EPA/600/R-10/146 

September, 2010 

GENERIC PROTOCOL FOR THE
 

VERIFICATION OF BALLAST WATER
 

TREATMENT TECHNOLOGY
 

Produced by
 

NSF International
 

Ann Arbor, MI
 

For
 

U.S. Environmental Protection Agency
 

Environmental Technology Verification Program
 

In cooperation with 

U.S. Coast Guard
 

Environmental Standards Division
 

Washington, DC
 

And 

U.S. Naval Research Laboratory
 

Center for Corrosion Science and Engineering
 

Washington, DC
 
 

 

 

 

 

 

 

 

Protocol for the Verification of Ballast Water Treatment Technologies September 2010 

Version 5.1 

NOTICE 

The U.S. Environmental Protection Agency, through its Office of Research and Development, 

funded and managed, or partially funded and collaborated in, the research described herein. It has 

been subjected to the Agency’s peer and administrative review and has been approved for 

publication. Any opinions expressed in this report are those of the author (s) and do not 

necessarily reflect the views of the Agency, therefore, no official endorsement should be inferred. 

Any mention of trade names or commercial products does not constitute endorsement or 

recommendation for use. 

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Protocol for the Verification of Ballast Water Treatment Technologies September 2010 

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FOREWORD
 

The EPA is charged by Congress with protecting the nation’s air, water, and land resources. 

Under a mandate of national environmental laws, the Agency strives to formulate and implement 

actions leading to a compatible balance between human activities and the ability of natural 

systems to support and nurture life. To meet this mandate, the EPA’s Office of Research and 

Development provides data and science support that can be used to solve environmental problems 

and to build the scientific knowledge base needed to manage our ecological resources wisely, to 

understand how pollutants affect our health, and to prevent or reduce environmental risks. 

The Environmental Technology Verification (ETV) Program has been established by the EPA to 

verify the performance characteristics of innovative environmental technology across all media 

and to report this objective information to permitters, buyers, and users of the technology, thus 

substantially accelerating the entrance of new environmental technologies into the marketplace. 

Verification organizations oversee and report verification activities based on testing and quality 

assurance protocols developed with input from major stakeholders and customer groups 

associated with the technology area. ETV consists of six environmental technology centers. 

Information about each of these centers can be found on the Internet at http://www.epa.gov/etv 

Under a cooperative agreement, NSF International has received EPA funding to plan, coordinate, 

and conduct technology verification studies for the ETV “Water Quality Protection Center” and 

report the results to the community at large. The WQP Center’s primary technology areas address 

surface water pollution concerns such as ship ballast water treatment, wastewater treatment, 

stormwater runoff treatment, confined animal feeding operations (CAFOs), and urban 

infrastructure rehabilitation. 

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Protocol for the Verification of Ballast Water Treatment Technologies September 2010 

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ACKNOWLEDGEMENTS
 

EPA and NSF acknowledge and thank those persons who participated in the preparation and 

review, subsequent validation, and the revision of this Generic Protocol for Verification of Ballast 

Water Treatment Technologies. Without their hard work and dedication to the project, this 

document would not have been possible. The original draft was prepared by Battelle, Duxbury, 

MA, while significant input in later drafts was provided in a multi-year effort by the Naval 

Research Laboratory, Key West, FL. Much input was also provided by the biological sub-group 

of the Ballast Water Treatment Protocol Technical Panel as well as the Technical Panel in whole. 

Revised Protocol Authors 

Edward Lemieux Naval Research Laboratory 

Jonathan Grant Battenkill Technologies, Inc. 

Timothy Wier EXCET, Inc. 

Lisa Drake Science Applications International Corporation 

NRL Technical Staff 

Stephanie Robbins Science Applications International Corporation 

Kevin Burns Science Applications International Corporation 

Scott Riley Science Applications International Corporation 

Luke Davis Science Applications International Corporation 

Wayne Hyland Azimuth Technical Consultants, Inc. 

Bruce Nelson Battenkill Technologies, Inc. 

Tiffanee Donowick Science and Engineering Technologies, Inc. 

Robert Brown Azimuth Technical Consultants, Inc. 

Original Protocol Writers 

Deborah Tanis Battelle 

Carlton D. Hunt Battelle 

NSF Staff 

Thomas Stevens Project Manager, ETV Water Quality Protection Center 

EPA Staff 

Raymond Frederick Project Officer, ETV Water Quality Protection Center 

ETV Water Quality Protection Center – Technical Panel Participants for Current and Past 

Versions 

Donald Anderson Woods Hole Oceanographic Institute 

Allegra Cangelosi Northeast-Midwest Institute 

Dorn Carlson National Oceanic and Atmospheric Administration 

Fred Dobbs Old Dominion University 

Richard Everett U.S. Coast Guard 

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Protocol for the Verification of Ballast Water Treatment Technologies September 2010 

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Maurya Falkner	 California State Lands Commission 

Richard Fredricks	 Maritime Solutions, Inc. 

Stephan Gollasch GoConsult and Chairman, ICES/IOC/IMO Working Group 

on Ballast and Other Ship Vectors (WGBOSV) 

Frank Hamons	 American Association of Port Authorities 

Richard Harkins	 Formerly with the Lake Carriers Association, currently 

with Keystone Shipping Company 

Penny Herring	 U.S. Coast Guard R&D Center 

Russell Herwig	 University of Washington 

Brian Howes	 University of Massachusetts 

James Hurley	 U.S. Coast Guard R&D Center 

Thomas Mackey	 Hyde Marine, Inc. 

Lucie Maranda	 University of Rhode Island 

Kathy Metcalf	 Chamber of Shipping of America 

Richard Mueller	 Northeast Technical Services Company, Inc. 

Gail Roderick	 U.S. Coast Guard R&D Center 

Andrew Rogerson	 California State University, Fresno 

Terri Sutherland	 Fisheries and Oceans Canada 

Mario Tamburri	 University of Maryland 

Fred Tsao	 U.S. Navy 

Thomas Waite	 Florida Institute of Technology 

Nick Welschmeyer	 Moss Landing Marine Laboratories 

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Table of Contents
 

Chapter 1 Introduction ....................................................................................................................1
 

1.1 The ETV Program............................................................................................................... 1
 

1.2 Objectives of Verification Testing...................................................................................... 1
 

1.3 Purpose and Scope of the Protocol ..................................................................................... 2
 

1.4 Verification Testing Process............................................................................................... 2
 

1.5 Policies and Program Specifications and Guidelines.......................................................... 2
 

Chapter 2 Responsibilites of Involved Organizations ....................................................................3
 

2.1 Vendor................................................................................................................................. 3
 

2.2 Testing Organization (TO).................................................................................................. 3
 

2.3 TF Owner (Owner) ............................................................................................................. 3
 

2.4 Verification Organization (VO).......................................................................................... 4
 

2.5 Environmental Protection Agency (EPA)........................................................................... 4
 

2.6 Stakeholder Advisory Group (SAG)................................................................................... 4
 

2.7 Technology Panel................................................................................................................ 4
 

Chapter 3 Ballast Water Treatment System Capabilities and Description .....................................5
 

3.1 Ballast Water Treatment System Definition....................................................................... 5
 

3.2 Technology or Treatment Performance Claims.................................................................. 5
 

3.3 Acceptability for Testing .................................................................................................... 6
 

3.4 Test BWTS Requirements .................................................................................................. 6
 

3.5 Operating and Maintenance (O&M) Evaluation................................................................. 7
 

3.6 Biological Efficacy Evaluation with Standard Test Organisms ......................................... 9
 

3.7 Calibration and Test Requirements................................................................................... 10
 

3.8 System Documentation Evaluation................................................................................... 10
 

3.9 Technical Data Package Submission ................................................................................ 11
 

3.10 Format for the BWTS Technical Data Package................................................................ 12
 

Chapter 4 Treatment Verification TQAP Development ...............................................................14
 

4.1 Description of Ballast Water Treatment System .............................................................. 14
 

4.2 Required Elements of the TQAP ...................................................................................... 14
 

Chapter 5 Experimental Design ....................................................................................................16
 

5.1 Test Verification Factors................................................................................................... 16
 

5.1.1 Biological Treatment Efficacy...................................................................................... 17
 

5.1.2 Operation and Maintenance .......................................................................................... 17
 

5.1.3 Reliability...................................................................................................................... 17
 

5.1.4 Cost Factors .................................................................................................................. 17
 

5.1.5 Environmental Acceptability ........................................................................................ 17
 

5.1.6 Safety Factors................................................................................................................ 17
 

5.2 Challenge Conditions........................................................................................................ 18
 

5.2.1 Challenge Water – Water Quality Characteristics........................................................ 18
 

5.2.2 Challenge Water - Biological Organism Conditions .................................................... 21
 

5.2.3 Challenge Water – Flow Rates and Volumes ............................................................... 22
 

5.3 TF Physical Configuration................................................................................................ 22
 

5.3.1 Overall experimental configuration .............................................................................. 22
 

5.3.2 Sampling Methodology................................................................................................. 23
 

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5.3.3 Test Organism & Water Quality Augmentation ........................................................... 29
 

5.3.4 Control & Instrumentation............................................................................................ 29
 

5.4 Verification Testing .......................................................................................................... 30
 

5.4.1 Treatment System Commissioning............................................................................... 31
 

5.4.2 Operation and Maintenance (O&M) Manual................................................................ 32
 

5.4.3 Vendor and Test Organization Requirements............................................................... 32
 

5.4.4 Toxicity Testing for Biocide Treatments...................................................................... 33
 

5.4.5 BE and O&M Verification Strategy: Test Duration and Coordination ........................ 33
 

5.4.6 Biological Efficacy (BE) Verification Testing ............................................................. 35
 

5.4.7 BE Validity Criteria ...................................................................................................... 48
 

5.4.8 Alternative and Emerging Methods.............................................................................. 50
 

5.4.9 Operation and Maintenance Verification Factor........................................................... 51
 

Chapter 6 Reporting Verification Testing Results........................................................................57
 

Chapter 7 Quality Assurance/Quality Control (QA/QC)..............................................................58
 

7.1 Verification of Test Data .................................................................................................. 58
 

7.2 Project Management ......................................................................................................... 58
 

7.3 Measurement and Data Acquisition.................................................................................. 58
 

7.4 Assessment........................................................................................................................ 59
 

Chapter 8 Data Management, Analysis and Presentation.............................................................60
 

8.1 Data Management ............................................................................................................. 60
 

8.2 Data Analysis and Presentation ........................................................................................ 60
 

Chapter 9 Environmental, Health, and Safety Plan ......................................................................61
 

References......................................................................................................................................62
 

Appendix A Quality Assurance Project Plan............................................................................... 65
 

Appendix B Approach for Evaluating standard Test Organisms.....................................70
 

Appendix C Report on Discharge Sampling.............................................................96
 

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List of Tables 

Table 1. Acceptable Variations for Frequency and Voltage...........................................................7
 

Table 2. Recommended Standard Test Organisms for Bench-Scale Testing ...............................10
 

Table 3. Water Quality Challenge Matrix for Verification Testing..............................................19
 

Table 4. Minimum Criteria for Challenge Water Total Living Populations.................................22
 

Table 5. Accuracy and Precision Requirements for Potential Sensors.........................................24
 

Table 6. Likely Treatment Sequences and Applications Inherent to Ballast Operations .............31
 

Table 7. Criteria for Concentrations of Living Organisms in Control Tank Discharge ...............34
 

Table 8. Core and Potential Auxiliary Parameter and Measurement Techniques ........................36
 

Table 9. Sample Volumes, Containers and Processing.................................................................38
 

Table 10. Recommendation for Water Quality Sample Analysis Methods..................................39
 

Table 11. Data Quality Objectives for Water Quality Samples....................................................40
 

Table 12. Density Confidence Intervals for Poisson Distributions Using the Chi-Square Statistic
 

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

Table 13. Sample Volume Required Relative to Treatment Standards–Organisms ≥ 50 μm ......44
 

Table 14. Sample Volume Required Relative to Treatment Standards Organisms ≥ 10 μm and <
 

50 μm .....................................................................................................................................44
 

Table 15. Challenge Test Validation Criteria by Location ...........................................................50
 

List of Figures 

Figure 1. Sampling design example for in-tank treatment............................................................26
 

Figure 2. Sampling design example for in-line treatment.............................................................27
 

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GLOSSARY OF TERMS 

Accuracy: The degree of agreement between an observed value and an accepted reference value, 

including a combination of random error (precision) and systematic error (bias) components that 

are due to sampling and analytical operations (EPA, 1992). 

