher on intake and 2 - 33 times higher in the 

control discharge (Table 9), but this difference does not itself account for the disparity. 

Operational sources of error were likely not the cause. Cross contamination was prevented in 

Trial C, as well as the other three trials, and samples 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 addition, a thorough review of the raw data did not 

reveal any potential contamination from the plankton net or sampling equipment, nor did the 

procedural audits conducted during the NaOH Trials point out any potential contamination from 

those sources. The density estimates 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 here by sample size as well (Miller et al., 2011). 

WET test findings were indicative of chronic toxicity and behavioral effects of 100 % treatment 

discharge effluent water. Undiluted discharge from the treatment process led to inhibition of 

cladoceran reproduction, accelerated algal growth, and 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 for residual toxicity. More WET 

testing using additional dilution levels should be undertaken in any follow-up land-based tests to 

corroborate the findings and determine the percent dilution at which 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 increased conductivity or residual CO2 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 contribute to an effect in order to 

better isolate the causal agent(s). 

6.0. CONCLUSIONS

The NaOH (Sodium Hydroxide, Lye) BWTS performed very well operationally and well enough 

biologically to warrant additional testing at the bench, land and ship-based scales. The system 

successfully treated ballast water without interruption, and successfully neutralized treated 

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 
GSI/LB/F/A/3 

May 13, 2011 

Page 51 of 54 

discharge target values that were approximately consistent with the IMO Convention’s Annex D2 discharge standards, though precision in this estimate was not possible given the research and 

development testing parameters. This testing revealed that the water discharged after two- or 

three-day retention periods was not entirely environmentally benign (i.e., with no residual 

toxicity at the 100 percent effluent dilution), though the level of residual toxicity in 100 % 

effluent evident from these tests may not be of regulatory concern. 

REFERENCES

Great Ships Initiative (2009). Technical Report 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 Institute, Washington, DC. 

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

 

International Maritime Organization (IMO), 2004. International Convention for the Control and 

Management of Ships Ballast Water and Sediments. As adopted by consensus at a Diplomatic 

Conference at IMO, London, England, February 13 2004. 

International Maritime Organization (2008a). Guidelines for Approval of Ballast Water 

Management Systems (G8), Resolution MEPC.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). Generic Protocol for the Verification of Ballast Water Treatment 

Technology. Prepared for the U.S. Environmental 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, Washington, DC. Version 5.1., September 2010.

USEPA, Office of Water (2002). Short-term Methods for Estimating the Chronic Toxicity of 

Effluents and Receiving Waters to Freshwater Organisms, 4th Edition. Accessed from: 

http://www.epa.gov/waterscience/methods/wet/disk3/index.html. 
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APPENDIX 1. 

List of GSI SOPs Relevant to the Land-Based 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 Determining Percent Transmittance 

(%T) of Light in Water at 254 nm 

Research 

Activities Chemistry 
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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 of 

the 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 Analysis 

   
GSI/LB/F/A/3 

May 13, 2011 

Page 54 of 54 

APPENDIX 2. 

Trimmed, Raw Microbial Analysis Data from 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