Ambient Populations: The biological organisms, including bacteria, protists, and zooplankton 

that are naturally occurring in the water at the TF location. 

Ballast Water Treatment System (or System): Prefabricated, commercial-ready, treatment 

systems designed to remove, kill or inactivate (prior to discharge) organisms in ballast water. The 

entirety of a vendor’s ballast water treatment product will be used to achieve the vendor claims for 

treatment efficacy or operational performance, and includes all components, in an integrated 

fashion. 

Bias: The systematic or persistent distortion of a measurement process that causes errors in one 

direction. 

Challenge Water: Water supplied to a treatment system under test. Challenge water must meet 

specified ranges for living organism densities and water quality parameters and is used to assess 

the efficacy of the treatment equipment under full-scale operational conditions. 

Comparability: The measure of the confidence with which one data set can be compared to 

another. 

Completeness: The amount of data collected as compared to the amount needed to ensure that 

the uncertainty or error is within acceptable limits. 

Core Parameters: The measurements that are required as part of the ETV verification. 

Cyst: The dormant cell or resting stage of microalgae, heterotrophic protists, and metazoans, 

including but not limited to cysts of dinoflagellates, spores of diatoms, cysts of heterotrophic 

protists, and cysts of rotifers. 

Effluent: The treated discharge water produced by a ballast water treatment system. 

Equipment: The ballast water treatment system, defined as either a package or a modular system, 

which is tested in the Verification Testing Program. 

ETV Testing: Testing of a technology under the EPA Environmental Technology Verification 

Program following provisions of an established protocol and/or TQAP, with the final outcome 

being a Verification Report, containing all findings of the test, and a Verification Statement, 

signed by the US EPA and the Verification Organization (VO). 

In-Line Treatment: A treatment system or technology used to treat ballast water during normal 

flow of ballast during uplift or discharge. 

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In-Tank Treatment: A treatment system or technology used to treat ballast water during the 

time that it resides in the ballast tanks. This may involve treatment steps during uptake. 

Mean Time Between Failure (MTBF): The predicted elapsed time between inherent failures of 

a system during operation. MTBF can be calculated as the arithmetic mean (average) time 

between failures of a system. The MTBF is typically part of a model that assumes the failed 

system is immediately repaired (zero elapsed time), as a part of a renewal process. This is in 

contrast to the mean time to failure (MTTF), which measures average time to system failure with 

the modeling assumption that the failed system is not repaired. 

Normally distributed data: Data that meet the following criteria: the data forms a bell shaped 

curve when plotted as a graph, the mean is at the center of the distribution on the graph, the curve 

is symmetrical about the mean, the mean equals the median, and the data are clustered around the 

middle of the curve with very few values more than three standard deviations away from the mean 

on either side. 

Owner: The owner of a test site used for verification testing of a ballast water treatment system. 

Performance Data: Removal efficacy and effluent concentration data for core and supplemental 

parameters for a given set of Challenge conditions. 

Precision: The degree to which a set of observations or measurements of the same property, 

obtained under similar conditions, conform to themselves. Precision is usually expressed as 

standard deviation, variance, or range, in either absolute or relative terms (NELAC, 1998). 

Protocol: A written document that clearly states the objectives, goals, scope, and procedures for 

the study of a particular group of similar technologies. A protocol shall be used for reference 

during vendor participation in the verification testing program. 

Proxy Measurement: A parameter used in lieu of another measurement (i.e., chlorophyll a as a 

bulk measure of phytoplankton). 

Quality Assurance Project Plan (QAPP): A written document that describes the 

implementation of quality assurance and quality control activities during the life cycle of the 

project (also see Test/quality assurance plan). 

Representativeness: The degree to which data accurately and precisely represent a characteristic 

of a population. 

Sensitivity: The capability of a test method or instrument to discriminate between different levels 

(e.g., concentrations) of a variable of interest. 

Stakeholder Advisory Group (SAG): A group overseen by a Verification Organization (VO) 

consisting of representatives from verification customer groups, technology developers and 

vendors, the consulting engineer sector, the finance and export communities, and government 

permitters and regulators. 

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Standard Operating Procedure (SOP): A written document containing specific instructions and 

protocols to ensure that quality assurance requirements are maintained. 

Standard Test Organisms: Biological organisms of known types and abundance that have been 

previously evaluated for their level of resistance to physical and/or chemical stressors representing 

ballast water technology. The organisms are added to the challenge water during testing of ballast 

water treatment technologies to determine treatment system effectiveness. 

Start-Up: The period between the time the ballast water treatment system is activated and when 

stable operating conditions are achieved. 

Stable Operation: The time interval following a start-up period that the ballast water treatment 

system performs consistently within the range of vendor-specified operating conditions. 

Supplemental Parameters: A measurement taken that is specific to a particular treatment and 

augments the results of the core parameter measurements. 

Technical Panel: A group comprised of a subset of stakeholders and other individuals with a 

technical expertise in various ballast water issues, such as fresh water and marine biologists, 

environmental scientists, engineers, and ship architects. 

Test Cycle: One fill/discharge cycle (including appropriate holding periods) designed to gather 

data on treatment efficiency. 

Test Facility: A site that provides the necessary infrastructure, systems and personnel to 

complete the verification testing described in this protocol. The facility may be part of the Testing 

Organization or may be independent from the Testing Organization, but in any case shall be 

totally independent from technology vendors testing at their site. 

Test/Quality Assurance Plan (TQAP): Also called a Quality Assurance Project Plan (QAPP), 

this is a written document that describes the procedures for conducting a test or study according to 

the verification protocol requirements for the application of a particular ballast water treatment 

system at a particular site. At a minimum, the TQAP shall include detailed instructions for 

sample and data collection, sample handling and preservation, precision, accuracy, goals, and 

quality assurance and quality control requirements relevant to the particular site. 

Testing Organization (TO): An organization qualified to conduct studies and testing of ballast 

water treatment technologies in accordance with protocols and TQAPs. 

Upset Conditions: Deviation or exception from normal or vendor-defined operating conditions, 

for example, system faults or hardware failures. 

Vendor: A business that manufactures, assembles, or sells ballast water treatment technologies. 

Verification: The establishment of evidence on the performance of a ballast water treatment 

system under specific conditions, following a predetermined study protocol(s) and TQAP(s). 

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Verification Organization (VO): The party responsible for overseeing TQAP development, 

overseeing testing activities in conjunction with the Testing Organization, and overseeing the 

development and approval of the Verification Report and Verification Statement for the ballast 

water treatment system. Within the ETV Program, verification organizations are the managers 

and operators of the various technology centers under cooperative agreements with the EPA. 

Verification Report: A detailed report on the testing results of a particular technology according 

to an approved Test /Quality Assurance Plan and conducted under the ETV Program. The report 

is typically prepared by the TO and contains a description of the test facility, photographs of 

technology being tested methods and procedures, presentation of analyzed data including all 

QA/QC data obtained during the test. Appendices include raw data sets and lab audit information, 

TQAP, O&M Manual and other relevant information. Both the verification report and verification 

statement are publically available on the ETV Program’s web site and NSF’s web site . 

Verification Statement: An executive summary of the verification report, usually 4-6 pages in 

length which is signed by EPA and the verification organization.. The verification statement is 

intended to be used by the vendor for sales and marketing purposes. 

Verification Test: A complete test of a treatment system, following a well defined TQAP which 

includes enumeration of ambient and test populations in the challenge water to determine the 

efficacy of the technology. Also see ETV Testing. 

Viable: According to the IMO G8 guidelines, “organisms and any life stages thereof that are 

living”. 

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Abbreviations and Acronyms
 

ATP Adenosine triphosphate 

BE Biological efficacy 

BWTS Ballast water treatment system(s) 

CT Concentration-time relationship (curve) demonstrating the relationship between 

concentration and time that achieves desired treatment effect. 

m3 Cubic meter 

CFR Code of Federal Regulations 

DOC Dissolved organic carbon 

DOM Dissolved organic matter 

EPA U.S. Environmental Protection Agency 

ETV Environmental Technology Verification 

FRU Field replaceable unit 

μg/L Micrograms per liter 

mgd Million gallons per day 

mg/L Milligrams per liter 

MAWP Maximum allowable working pressure 

MM Mineral matter 

MOA Memorandum of agreement 

MSDS Material safety data sheets 

MTBF Mean time between failures 

NRL U.S. Naval Research Laboratory 

NSF NSF International (formerly National Sanitation Foundation) 

NTU Nephelometric turbidity unit 

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O&M Operations and maintenance 

OSHA Occupational Safety and Health Administration 

Owner TF owner, if different from the Testing Organization (TO) 

POM Particulate organic material 

PSU Practical salinity units 

QA Quality assurance 

QAPP Quality assurance project plan 

QC Quality control 

QMP Quality management plan 

SAG Stakeholder Advisory Group 

SOP Standard operating procedure 

STO Standard test organism 

TF Test Facility 

TO Testing Organization 

TQAP Test/quality assurance plan 

TSS Total suspended solids 

USCG U.S. Coast Guard 

VO Verification Organization 

WQPC Water Quality Protection Center 

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Chapter 1
 

Introduction
 

1.1 The ETV Program 

The U.S. Environmental Protection Agency (EPA) established the Environmental Technology 

Verification (ETV) Program in 1995. The goal of the ETV Program is to promote environmental 

protection by accelerating the development and commercialization of improved and more costefficient environmental technologies through third-party verification, performance reporting, and 

information dissemination. The ETV Program neither certifies nor endorses environmental 

technologies, but rather provides objective, high-quality, peer-reviewed performance data that 

can be utilized by customer groups and regulators when selecting, permitting, or certifying the 

use of environmental technology. The ETV Program’s Water Quality Protection Center 

(WQPC) addresses technologies to protect surface and ground water from chemical or biological 

contamination and conducts performance verifications of technologies resulting in 

comprehensive reports that are publically available on the ETV Program web site. Further 

information on the ETV Program can be obtained at http://www.epa.gov/etv. 

Through a formal Memorandum of Agreement (MOA) signed in 2001, the U.S. Coast Guard 

(USCG) and EPA formed a partnership to develop procedures for evaluating the performance of 

ballast water treatment systems (BWTS). The partnership also provided the Coast Guard a 

pathway to begin the development of technical procedures for approving BWTSs for installation 

on ships. EPA’s interest includes the ecological, economic and public health impacts of ballast 

water discharges. Ballast water treatment is viewed as an important step in mitigating the 

proliferation of aquatic invasive species in U.S. coastal waters and the Great Lakes. 

1.2 Objectives of Verification Testing 

The objective of ETV ballast water treatment technology testing is to evaluate the performance 

characteristics of commercial-ready treatment technologies with regard to specific verification 

factors, including biological treatment performance, predictability/reliability, cost, environmental 

acceptability, and safety. Given the variety of ship and ballast tank types, and potential treatment 

system configurations, this protocol addresses the use of a land-based testing facility (TF) rather 

than shipboard testing, to provide controlled conditions for verifying treatment performance. 

Land-based BWTS verification testing will be conducted in a manner providing information that 

is comparable to the maximum practical extent, to ensure that consumers and other stakeholders 

can make informed choices in selecting appropriate ballast water treatment technology for 

shipboard installations. 

It is believed that ballast water treatment systems performing well under the controlled but 

challenging conditions specified in this protocol at land-based testing facilities will have a 

reasonable chance of performing as well in a shipboard installation. However, because of the 

various designs used in ship ballasting systems and the water quality conditions encountered by 

vessels in seaports around the world, any assumptions of shipboard technology performance 

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based solely on land-based testing results should be avoided. Thorough evaluation of ballast 

water treatment technology must also include shipboard trials to monitor biological performance 

and other ship-related verification factors over an extended period of time. The U.S. Coast 

Guard’s Environmental Standards Division http://www.uscg.mil/environmental_standards/ 

should be contacted for information concerning procedures for shipboard testing of ballast water 

treatment technologies. 

1.3 Purpose and Scope of the Protocol 

The parties involved with ETV testing, including vendors, testing organizations, testing site 

owners, and verification organizations, can use the information provided in this protocol as 

guidance for BWTS verification testing. This protocol provides guidance on the necessary 

elements of verification testing including: technology acceptability; vendor provided 

specifications and information; and test/quality assurance plan (TQAP) development and 

content. The protocol is intended for verification testing of entire BWTSs, not individual 

component technologies that could be combined to form a system. The systems addressed by the 

protocol could be in many configurations, such as treatment on uplift or discharge, treatment intransit (in-tank), or combinations of these options. 

Periodic review and revision of protocols is a critical aspect of the ETV Program. As such, this 

protocol will be reviewed periodically and revised as necessary. These efforts will keep the 

protocol scientifically and functionally up to date. 

1.4 Verification Testing Process 

Verification testing is a three-step process, consisting of planning, verification, and data 

assessment/reporting phases. The planning phase includes development of standardized 

challenge conditions and the specific experimental design as it will be applied to the testing of 

the vendor’s BWTS. A site and treatment system-specific TQAP are prepared during the 

planning phase in accordance with the guidance provided in Chapter 4 of this protocol. The 

BWTS vendor, Testing Organization (TO), and Verification Organization (VO) collaborate on 

the planning phase documents. The verification phase involves the testing of the BWTS by the 

TO under the conditions and standard operating procedures specified in the TQAP. In the data 

assessment and reporting phase, data are processed and analyzed by the TO, who prepares the 

draft verification report and verification statement. The VO is responsible for QA review of the 

data generated during the testing and coordination of the finalization of the verification report 

and statement. 

1.5 Policies and Program Guidelines 

Treatment system verification testing will be conducted in accordance with an approved TQAP 

(for test specific activities) and with the policies and guidelines set forth by an established 

Quality Management Plan (QMP) for the testing facility. Examples of ETV Center QMPs and 

quality assurance plan documents for other testing activities can be viewed on the ETV 

Program’s web site. EPA also provides guidance documents for preparing QMPs and quality 

assurance project plans (QAPPs) at http://www.epa.gov/quality 

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Chapter 2 

Responsibilities of Involved Organizations 

Verification testing will involve several organizations with responsibilities divided among them. 

These organizations may include the vendor of the treatment system, the TO (TO), the Test 

Facility (TF) owner, the Verification Organization (VO), EPA, and sometimes the Technology 

Panel and Stakeholder Advisory Group. 

2.1 Vendor 

The vendor of the ballast water treatment system will apply to the VO for verification testing. 

The vendor must provide the VO and TO verification testing objectives and any existing relevant 

performance data, along with the information required in Chapter 3. This information will be 

considered during the development of the TQAP, which will be reviewed and approved by the 

vendor. The vendor will provide a complete System along with any relevant operation and 

maintenance manuals. Additionally, the vendor will be responsible for assuring proper 

installation and set up of the equipment at the test site, training of TO personnel on BWTS 

operation, and confirmation of the system’s proper operation prior to commissioning and 

commencement of maintenance or treatment efficacy testing. It is strongly recommended that 

the vendor inspect the installation and operation of the system prior to the initiation of the 

testing. The vendor will be available for logistical and technical support as required during the 

planning and verification phases, but will not be directly involved in the testing. The vendor will 

also be responsible for reviewing the verification report and statement generated from the TO. 

2.2 Testing Organization (TO) 

The TO is responsible for preparing the TQAP and working with the vendor and VO to assure 

EPA approval of the TQAP, conducting the verification testing and all aspects of test data 

management, and may be responsible for preparing drafts and final versions of the verification 

report and verification statement. The TO is also responsible for coordinating all personnel and 

testing activities, operating the vendor’s equipment as specified in the equipment operations and 

maintenance manual(s), and evaluating and reporting on the performance of the equipment. 

Maintaining security for testing activities and site safety for all personnel is also the 

responsibility of the TO. 

2.3 TF Owner (Owner) 

If different from the TO, the Owner of the verification TF may provide logistical and technical 

support during planning and verification phases, as agreed upon by the TO, vendor, and Owner. 

The Owner must notify the TO of any logistical or operational developments that may affect the 

verification testing process and results. 

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2.4 Verification Organization (VO) 

The VO is responsible for the technical and administrative operation of the ETV Program’s 

Water Quality Protection Center and all verification activities conducted on behalf of the ETV 

Program. The VO is responsible for overseeing the development and approval of the TQAP, and 

collaborating with the TO to administer testing activities at the TF. The VO is also responsible 

for reviewing, revising and submitting the Verification Report and Statement to the EPA project 

officer for final QA and technical review. The Report and Statement are typically drafted by the 

TO, but they may be drafted by the VO or a contractor to the VO. The VO is also responsible 

for initiating and coordinating periodic review and revision of this protocol. 

2.5 Environmental Protection Agency (EPA) 

The EPA Office of Research and Development (ORD), through the National Risk Management 

Research Laboratory (NRMRL) in Cincinnati, Ohio oversees the Environmental Technology 

Assessment, Verification and Outcomes Staff (ETAVOS) where the ETV Program is 

headquartered. The ETV Program’s Water Quality Protection Center is managed 

administratively from ETAVOS. The Project Officer (PO) for the WQPC is assigned to the 

Water Supply and Water Resources Division, Urban Watershed Management Branch. The 

Project Officer is responsible for administrative and technical management of the cooperative 

agreement with the VO. The PO is also responsible for obtaining EPA reviews of TQAPs for 

BWTS verification testing, the verification report and statement generated from the testing, and 

for assuring that the report and statement are posted on the EPA/ETV web site. EPA is also 

responsible for coordinating review and approval of revisions that may be proposed to this 

protocol. 

2.6 Stakeholder Advisory Group (SAG) 

Stakeholder Advisory Groups (SAGs) are established in each of the ETV Program’s six Centers, 

and consist of representatives from verification customer groups, such as buyers and users of 

technology, developers and vendors, the consulting engineering sector, the finance and export 

communities, and government regulators. The SAGs support generic verification protocol 

development, prioritizing the types of technologies to be verified, and defining and conducting 

outreach activities appropriate to the technology area and customer groups. In addition, the 

SAGs may review WQPC-specific procedures and selected ETV verification reports emerging 

from the ETV WQPC and serve as information conduits to the particular constituencies that each 

member represents. The Ballast Water SAG, of the WQPC, is charged with addressing ballast 

water treatment technologies. 

2.7 Technology Panel 

The Technology Panel is comprised of a subset of stakeholders and other individuals with 

technical expertise in ballast water and environmental technology issues. Scientists, engineers, 

technology vendors, naval architects, and regulators supported the development of this 

Verification Protocol as participating panel members. In the future, the Technology Panel may 

be responsible for reviewing TQAPs and verification reports and statements. The Panel will also 

play a key role in working with the VO in reviewing and revising this protocol as needed. 

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Chapter 3 

Ballast Water Treatment System Capabilities and Description 

3.1 Ballast Water Treatment System Definition 

For the purposes of this verification testing program, ballast water treatment systems (BWTS) 

are defined as: 

Prefabricated, commercial-ready, treatment systems designed to remove, kill or 

inactivate (prior to discharge) organisms in ballast water. This includes all components, 

in an integrated fashion, required for shipboard operation. 

Note that it is understood that many of the proposed regulatory discharge standards, and in fact 

the desired effect of BWTSs, is that these technologies should render organisms unviable or 

incapable of reproduction. In other words, to “kill, remove or inactivate” is technically 

unnecessary when the objective is to eliminate the organism’s capability for reproduction. 

However, as the introduction of “viability” as a measure of efficacy significantly complicates the 

Protocol and test methods, and since “kill, remove or inactivate” is a conservative approach, the 

latter has been adopted as the measure of biological efficacy in this Protocol. 

This definition includes both in-line (systems that treat the flow of ballast water either on uplift 

or discharge) and in-tank systems (systems that treat ballast water during the time it resides in the 

ballast tanks). Typically, BWTSs treat an average design flow between 1.4 – 17 m3 per minute 

(370 – 4,490 gpm) or a total tank volume within a range of 20 – 14,500 m3 (5,280 – 3,830,000 

US gal). 

Systems that will be tested under this program will be capable of treating the entire discharge or 

ballast water volume for biological organisms, either through a one-step treatment process or 

through multi-step treatment processes, and will be capable of treating a wide range of source 

water typical of ballast uplifted from fresh, coastal, estuarine and/or marine origins. These 

technologies may be biological, physical, or chemical in nature or a combination of any or all of 

the technologies. Treatment systems, or components of systems, that provide only partial 

treatment of the discharge are excluded from verification testing. 

3.2 Technology or Treatment Performance Claims 

The vendor will supply a statement of treatment performance claims for the treatment or 

technology. Discharge water quality specifications should reference current EPA regulations or 

recommendations for shore discharge standards. The statement should include, as a minimum: 

� Quantitative measures of biological treatment efficacy expressed as a concentration upon 

discharge for a range of biological size groups as defined in Section 0; minimum 

reporting parameters are specifically detailed in Section 5.4.6; 

� Quantitative measures of operational performance requirements to achieve the biological 

treatment performance stated above; these should include, as a minimum, the allowable 

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and treatable flow rate range and water quality (dissolved and particulate matter 

concentration and particulate size range, salinity, water temperature, turbidity and 

dissolved oxygen content); 

� Treatment capabilities over the anticipated range of maritime environmental conditions 

must be identified by the vendor; the effects of extremes in temperature, turbidity, 

biomass density, or other environmental conditions that may impact the treatment system 

must be noted where these may cause variations in Vendor performance specifications; 

� Quantify the concentration of disinfection residuals, by-products and toxicity for relevant 

systems; 

� The required operational and maintenance conditions (operator time, power requirements, 

chemical consumption requirements, reliability, etc.) to achieve the biological 

performance under a range of source water conditions typical to fresh, coastal, estuarine, 

and marine ballast water (water conditions are detailed in Section 0); and 

� The projected mean-time between failure (MTBF) for the technology given the operation 

and maintenance schedules provided for the technology. 

3.3 Acceptability for Testing 

The treatment system must meet the definition of a BWTS and all existing environmental 

regulatory requirements for operation and treatment byproduct discharge (including EPA 

Registration under FIFRA for any antimicrobial chemical used in the system as active 

substances). The system must be safe for the crew to operate and be compatible with other 

shipboard systems as defined by marine equipment certification procedures by the American 

Bureau of Shipping (ABS), or Det Norske Veritas (DNV). Only complete treatment systems will 

be accepted for verification testing. Moreover, it is anticipated that a BWTS will have undergone 

bench-scale testing with standard test organisms (STO) to validate treatment efficacy under 

controlled laboratory conditions prior to the full scale standardized testing within the ETV 

Program. 

The VO has the right to reject a proposed system that does not satisfy the definition of a BWTS 

in Section 3.1. A proposed treatment system may also be denied acceptance to the verification 

testing program if, for technical or logistical reasons, it cannot be accommodated at the TF or its 

use will result in non-compliance with the discharge requirements for the TF. 

3.4 Test Requirements for BWTS 

All piping, valves and fittings shall comply with regulations and marine industry standards as 

contained in applicable sections of 46 CFR Subchapter F. Pressure piping shall be fitted with 

relief valves set not to exceed maximum allowable working pressure (MAWP). 

Electrical and electronic components in alternating current (AC) systems must be capable of 

operating satisfactorily under normally occurring variations in voltage and frequency. Unless 

otherwise stated, the variations from the rated value may be taken from Table 1. Direct current 

(DC) system devices must be capable of operating satisfactorily at minus 15% voltage. 

Conductors, power supply, and over-current protection shall be provided in accordance with 46 

CFR Subchapter J and appropriate marine industry standards. 

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Table 1. Acceptable Variations for Frequency and Voltage 

Quantity in Operations Permanent Variation Transient Variation 

Frequency ±5% ±10% (5 s)
 

Voltage +6%, -10% ±20% (1.5 s)
 

Operating conditions and tolerances for TO supplies of water pressure and flow, power 

conditions, air pressure and flow, or any other requirements specific to the BWTS must be 

clearly identified in system documentation. 

 

System design should provide for appropriate lift and/or hoist points during installation. Center 

of gravity, no step areas and other installation specific information should be clearly identified. 

Any areas presenting a hazard to personnel during installation, checkout, and operation should be 

visibly marked. 

Recommendations to ensure post-installation operator access to maintenance ports, access 

panels, and field replaceable units (FRUs) should be clearly identified in an installation guide 

with appropriate layout diagrams. 

3.5 Operating and Maintenance (O&M) Evaluation 

The BWTS will be evaluated during the testing to determine if the system is: 

� Designed and constructed to ensure that user access is restricted to essential controls for 

normal operation of the system; 

� If access beyond these controls is available for emergency maintenance and temporary 

repair, and requires the breaking of security (lockout) seals or activation of another 

device indicating an entry to the equipment; 

� Provides capability for efficient maintenance and repair operations and provides a high 

mean-time between failures (MTBF); 

� If minor and major maintenance schedules, pre-requisite training, level of effort, and 

recommended spares/supplies are detailed in the appropriate sections of the O&M 

manual; 

� If adequate documentation, including drawings, diagrams and instructions necessary for 

routine maintenance, troubleshooting, and repairs, are provided; 

� Designed to ensure any potential exposure to hazards or hazardous materials that are 

involved in the maintenance or operation of the equipment are minimized; 

� If explicit warning labels identifying the hazard are installed in accordance with OSHA 

and/or other appropriate federal regulations; 

� If procedures for working with stated hazards are clearly identified in the operating 

instructions; 

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� If by-product, disposable component, or field replaceable unit (FRU) that presents a 

safety or environmental hazard are explicitly identified, along with procedures for 

material handling and disposal according to relevant regulations; and 

� If the vendor provides technical support for this system via phone and internet, including 

contact information for both methods. 

The BWTS operation and control capability will be evaluated to determine the following: 

� The control system ensures that services needed for the proper operation of the BWTS 

are provided through automatic arrangements and operators are promptly alerted when 

conditions warrant human intervention; 

� The operator is able to control all BWTS functions through a single control unit; 

� The control unit automatically monitors and adjusts optimal treatment dosages or 

intensities, or other aspects of the BWTS, and/or provides control signals to the ballast 

water system of the vessel to properly provide the necessary treatment; 

� The control unit provides a continuous self-monitoring function when the BWTS is in 

operation; 

� The control unit includes a tamper-proof or tamper-evident recording device, located in a 

position easily accessible to the person in charge of the BWTS, that provides the operator 

the parameters listed below during ballast water treatment while continuously logging the 

data: 

o	 Proper functioning and status of all the services needed for the operation of the 

BWTS; 

o	 All parameters necessary to ensure the proper operation of the BWTS; 

o	 Status of the valves present in the BWTS, including those leading to overboard 

discharge; 

o	 Total quantity of ballast water treated; 

o	 Ballast water treatment rates; 

o	 Alarm conditions; 

o	 Date and time of start and end of the treatment operation; 

o	 Ballast operation monitored (upload, discharge); 

o	 Calibration and maintenance events; 

o	 Other system events of interest; 

o	 Relevant and necessary measurement information required for control and monitoring 

operation of the BWTS; 

o	 Meter and sensor accuracy to measure the suite of parameters appropriate and 

necessary for control of the BWTS, representing the actual value of the parameters 

being monitored within 10% despite the presence of contaminants normally expected 

in ballast water and the operational environment of the BWTS; 

o	 Diagnostics to enable the local operator to check the functioning of the electrical and 

electronic circuitry, as well as the calibration of meters and sensors according to the 

manufacturer’s specifications; 

o	 An emergency manual override function to be used in the event of failure of the 

control unit; 

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o	 Audio and visual alarms and a recording in the event there is discharge of any 

effluent or a component failure whenever the control unit is not fully operational; and 

o	 The capability to print reports and logged data, as applicable, or stored electronically 

with printout capability, upon the following events: 

− the BWTS is started 

− the BWTS is stopped 

− an alarm condition develops 

− normal conditions are restored 

− manual override is engaged 

� In case of a single failure compromising the proper operation of the BWTS, audible and 

visual alarm signals are given in all stations from which ballast water operations are 

controlled, including, but not limited to, the following conditions: 

o	 Power failure to the BWTS or any subsystem; 

o	 Failure of any sensor, meter, or recording device; 

o	 Hazardous condition detected by control system; and 

o	 Operation outside set points of the BWTS for proper treatment. 

3.6 Biological Efficacy Evaluation with Standard Test Organism 

Standard test organisms (STOs) shall be used in bench-scale tests to mimic and assess the 

efficacy of the ballast water treatment system. Such tests occur in the laboratory prior to fullscale testing. Recommended STOs are identified in Table 2 along with the recommended 

densities to be added to the experimental water in the laboratory experiments. 

The viability of STOs used in bench-scale tests should be determined with the following 

parameters: using one organism from each size class listed in Table 2, treating the STOs in 

conditions identical to the ballast water treatment system being tested (e.g., 18 ppm of sodium 

hypochlorite) and following the experimental replication and use of controls as well as the 

guidance for synthetic water preparation described in Anderson et al. (2008, which is included in 

Appendix B of this document; e.g., tests are run in quadruplicate for bacteria and protists and run 

at least in triplicate for zooplankton). If STOs are cultured rather than purchased from a vendor, 

the methods described in Anderson et al. (2008) should be followed. Bench-scale tests may be 

completed by the test facility or another organization; results should be included in the Technical 

Data Package (Section I.8, Test Results/Qualification Data) that is submitted to the TO following 

full-scale testing. 

If the STOs identified in Table 2 are unsuitable for use, alternatives may be considered and 

utilized with completion of validation experimentation and the concurrence of the VO. Test 

facilities wishing to replace any of the recommended STOs with other organisms should conduct 

sufficient experimentation and provide evidence indicating a broad resistance to treatments as 

outlined by Anderson, et al. (2008). The Anderson research identified the recommended 

standard test organisms as a function of biological functional group and salinity. Similar 

methods, as described in Appendix B, should be used by the TF to determine replacements for 

those STOs. 

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Table 2. Recommended Standard Test Organisms for Bench-Scale Testing 

Minimum Size Class Marine/Brackish Water Fresh Water Concentration 1 

Zooplankton Artemia franciscana Ostracod 105 organisms/m3 

Protists Tetraselmis sp. Green microalgae 103 organisms/mL 

Bacteria Geobacillus sp. Geobacillus sp. 103 organisms/mL 

1 The volumes of water used in the laboratory do not have to match those in the table, but the concentration of 

organisms should be equivalent (e.g., 102 zooplankton/l is acceptable for 105 zooplankton/m3

). 

3.7 Calibration and Test Requirements 

The BWTS will be evaluated during the testing to determine if the system provides: 

� Diagnostic routines and procedures to maintain accuracy of measured process 

parameters, including: 

o	 The degree to which diagnostics are automated; 

o	 If self test routines are incorporated as part of the control unit; 

o	 If the manufacturer specified appropriate diagnostic intervals; and 

o	 If the diagnostics confirm that parameters are within specifications or that 

calibration is required. 

� Diagnostics for fault checking, system maintenance and repair; 

� Automated diagnostics that also may be manually initiated by the operator; 

� Diagnostics that isolate faults down to field replaceable units (FRUs); 

� If the accuracy of the system components that take measurements are verifiable according 

to the manufacturer’s instructions; and 

� If only the manufacturer or persons authorized by the manufacturer do the accuracy 

checks. 

3.8 System Documentation Evaluation 

The documentation provided for the BWTS will be evaluated during verification to determine if 

the specifications provide detailed requirements and tolerances for the following system 

parameters: 

� Ballast water turbidity, pressure, temperature and flow rate ranges (include any other 

applicable criteria); 

� Electrical power requirements; 

� Air/pneumatic pressure and flow rate ranges; 

� Weight; 

� Dimensions; 

� Environmental limitations (e.g., ambient temperature); 

� Treatment limitations; 

� Safety hazards; and 

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� The vendor provided list of procedures for unpacking and verifying contents of shipped 

items. 

The documentation of the installation procedures and requirements in the installation guide will 

be evaluated to determine if: 

� All areas of mechanical, electrical, hydraulic, pneumatic, and any other interface 

requirements are addressed; 

� Time estimates in man-hours provided for installation procedures are appropriate; 

� If applicable standards are referenced and special precautions and hazards identified; and 

� Appropriate diagrams, photographs and/or assembly drawings detail footprints, 

attachment points, interfaces, and any referenced components or subassemblies. 

The adequacy of the O&M manual(s) provided with the system will be evaluated during the 

verification. If not included in the O&M manual, ancillary documentation provided with the 

BWTS will be evaluated for the detail provided for the following items: 

� Piping and instrumentation diagrams; 

� Electrical schematics and wiring diagrams; 

� Photographs; 

� Guides for diagnostics and troubleshooting; 

� Parts lists; and 

� Operator training – minimal additional special training required to operate the system 

(identified and supplied). 

3.9 Technical Data Package Submission 

A technical data package must be submitted to the TO by the Vendor of a BWTS to be 

considered for verification. Vendor-specific performance claims should be identified along with 

relevant existing performance data. 

The information in the technical data package should demonstrate that the treatment processes 

are well characterized and the equipment is designed to meet specific ballast water treatment 

performance criteria at the intended operational scale. Photographs with appropriate reference 

scales should be included. The data package shall also document operational and maintenance 

requirements and conditions. At a minimum, the technical documentation provided by the 

Vendor should address the items identified in the format outline in Section 3.9. 

Much of the required information will likely be available in the Vendor O&M manual(s), which 

are part of the required documentation. The information presented in an O&M manual will, 

however, vary by vendor. To be considered for verification testing under this protocol, vendors 

are required to submit a technical documentation package. This allows each vendor the 

opportunity to incorporate those data most appropriate to the content topic. In addition to the 

technical data package and the O&M manual(s), vendors may also provide ancillary reference 

information through any combination of manuals, product literature, and electronic files. Any 

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ancillary information or proprietary information must be clearly identified as such, and the 

intended purpose/relevance of providing the information must be clearly stated. 

While not required for verification, but likely to be part of a submittal for regulatory compliance, 

the manufacturer may provide certifications or quality assurance documentation for all vendor 

QA/QC and factory testing that occurs during the manufacture of the equipment. If provided, 

relevant standards traceability data should also be provided. 

3.10 Format for the BWTS Technical Data Package 

A	 Cover Page 

B	 Table of Contents 

C	 General Description & Capabilities (Marketing and technical specifications, and other items 

below) 

C.1 System volume, weight, power & mechanical interface requirements 

C.2	 Vendor performance objectives (vendor should describe primary and non-primary 

objectives of ETV testing, i.e., verification testing, or full scale evaluations) 

D	 Target operating environments and conditions 

D.1 General Features 

D.2 Permitting and Certifications 

D.3	 Scalability (no specified requirement – please address range of applicable ballast system 

volumes and rates for the described treatment system) 

E	 Installation Requirements and Instructions 

E.1 Hydraulic and mechanical connections 

E.2 Electrical connections to mains 

E.3 Hazard locations 

E.4 Other special installation criteria / handling 

E.5 Considerations for maintenance / consumables / repair 

E.6	 Shipping and delivery considerations (no specified requirement – vendor should 

describe ability / methods to transport treatment system) 

E.7	 Interfacing for performance monitoring, alarms & controls (no specified requirement – 

vendor should describe available options) 

F	 Operating and Maintenance Instructions 

F.1	 Operating and Maintenance Manual (may provide as standalone document(s), but – any 

references in the text of the technical data package to the separate O&M manual 

must/should be specific to page and paragraph.) 

F.2 Training Materials 

F.3 Repairs and Troubleshooting 

F.4 Recommended Spares (and sources) 

F.5 Safety Precautions and Issues 

F.6 Environmental Hazards and Issues, Including By-Products 

F.7 Expendables, Materials Handling, and Waste Disposal 

F.8 Technical Support contact information 

G	 System Performance Specifications 

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G.1 Discharge water quality
 

G.2 Treatment capabilities vs. environmental conditions
 

G.3 Control features and capabilities
 

G.4 Factory testing criteria and procedures (for entire system and ancillary equipment)
 

G.5 Human operator requirements (special skills or training required to operate the system)
 

G.6 Data Storage
 

G.7 Automated capabilities
 

G.8 Alarms and safety capabilities
 

H Calibration and System Test Procedures 

H.1 Diagnostics
 

H.2 Quality assurance during operation
 

H.3 Calibration schedules and procedures
 

I Detailed Description of System Operation 

I.1	 Theory, processing and principles of operation (no specified performance requirement –
 

vendor should provide background on how and why treatment system works, including
 

explanation of any environmental limiting factors)
 

I.2 Selection of materials used in fabrication
 

I.3 Design considerations for marine applications
 

I.4	 Ancillary Documentation Package (this section is for documentation not referenced
 

elsewhere)
 

I.5 Reference drawings and photographs
 

I.6 Materials / parts lists
 

I.7 Certifications (such as American Bureau of Shipping certifications)
 

I.8	 Test Results / Qualification Data (no specified requirements – this should be results of
 

vendor and/or independent testing of system performance)
 

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Chapter 4
 

Treatment Verification TQAP Development
 

4.1 Description of Ballast Water Treatment System 

Each ballast water treatment verification test will be completed following a written TQAP. From 

the vendor-supplied treatment system documentation submitted as outlined in Chapter 3, the 

TQAP should include those materials, data, and information that are necessary to describe the 

treatment system’s principle of operation, physical properties, installation and commissioning, 

startup and operation, data collection, required actions during upset conditions and necessary 

consumables. These may include, but are not limited to: 

� Vendor treatment and operation claims as identified in Section 3.2 

� Engineering description 

� Process description including performance ranges and expectations 

� Discharge characteristics 

� Footprint 

� Photographs 

� TO physical and electrical interfaces 

� Safety and Environmental Hazards and Precautions 

4.2 Required Elements of the TQAP 

The TQAP will detail test objectives, specific test procedures (including sample and data 

collection, sample handling, analysis and preservation) and quality control and assurance 

requirements (including measures of precision, accuracy, comparability, and representativeness). 

The experimental approach for the ballast water treatment test, treatment system start-up, and 

verification procedures will be presented. The TQAP will include a summary description of the 

standardized water quality and biological challenge conditions established by the experimental 

configuration as described in Section 5.3. The TQAP will summarize how the challenge 

conditions will be implemented at the TF relative to the ballast water treatment system being 

tested. Any modifications or supplements to the treatment verification protocols will be defined 

and discussed in the Plan. The TQAP will also address quality assurance/quality control 

(QA/QC) requirements, data handling and presentation, and environmental, health, and safety 

issues. 

The TO, with input from the vendor, is responsible for preparing the TQAP. If the vendor desires 

data from ETV testing to be made available for type approval or other regulatory purposes, the 

data required should be clearly identified in the TQAP. The VO shall review and coordinate the 

approval of the TQAP prior to the start of verification testing. 

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The TQAP shall include: 

� Title page/approval page with all project participants 

� Table of contents 

� Project description and treatment performance objectives 

� Project organization and personnel responsibilities 

� TF description 

� Treatment system description 

� Experimental design (including installation/start-up plan) 

� Challenge water conditions and preparation (including TF standard operating procedures 

(SOPs) for preparation) 

� Sampling and analysis plan including sampling and analytical procedures 

� Data management, analysis and reporting 

� Environmental, health and safety plan 

� References 

� Appendices 

o	 Quality Assurance Project Plan (QAPP) 

o	 Vendor operation and maintenance manual 

Content requirements for the QAPP are discussed in more detail in Appendix A. 

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Chapter 5 

Experimental Design 

The primary purpose of ETV verification of BWTSs is to verify the biological treatment 

performance according to an established protocol and specified challenge conditions identified in 

an approved TQAP. Other factors pertinent to the treatment system’s performance will also be 

evaluated, including engineering and environmental metrics. To enable purchasers and other 

stakeholders to make informed choices in selecting appropriate treatment systems, land-based 

verification testing conducted in accordance with this protocol is intended to provide comparable 

data sets for each technology or system to the maximum extent practical Standardized challenge 

conditions included in this protocol address both water quality and the biological organism 

concentrations used to evaluate treatment performance. Key water quality challenge conditions 

are standardized under this protocol because the effectiveness of various treatment processes 

may be influenced by certain water quality characteristics (e.g., salinity, turbidity, color, etc.). 

Moreover, the natural environment (as would be encountered during shipboard BWTS 

performance testing) has a large range of conditions, which may or may not provide adequate 

information on a system’s ability to perform in accordance with the Vendor’s specifications 

under non-ideal water quality conditions. Therefore, non-ideal water quality conditions form the 

basis for challenging the treatment systems under this land-based verification testing protocol. 

To this end, the protocol also includes the requirement for vendors to produce BWTS test data 

using STOs at an appropriate scale in a controlled environment, and verification testing using 

robust ambient species during full-scale tests to measure biological treatment efficacy. 

The general objectives of the verification testing are to: 

� Provide a comprehensive set of water quality and biological challenge conditions against 

which treatment effectiveness can be quantitatively evaluated. 

� Develop adequate data to document system performance against the verification factors. 

The requirements for testing are described in the following sections, which provide guidance on 

the four key elements of the protocol: 1) verification factors, 2) water quality and biological 

challenge conditions, 3) the TF experimental configuration, and 4) verification testing, including 

commissioning of the equipment and the measurement programs required under this protocol. 

Variations in the protocol for specific treatment system types (e.g., in-line treatment versus intank treatments) are also described. 

5.1 Verification Factors 

All treatment systems will be verified according to the following factors: 

� Biological treatment efficacy 

� Operation and maintenance 

� Reliability 

� Cost factors 

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� Environmental acceptability 

� Safety 

5.1.1 Biological Treatment Efficacy 

Biological treatment efficacy is defined as the removal, inactivation, or death of organisms and 

will be measured in terms of the concentration of selected organism size classes in the treated 

discharge. Additional measures of efficacy may include measurements in terms of removal 

efficiency (e.g., a percentage reduction of organisms present at uptake), against a threshold (e.g., 

a water quality standard), or in relation of treatment vs. control discharge concentrations. The 

measurement program required by the protocol evaluates the primary treatment efficacy criteria 

by measuring the quantity of living organisms in both the challenge water and the treated 

discharge. 

5.1.2 Operation and Maintenance 

Operation and maintenance includes the labor expertise, equipment, and consumables required to 

operate the system to achieve the stated performance goals and objectives. The quantitative 

indicators to be considered during verification are described in detail in Section 5.4.9.1. 

5.1.3 Reliability 

Reliability is a statistical measure of the number of failures (either qualitative or quantitative) per 

known quantity of test cycles. This is described in greater detail in Section 5.4.9.8. 

5.1.4 Cost Factors 

Cost factors include only those factors that can be verified, such as labor hours to operate and 

maintain the system, expendable material, such as filter cartridges, and pounds or gallons of 

chemicals consumed by the treatment system. Data is collected in units, to which unit prices, 

which are likely to vary from location to location, can be applied to determine costs. These are 

discussed further in Section 5.4.9.2.9. 

5.1.5 Environmental Acceptability 

Environmental acceptability assesses ballast water quality following treatment for factors other 

than the abundance and viability of organisms. For example, this will determine if the treated 

water meets acceptable water quality characteristics for such measures as dissolved oxygen, 

temperature, treatment residuals, pH, etc. This is discussed in further detail in Section 5.4.4. 

5.1.6 Safety Factors 

Safety factors include any treatment-specific considerations that may pose a threat to the safety 

of the operator or shipboard operations. These are not intended to be comprehensive in nature, 

which is best evaluated by Classification Societies, such as the American Bureau of Shipping, 

but are included as observations that can be made during the verification testing. Further 

discussion of these observations are discussed in Section 5.4.9.2.11. 

Performance test results will be reported using standard ETV formats to make certain the 

reported information among treatment technologies tested is comparable. Flexibility is 

permissible to ensure reporting for a specific treatment system type is appropriate and accurate. 

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Some information supplied by vendors may not be verified under the protocol. This information 

may be included in the verification test report and clearly identified as non-verified information. 

Vendor-provided information may include shipboard compatibility (e.g., corrosion resistance, 

system weight, system volume including clearances needed to perform maintenance and replace 

vital components, and compatibility with other common shipboard systems such as operational 

flow rates). Submission and reporting requirements for non-verified, Vendor-supplied 

information is included under Chapter 3. 

5.2 Challenge Conditions 

This protocol recognizes that land-based testing cannot fully replicate actual treatment system 

performance onboard ship. However, land-based verification testing can provide sufficient 

information to verify the expected performance of treatment in the shipboard environment. It is 

understood that all treatment technologies will face a range of physical/chemical water quality 

conditions and biological organisms when operated onboard a ship. Therefore, each treatment 

system’s performance will be verified using a set of standard challenge conditions. This protocol 

defines the following objectives for the challenge conditions: 

� To verify a treatment system’s performance using a set of challenging, but not rare, water 

quality conditions representative of the natural environment. 

� To verify removal or kill of bacteria, protists, and zooplankton using ambient organisms 

as defined by size classes and analytical techniques that identify living quantities for 

these organisms. 

The standard challenge conditions are specified using two groups of factors that must be 

addressed to properly challenge treatment technologies: water quality and living organisms. The 

requirements for each group are presented in the following sections. 

5.2.1 Challenge Water – Water Quality Characteristics 

Since water quality conditions in ports and harbors around the world vary greatly, treatment 

systems may encounter a wide range of conditions. Also, certain water quality conditions may 

interfere with the ability of some treatment processes. It is therefore critical to evaluate the 

effectiveness of a treatment system under water quality conditions that are challenging to the 

technology being tested. Simulating all potential water quality conditions in a land-based testing 

design would be prohibitively expensive1 and not essential for verifying the performance of a 

treatment system. Because water quality parameters that can interfere with various treatments 

are generally understood and few in number (e.g., salinity, turbidity, organic matter either as 

dissolved or particulate forms), the number of water quality metrics that must be explicitly 

included in the protocol can be limited. This protocol defines three possible challenge conditions 

that represent some of the more challenging, natural conditions that may be encountered by 

 

 

 

 

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1 Similarly, shipboard testing of all potential water quality conditions will require extensive logistics to move a 

treatment system to a matrix of natural conditions, as well as investment in methods and protocols by which the 

treatment effectiveness is established using natural populations of organisms. 

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ballast water treatment systems. Challenge water quality characteristics to be used during testing 

events are presented in Table 3. 

Table 3. Water Quality Challenge Matrix for Verification Testing 

Water Types Minimum Water Characteristics 

DOM: 6 mg/L as DOC Fresh (Salinity <1 PSU) 

POM: 4 mg/L as POC 

MM: 20 mg/L Brackish (Salinity 10-20 PSU) 

TSS = POM + MM: 24 mg/L 

Marine (Salinity 28-36 PSU) Temperature: 4 – 35 °C 

Another basic premise in the design for this protocol is that ballast water treatment systems are 

designed to function effectively in the full range of water quality characteristics that will be 

encountered under shipboard operational conditions. By challenging the treatment systems with 

these conditions, it is assumed treatment will be effective under less demanding conditions. 

Challenge waters have been tailored to a minimum set of water quality conditions that may be 

achieved either through naturally occurring conditions or through augmentation, if appropriate, 

and validated by the TF. The challenge conditions are specified for three possible levels of 

salinity, <1, 10 to 20, and 28 to 36 PSU (practical salinity units), and water quality characteristics 

problematic for the range of technologies being developed to treat ballast water, namely, 

suspended solids and dissolved organic matter (DOM).2 

Suspended solid material that can interfere with treatment effectiveness is composed of several 

types of particles, which can be of biological or mineral origin, specifically clay and silt. The 

water quality challenge conditions defined by the solids content of the matrix include particulate 

organic matter (POM) and mineral matter (MM). These two types of particles are both present 

in natural waters at a range of concentrations and size distributions. Therefore, both forms are 

included in the challenge conditions to address issues of particulate removal and turbidity, which 

can interfere with transmission of UV light or other treatment processes. 

Various forms of dissolved chemicals and compounds, particularly organic material, can directly 

affect the efficiency of some treatment processes. Dissolved organic matter (DOM) and 

dissolved organic carbon (DOC) are two terms used to describe this component of natural water. 

2 The protocol does not explicitly call for verification at a series of temperatures, even though some treatments may 

have strong temperature dependence or may include temperature manipulation as part of the treatment procedure. 

Rather than include temperature as a controlled water quality condition, which can have significant cost implications 

for the TF, accurate and continuous monitoring of the source and treated water temperatures is required for all test 

cycles. If temperature manipulations are to be included, the Test Plan will include protocols for these 

manipulations. Temperature challenges should be addressed in shipboard testing, and in bench-scale tests of 

treatment process. 

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DOM/DOC often contains many chromophores that contribute substantially to the color of the 

water, another potential interference for treatments. Thus, the color of a water and DOM/DOC 

concentration are often interrelated. 

The measurement methods for evaluating the status of the challenge water quality conditions are 

described in Section 5.4.6. They include standard analytical methods to document the 

concentration of total suspended solids, particulate organic matter or dissolved organic matter, 

and methods that indirectly measure these parameters (e.g., turbidity measured by 

electronic/optical measurement such as nephelometery (NTUs) or transmissometry (beam 

attenuation) or fluorescence (color /DOM)). 

Standardization of the water quality conditions for the verification testing requires a consistent 

set of source water (e.g., fresh or marine water), as well as the use of well-characterized organic 

matter and mineral matter. The TF will be responsible for providing these materials and 

ensuring the water quality conditions are as described under this protocol. The water quality test 

conditions will be standardized for salinity, particulate organic matter, mineral matter, and 

dissolved organic matter as described in the following sections: 

5.2.1.1 Salinity 

Natural water of less than 1 PSU will be used for fresh water conditions, while natural seawater 

will be used for marine conditions. Testing at multiple salinities at a given TF should only be 

conducted if there are natural water sources with the differing salinities (e.g., fresh and brackish 

waters). Artificial modification of the salinity of the waters should be used only if it can be 

demonstrated that the concentrations, diversity and condition of organism populations required in 

Section 5.2.2 will not be impacted by adjustment of the salinity. 

5.2.1.2 Particulate and Dissolved Organic Matter 

In the case of POM, if the natural waters have insufficient concentrations, the TO may augment 

them through the addition of humic material (e.g., Micromate humates [Mesa Verde Resources, 

Placitas, New Mexico]). Other sources include particulate carbon from sources such as ground 

up seaweed or plankton detritus. DOM can be very difficult to adjust or augment if the natural 

waters have insufficient content. There has been some success using Camellia sinesis 

(decaffeinated iced tea mix) to augment natural DOM content, but a TF must assess the effect of 

additives on the ambient and test organisms (if used) before using. 

5.2.1.3 Mineral Matter (MM) – Clays and Silts 

Mineral particles in the size range typically found in coastal and estuarine waters are readily 

obtained from commercial sources and will be used as the source of the mineral matter. A study 

of sediment size in ballast tanks suggests that particles are mostly fine grained (less than 63 μm) 

and most vessels contain <10% sand (F. Dobbs, Pers. Com.). Thus, addition of the commercially 

available clay minerals (with a majority of particles in the 10 to 50 μm size) addresses the 

objective of having a prescribed level of non-biological particles as part of the water quality 

challenge conditions. Specifically, ISO 12103-1, A3 MEDIUM TEST DUST and ISO 12103-1, 

A4 COARSE TEST DUST can be used for this purpose. As these particles will tend to settle out 

over time within the augmentation storage vessels, the test protocols must include a means of 

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maintaining any sediments in a homogeneous suspension prior to addition of the challenge water 

(e.g., continuous mixing of the sediment augmentation tank). 

The TO should verify that, whatever source of augmentation or delivery system is used, the 

addition of that material should minimize to the extent possible biocidal or growth stimulant 

response to the ambient organisms. The TF will be responsible for preparing the challenge water, 

documenting the challenge conditions, and validating that the conditions are maintained at the 

treatment system or control entry point. Challenge waters will be prepared under standard 

operating procedures developed by the TF. The TQAP will include these SOPs and describe any 

planned deviations from the SOPs. 

5.2.1.4 Challenge Water – Water Quality Deviations 

In some cases, a specific ballast water treatment system may be unable to operate with all of the 

prescribed challenge water quality conditions as specified in Table 3. This may be either due to 

mechanistic limitations of the technology (e.g., electrolytic chlorination (without brine addition) 

is inoperable in fresh water) or by design (e.g., scale). In such cases, deviations may be 

permitted provided that significantly challenging and realistic conditions are identified and 

justified by the TO, and that the VO approves the deviation. In no case, however, shall the total 

number of test cycles be reduced. All deviations will be specified in the verification report as 

limiting conditions of the technology. 

5.2.2 Challenge Water – Biological Organism Conditions 

The death or removal of living aquatic organisms is central to the need to treat ballast water. To 

ensure proper evaluation of a BWTS’s performance, the effects on biological organisms living in 

the challenge water will be measured for each treatment system tested. Biological efficacy will 

be evaluated as function of a system’s ability to kill or remove organisms that are naturally 

occurring and represent the more robust ambient populations at the test site. 

5.2.2.1 Organism Concentrations 

A minimum total input concentration of living organisms, by size class, is defined in Table 3. 

The two larger size classes must contain at least 5 different species from at least 3 

phyla/divisions. Challenge water meeting these criteria shall be demonstrated for each test cycle 

at 1) the influent point of the control tank and 2) immediately prior to the point of treatment for 

systems that treat upon uptake or at the treatment tank influent point for systems that treat either 

wholly in-tank or upon discharge. 

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Table 4. Minimum Criteria for Challenge Water Total Living Populations 

1 Organism Size Class Total Concentration Diversity 

≥ 50 μm 

≥ 10 μm and < 50μm 

<10 μm 

105 3 organisms/m

103 organisms/mL 

10 3/mL as culturable aerobic 

2 heterotrophic bacteria 

5 species across 3 phyla 

5 species across 3 phyla 

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1 Size is determined by the maximum dimension on the smallest axis. 

2 Note it is assumed that the effects on culturable aerobic heterotrophic bacteria will be indicative of the effects on 

all bacteria. 

3 Diversity of bacteria by species or phyla is not applicable, and there is no diversity requirement for this size class. 

5.2.3 Challenge Water – Flow Rates and Volumes 

5.2.3.1 Flow Rate 

Treatment tests will evaluate equipment at operational flow rates defined by the vendor’s O&M 

manual. The TF shall be capable of providing flow rates of at least 200 m3 per hour (880 gallons 

per minute) and an available volume per test cycle of at least 400 m3. The TF shall provide 

sufficient challenge water volume to meet these requirements, and the TQAP will identify the 

rates that will be tested. 

5.2.3.2 Volume 

A minimum of 200 m3 shall be processed in each BE test cycle. The recommended minimum 

volume for in-tank testing is 200 m3 (~52,800 gallons). The TF shall provide test and control 

ballast tank configurations of at least 200 m3. Larger volumes may be used depending on vendor 

specifications and availability of tanks at the TF. 

5.3 Test Facility Physical Configuration 

5.3.1 Overall experimental configuration 

As a minimum, the TF should encompass four components: (1) fluid delivery capacity, (2) a 

control tank and piping system, (3) a treatment tank and piping system, and (4) a discharge 

collection tank and post-test treatment system. The fluid delivery systems include pumps, 

piping, flow distribution controls, flow rate controls and relevant instrumentation to support the 

challenge water requirements described in Section 5.2. The control tank shall be utilized to hold 

untreated challenge water for each biological efficacy test cycle. The treatment tank will be 

utilized to hold all test water subject to the BWTS during the test cycle. Both tanks shall be a 

minimum of 200 m3 and suitably constructed to hold such volumes for at least one day. The tank 

drains shall be located, to the extent possible, to minimize the retention of water following 

discharge. Tank intake and discharge piping, fittings and relative configurations shall be 

identical or the equivalent as validated by the TO. Finally, the discharge tank may be necessary 

if the TF is required to post-treat on-site the control and treated challenge or test waters to 

remove added inorganic and organic matter, disinfection by-products, or other regulated 

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substances prior to discharge back to the environment. The discharge tank should be of 

sufficient volume to store at least 200 m3, but preferably large enough to store the cumulative 

volume of the control and treatment tank. 

There are multiple potential locations of ballast water treatment systems when used onboard 

vessels. The TF must be arranged to support testing of systems, which operate at uptake, 

discharge, in-tank or a combination of these. Examples of the arrangement for in-tank and inline treatment are shown in Figures 1 and 2. As shown, the test configuration includes a flowsplitter such that challenge or test water is provided to both the control and treated legs 

simultaneously. Note that in such an arrangement, the fluid pumping capacity of the TF would 

be a minimum of 400 m3

/hr. A sequential fill configuration may also allowable, in which the 

treatment and control are filled or drained sequentially. The latter may result in reduced pump 

capacity needs (but still requires a minimum pump capacity of 200 m3 per hour), less overall 

logistic complexity, and reduced piping through the dual use of sampling apparatus, feed and 

discharge plumbing, instrumentation and so on. In either case, the TF shall validate, to the 

satisfaction of the VO, that significant differences between treatment and control lines in 

biological and physical responses are minimal, and that there is no cross contamination by dual 

use of the site infrastructure. 

5.3.2 Sampling Methodology 

Several types of samples are to be acquired during the verification testing of a ballast water 

treatment system. During biological efficacy tests, discrete samples for water quality and 

biological enumeration shall be acquired over the course of the test on a time averaged basis. A 

minimum volume of 3 m3 shall be collected per location. In situ instrumentation to monitor water 

quality and physiochemical parameters are also included. All sampling is assumed to be in-line, 

whether discrete or in situ. Characterization of ambient waters may require discrete grab 

samples, as described in Table 8 

5.3.2.1 Sampling Locations 

Required sample locations for various treatment scenarios are shown in Figures 1 and 2; samples 

should be collected according to one of these test designs, unless otherwise accepted by the VO 

in the TQAP. Samples (data) from the challenge water must be obtained, in accordance with the 

guidance in Section 5.3.2.4, immediately prior to water entry to the control tank, and 

immediately before entry to the BWTS (in-line treatment) or the ballast tank in the case of intank treatments (if demonstrated as representative of the control and challenge water, a single 

sample collected ahead of the splitter shown in Figures 1 and 2). For in-line BWTSs, samples of 

treated water must be collected (1) immediately following the treatment system and (2) 

following the holding tank at the end of the one-day hold time. For in-tank treatments, samples 

of treated water must be collected from the ballast tank discharge following the vendor-defined 

contact period. Further definition of hold times is described in Section 5.4.5. Systems that 

incorporate treatment at multiple locations (e.g., upon uptake and discharge) will only require 

sampling after the final stage of treatment. Sampling locations for the control tanks and BWTS 

must exactly mimic the treatment tanks and system. Finally, in-tank sampling (e.g., via plankton 

net tows) shall not be utilized for the purposes of verifying biological efficacy, as this method 

may not result in representative samples. The exact locations, frequency, and methods to be used 

to collect the samples must be defined in the TQAP. 

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5.3.2.2 Sample Collection Requirements – Frequency 

Continuously recording in situ sensors (as available and feasible) may be used to measure water 

quality and proxy parameters during verification testing. Description of the sensors, how they 

operate, and how they are calibrated shall be included in the TQAP. Minimum instrument 

performance requirements are provided in Table 5. Discrete samples for water quality 

characterization will also be obtained during verification testing as discussed above, and they 

will be collected at the time biological verification samples are collected. A higher frequency of 

collection for discrete samples may be used if additional samples for calibrating the sensors are 

necessary. The sample collection requirements and frequency of obtaining samples from the 

control tanks and piping system will identically match those of the treatment tanks and system. 

The appropriate frequency of discrete sample collections made in lieu of in situ sensing shall be 

described in the TQAP. 

Table 5. Accuracy and Precision Requirements for Potential Sensors 

Sensor Reporting Units Range Accuracy Precision 

Temperature oC 0 to 30 0.1 0.01 

Conductivity (salinity) MS/cm 0.5 to 65 0.1 0.01 

Transmissometer (20-cm) per m 0 to 40 0.20 0.01 

Dissolved oxygen mg/L 0 to 20 0.10 0.05 

Fluorometer μg/L 0.03 to 75 50% of reading 1 0.01 

1 When compared to wet chemistry results. 

5.3.2.3 Sample Replication 

Verification testing will include replication only in analysis. Sample collection replicates are 

based on the time integrated sample volumes collected during the test cycle (see examples shown 

in Figures 1 and 2. These sample volumes form the minimum sample collection replication 

required during each test cycle. Each of the integrated sample tanks will be sub sampled for the 

Core parameters, which are discussed later. The TQAP will describe each type of analytical 

replication planned, including acceptable ranges of variability. 

5.3.2.4 In-line Sampling for Biological Efficacy 

To obtain an accurate measurement of the organism concentration at the sample location, the 

installation of an isokinetic sampling facility at each of these locations is recommended. 

Isokinetic sampling is primarily intended for the sampling of water mixtures with secondary 

immiscible phases (i.e., sand or oil) in which there are substantial density differentials. In such 

conditions, convergence and divergence from sampling ports is of significant concern. Since 

most organisms are relatively neutrally buoyant, true isokinetic sampling is likely unnecessary 

for testing ballast water treatment systems. Nonetheless, the mathematics related to isokinetic 

sampling are deemed useful for describing and specifying appropriate sampling geometries. 

Isokinetic sampling is necessary to ensure that a sample contains the same proportions of the 

various flowing constituents as the flow stream being sampled. During isokinetic sampling the 

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sampling device does not alter the profile or velocity of the flowing stream at the moment or 

point at which the sample is separated from the main flow stream. To achieve isokinetic 

sampling conditions, a sampler is designed to separate a subsection of the total flow-stream in a 

manner that does not encourage or discourage water entry other than that which is otherwise in 

the cross-section of the sampler opening. In other words, flow streams in the main flow of the 

pipe should not diverge or converge as they approach the opening of the sampler. 

Recommendations for the design and installation of appropriate sampling facilities are given 

below. In any case, validation of the Test Facilities configuration should include verification that 

the chosen sampling design, geometry and installation result in representative samples and 

minimize organism mortality as a result of sample acquisition. 

5.3.2.5 Design of In-line Sampling Apparatus 

Through computational fluid dynamics modeling, it has been shown that the isokinetic diameter 

calculation can provide guidance for sizing of sample ports for sampling of organisms (Richard 

et al., 2008). Simulations showed that flow transitions from the main stream were best for sample 

port diameters between 1.5 and 2.0 times the isokinetic diameter. Ports sized in this range had 

smooth transitions and pressure profiles that allowed for direct sampling without the need of a 

pump to induce sample collection. The isokinetic sample port diameter should therefore be 

determined generally according to the equation: 

Qiso D iso m = D

Qm 

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Treatment Sample Tank 

BWTS 

Pump 

≥ 3 m3 

≥ 30 m3 ≥ 30 m3 

Challenge Water 450 tons 

Standard Test Organisms 

Ballast Tank 

≥ 200 m3 

Control Holding 

Tank 

≥ 200 m3 

Discharge Discharge 

Flow 

Splitter 

Control Sample Tank 

Challenge Water 

Sample 

Figure 1. Sampling design example for in-tank treatment. 

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Pump 

≥ 30 m3 ≥ 30 m3 

≥ 3 m3 

Standard Test Organisms 

Treatment 

Holding Tank 

≥ 200 m3 

Control Holding 

Tank 

≥ 200 m3 

Flow 

Splitter BWTS 

Challenge Water 

Sample 

Post Treatment 

Challenge Water 450 tons 

Post Treatment 

Sample 

(≥ 30 m3) 

Control Sample Holding Sample Tank Discharge Discharge Tank 

Figure 2. Sampling design example for in-line treatment. 

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where Diso and Dm are the diameters of the sample port opening and the main flow in the line to 

be sampled, respectively; and Qiso and Qm represent the respective volumetric flow rates through 

the two pipes. It is recommended that sample port size be based on the combination of maximum 

sample flow rate and minimum ballast flow rate that yields the largest isokinetic diameter. 

The opening of the sampling pipe should be chamfered to provide a smooth and gradual 

transition between the inside and outside pipe diameters. The length of the straight sample pipe 

facing into the flow can vary, but it should not usually be less than one diameter of the sampling 

pipe. The sampling port should be oriented such that the opening is facing upstream and its 

entrance leg flow is parallel to the direction of main pipe flow and concentric to the larger pipe, 

which may require sampling pipes to be “L” shaped with an upstream facing leg, if installed 

along a straight section of discharge pipe. 

The need to be able to service the sample pipe is important and should be considered, taking 

safety into consideration. Therefore, the sampling pipe should be retrievable either manually or 

mechanically, or it must be in a system that can be isolated. Because of the potential for the 

opening and interior of the sample pipe to become occluded by biological or inorganic fouling, it 

is recommended that samplers be designed to be closable at the opening, removed between 

sampling intervals, or be easily cleaned prior to sampling. 

The sample pipe and all associated parts of the sampler that come into contact or near proximity 

with the system piping should be constructed of galvanically compatible materials and generally 

corrosion resistant. Any corrosion of the sampling system will affect sample flow rates and 

potentially sample representativeness. 

If flow control of the sample flow rate is required, ball, gate, and butterfly valve types should be 

avoided as they may cause significant shear forces, which may result in organism mortality. For 

flow control, it is recommended that diaphragm valves or similar valve types be used to 

minimize sharp velocity transitions. For flow distribution, ball valves may be utilized only if 

they are either fully open or fully closed 

When sampling is conducted on the discharge of a tank through the use of a pump (i.e., a nongravity drain) and the sample port is located upstream of the pump, it may not be possible to 

draw an adequately sized sample since the line will be under suction with a variable hydrostatic 

pressure head. Therefore, maintenance of a time-averaged sample flow requires the sample to be 

drawn from the discharge utilizing a pump. In such cases, a diaphragm pump is recommended to 

minimize pump-induced organism mortality during sampling. 

5.3.2.6 Installation of an In-line Sample Point 

The sample taken should be removed from the main pipeline at a location where the flowing 

stream at the sample point is representative of the contents of the stream. The sample port 

entrance should be placed at a point where the flow in the main pipe is fully mixed and fully 

developed. 

The sampling point should be installed in a straight part of the system piping and the sampling 

fixture should be positioned such that a representative sample of ballast water is taken. It is 

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recommended that the position of the sample point be determined using methods such as 

computational fluid dynamics. 

5.3.2.7 Operation of an In-line Sample Points 

In-line biological samples will be collected on a time-integrated basis such that a composite 

sample of the entire period of uptake or discharge is acquired. The sample flow rate should be 

appropriately controlled to maintain an even distribution of samples acquisition over that time 

period. 

5.3.3 Test Organism & Water Quality Augmentation 

Where the addition of organisms for the augmentation of ambient organisms) is required for 

biological efficacy testing, a method for the injection or addition of organisms to the challenge 

water must be provided. Similarly, water quality parameters that require adjustment from the 

ambient conditions to the requisite challenge water properties will require some type of injection 

process. Various means are available to inject or add organisms to the challenge water, for 

instance, by a batch method to a large, discrete source volume or by injection into the flow 

stream. In any case, the following requirements are applicable: 

� Any organism addition or injection method must minimize, to the extent possible, 

organism mortality as a result of its addition/injection mechanism. 

� The method must result in a well-mixed and uniform distribution, spatially and 

temporally, of organisms within the challenge water and at its introduction at the point of 

treatment or tank intake. 

� The concentration of living test (if used) organisms at the point of treatment or tank 

intake must conform to the requirements given in Section 5.2.2. 

� The point of addition or injection must be situated such that the flow is well mixed at the 

subsequent point from which the first discrete sample is acquired to ensure a 

representative sample is obtained; inclusion of substantial pipe lengths and/or a static 

mixer may need to be considered. 

� All methods for the injection or addition of organisms must be validated by the TF to 

meet the above requirements. 

For water quality additions, for example sediments or dissolved organics, the addition should 

occur far enough upstream from the point of water quality sampling to ensure that the sample is 

well mixed. Furthermore, the apparatus used for addition should minimize the system related 

mortality on ambient and test organisms, to the extent possible. 

5.3.4 Control & Instrumentation 

The testing described throughout this protocol is complex and logistically challenging. 

Moreover, since these tests are designed to provide a repeatable and accurate verification of 

treatment system performance, it is important to ensure that each phase and measurement is 

conducted with a high degree of reliability, repeatability, and accuracy. The verification testing 

process is further complicated by the inclusion of biological organisms and related 

measurements, which result in a variety of design and timing complexities. As a result, it is 

recommended that the TF include a typical supervisory control and data acquisition (SCADA) 

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system to support the many operations and data acquisition tasks associated with this testing. A 

typical SCADA system provides the TF with the ability to: 1) provide automatic control of 

pumps, valves and sub-systems to maintain operational set points; 2) acquire and archive all 

events, data and conditions; 3) provide controllable process control algorithms which improve 

system efficiency, safety and repeatability; and 4) provide facility- and treatment-system 

diagnostics during commissioning, testing, and upset conditions. Instituting such a system can 

be expected to improve measurements, quality assurance, standardized reporting, and reduce 

labor and analysis time. 

Whether a SCADA system is utilized or not, the TF should include within its QAPP a discussion 

of how the instrumentation associated with TF operation, process control (either manual or 

automatic), and condition monitoring of the verification tests shall be operated, maintained and 

calibrated. Also, as a minimum, the TF shall include sufficient instrumentation and condition 

monitoring such that a substantive record is established which verifies that 1) challenge 

conditions were obtained and maintained, 2) the treatment system was operated in accordance 

with the Vendor’s requirements and 3) no system or environmental effects occurred to perturb 

the verification test or treatment system operation. The test instrumentation and test operating 

procedures shall be documented in the TQAP. 

5.4 Verification Testing 

Verification testing will be separated into three distinct phases, 1) treatment system 

commissioning, 2) biological efficacy (BE) tests, and 3) operating and maintenance (O&M) 

tests. Commissioning tests are intended to validate, prior to the commencement of either BE or 

O&M tests, that the treatment system is installed correctly and operating in accordance with the 

vendor’s requirements. A minimum of three BE tests shall be completed at each of two salinities 

selected by the vendor (the vendor may complete testing at all three salinities if desired) and with 

all of the challenge conditions specified in Section 5.2 to assess and verify the biological efficacy 

of the treatment system under pre-established conditions. O&M testing shall be conducted with 

ambient source water conditions, with the intention of operating the system with realistic 

physical conditions, to assess the systems engineering performance. 

Ballast water treatment system performance, operating conditions, and certain O&M criteria will 

be recorded and monitored during verification testing by the TO. Results will be presented in the 

verification report, described in Chapter 6. The factors to be verified during ballast water 

treatment system verification testing include: biological treatment performance, operation and 

maintenance, predictability/reliability, cost factors, environmental acceptability, and safety. 

Any of several treatment sequences may be used by a particular treatment system (see Table 6), 

including in-line treatment (during ballasting or deballasting), in-tank treatment, or a 

combination of the two. The stage in the ballasting cycle at which treatment is applied may also 

vary. This verification testing protocol accounts for these through flexibility in the TF and 

Verification TQAP. The guidance in the following section provides the basic test requirements 

and rationale for inclusion in the TQAP that will provide details specific to the treatment system 

and its operation. The final verification report shall document the system sequence(s) completed 

during testing. 

30
 
 

 

Table 6. Likely Treatment Sequences and Applications Inherent to Ballast Operations 

Sequence Number Ballast Operation Application 

1 Treatment applied during ballasting/ No treatment during 

deballasting. 

2 Treatment applied during ballasting/ Treatment applied during 

deballasting. 

3 No treatment applied during ballasting/ Treatment applied during 

deballasting. 

4	 No treatment applied during ballasting/ Treatment applied during 

transit/ No treatment during deballasting. 

5	 No treatment applied during ballasting/ Treatment applied during 

transit/ treatment during deballasting. 

6	 Treatment applied during ballasting/ Treatment applied during 

transit/ No treatment applied during deballasting. 

7	 Treatment applied during ballasting/ Treatment applied during 

transit/ Treatment applied during deballasting. 

The over-arching objectives of the verification testing (including all phases) are to: 

� Evaluate the treatment performance of the ballast water treatment system relative to the 

removal or kill of ambient and test organisms (if used), operating under vendor-specified 

conditions; 

� Evaluate the treatment system O&M criteria; 

� Determine and record cost factor data; and 

� Record and document test conditions, observations, and results. 

Other testing objectives may be defined by the vendor and included in the TQAP. The 

requirements for verification testing are described in the following sections and must be 

addressed in the TQAP. 

5.4.1 Treatment System Commissioning 

The TQAP shall describe all the tests and start-up requirements required to validate that the 

treatment system is installed correctly and operating in accordance with the vendor’s 

requirements. The objectives of the commissioning are to: 

� Install and start the ballast water treatment system in accordance with the vendor O&M 

manual; 

� Reach stable operating conditions; 

� Make modifications as needed to ensure stable operations under TF condition; and 

� Record and document all installation and start-up conditions, observations and results. 

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The treatment system shall be installed at the TF according to the vendor instructions included in 

the TQAP. Ideally, this phase of the verification will include close coordination between the 

vendor and TO to quickly resolve discrepancies or malfunctions. Commissioning tests may 

include small-scale tests of various vendor sub-systems or components, validation of treatment 

system integration into the TF (e.g., a leak test or communication tests), or any other vendorrequired installation tests that may be expected during a shipboard installation. However, at least 

one valid, full-scale verification test cycle, meeting all of the requisite challenge conditions, 

should be conducted successfully by the TO without vendor assistance. While the challenge 

conditions are to be employed during commissioning tests, it is not necessary to conduct a 

complete suite of analytical measurements to assess biological treatment efficacy. 

A successful commissioning is defined as one in which (1) all TF requirements and conditions 

defined by the challenge conditions were met and (2) all components of the treatment system 

operated in accordance with the vendor requirements. Subsequent BE and O&M verification 

testing cannot commence until commissioning is successfully completed and agreed upon by the 

TO and vendor. The verification report should document all of the small-scale, component-level 

tests conducted and their results, any treatment system or TF deficiencies or failures, and their 

successful resolution. Finally, the verification report should document in detail the challenge 

conditions during the full-scale commissioning verification test cycle. 

5.4.2 Operation and Maintenance (O&M) Manual 

The O&M manual shall be incorporated into the TQAP and will be essential to the development 

of the monitoring and maintenance plan incorporated in the TQAP. The vendor shall identify 

factors that affect the operation of the BWTS, including any warm up or other requirements that 

must be completed to achieve operational stability. The vendor’s O&M manual shall specify 

what constitutes stable operating conditions for the BWTS, factors that may affect operating 

conditions, and any adjustments required to reach or to maintain a stable operating condition. 

Adjustments made in the operating conditions will be presented in the final verification report. 

5.4.3 Vendor and TO Requirements 

An installation/start-up plan shall be prepared and included as part of the TQAP. The TO shall 

conduct start-up procedures for the BWTS in accordance with the installation/start-up plan and 

with the vendor O&M manual. At the end of the start-up period, the TO will assess whether the 

BWTS is in a stable operating state, as specified in the O&M manual, and the vendor will certify 

in writing that the system is installed and operating as intended. If the operation is stable, the 

verification testing can begin. If not, start-up procedures will be repeated no more than two 

additional times. If the BWTS does not achieve stable operating conditions after three start-up 

cycles, the TO, in conjunction with the vendor, will review the start-up work plan for 

applicability and determine where adjustments and modifications are required. In any case, the 

TO will have the option of concluding or postponing further testing at the conclusion of three 

failed start-up cycles. 

The vendor will identify any additional equipment, system maintenance, changes to operating 

conditions, or other modifications needed to ensure effective BWTS operation and to attain or 

maintain stable operational conditions. 

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5.4.4 Toxicity Testing for Biocide Treatments 

The Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), as amended, requires 

registration by the U.S. Environmental Protection Agency (EPA) of pesticides sold or used in the 

United States, which includes biocide products that might be used in BWTSs. The vendor is 

required to provide information regarding FIFRA registration of any biocide to be used in their 

BWTS. 

The residual toxicity in the discharge from BWTSs employing a biocide is of concern to the TF 

(as part of the TF’s NPDES permit requirements), as well as for the environmental acceptability 

of the treated ballast water from the BWTS in use. Toxicity testing of the water following 

treatment and hold time, as appropriate, shall be conducted during the commissioning phase of 

the verification testing according to the toxicity methods cited in Section 5.4.7.5. If the posttreatment effluent passes the toxicity tests, then verification testing can proceed. If, however, the 

effluent fails the toxicity test, verification testing shall not be initiated, and further toxicity tests 

shall be required. The vendor shall be allowed no more than two additional attempts to pass the 

toxicity tests within 30 days of the initial test. This may require modifications to the approach 

for verifying the technology in the TQAP or other investigations to understand the toxicity 

response. In the event a TF’s NPDES permit requires a toxicity evaluation of the treated waters 

at the end of each test with the addition of a biocide, or if the vendor requests additional toxicity 

testing during the verification, the TQAP shall address the additional testing. 

5.4.5 BE and O&M Verification Strategy: Test Duration and Coordination 

A minimum of three valid BE tests, described in detail below, are required for each salinity 

regime (defined in Section 5.2.1) under which the treatment system is verified. At a minimum, 

testing at two salinity conditions shall be conducted. In addition, O&M testing of the treatment 

system shall distribute testing of a minimum treated volume of 10,000 m3 amongst the BE test 

cycles. These O&M test cycles are equivalent to ~50 hours of operation at 200 m3 per hr (or 

~65 hours of operation at 150 m3 per hr). Upon completion of the commissioning verification 

tests, the next verification test shall be a BE test cycle. This sequence allows the testing to 

validate operation of a new unit prior to substantial operational testing. For example, for the case 

in which 6 BE test cycles will be conducted, each BE test cycle should be separated by 2,000 m3 

in O&M testing. This approach also involves a substantial duration for the testing period and 

associated range of ambient water conditions over this time. 

During actual shipboard operation, ballasting procedures may occur over time periods ranging 

from minutes to hours. For each in-line treatment BE or O&M verification cycle, a minimum 

operational period of one (1) hour is required, although this may be extended if flow rates are 

reduced from 200 m3

/hr. 

In addition to the uptake time, a minimum 1-day holding time within both the treatment and 

control tank is required for each BE test cycle to simulate the time that water would reside in a 

ballast tank. Thus, the duration of each test cycle will be defined by the operational approach 

used by the treatment system. The holding time of the required BE test cycles may be extended 

if the vendor requires such time as part of the BWTS or process. 

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The holding time included in this protocol is intended to provide a conservative assessment of 

the BWTS’s ability to treat ballast water according to the vendor’s claims. For in-tank treatment 

with additional in-line treatment during ballast water discharge, the duration will be equal to intank treatment requirements and the deballasting time. Regardless, subsequent to the one-day 

tank holding time, the control tank discharge must exhibit a minimum concentration of living 

organisms, as defined in Table 7. These criteria are necessary, in addition to the input challenge 

conditions, to constitute a valid BE test cycle. These control tank discharge concentrations are 

intended to make certain that treatment efficacy measurements attributed to the BWTS in any 

given BE test cycle are not the result of natural or non-treatment system related effects. 

Table 7. Criteria for Concentrations of Living Organisms in Control Tank Discharge 

Organism Size Class Minimum Concentration 

≥ 50 μm 100 organisms/m3 

≥ 10 μm and < 50μm 100 organisms/mL 

< 10 μm 5 x 102

/mL as culturable aerobic heterotrophic 

bacteria 

Shorter or longer tank hold times may be utilized but must be justified in the TQAP. 

Justifications for shorter tank hold times may include an inability to sustain organism 

populations in the control tank to achieve the requirements in Table 7 because of natural 

mortality. In such cases, tank hold times may be shortened, as appropriate and agreed upon, such 

that an adequate assessment of the BWTS treatment efficacy may be made. 

For in-tank treatments, test duration will include the minimum contact time the vendor prescribes 

for effective treatment, but not less than a cumulative one-day holding time for each of the 

required BE test cycles. As with the in-line approach, testing of the BWTS without active 

ingredients may be run in parallel with the challenge test to reduce the overall duration of the 

verification test. Modifications may be made according to vendor-specified requirements for 

treatment, but they must be justified in the TQAP. For example, if holding water for a specified 

time after the treatment’s minimum contact time is required by the vendor, that time interval 

would be added to each verification test incorporating challenge organisms. For combinations of 

in-tank and in-line treatment, test duration will be equal to treatment time (in-line plus in-tank). 

The O&M test cycles will provide data on the system’s operation and support the assessment of 

non-biological verification factors. In the case of in-tank treatment approaches, particularly 

those using biocides or other chemical/physical means of achieving treatment, the TQAP may 

elect to operate the BWTS during O&M cycle either eliminating or reducing dosage of the active 

agent (i.e., to verify the electro/mechanical aspects of the BWTS). In some cases, it may be 

possible to use inert substances in place of treatment chemicals to reduce the need for 

conditioning prior to discharge back to the environment. Any such substitution must mimic the 

operation of the BWTS when using treatment chemicals and must be agreed to by the VO. 

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Protocol for the Verification of Ballast Water Treatment Technologies September 2010 

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5.4.6 Biological Efficacy (BE) Verification Testing 

As discussed above, a minimum of three BE test cycles per salinity regime will be conducted; 

each having a minimum tank holding time of one-day and having input challenge conditions as 

described in Section 5.2. The BE verification test cycles are intended to measure the efficacy 

with which the treatment system removes or kills organisms under challenging conditions. The 

remainder of this section provides the detailed description of test parameters, measurements, and 

analyses related to assessing biological efficacy and monitoring challenge water conditions. Due 

to the nature of the verification tests, a set of core and auxiliary measurement parameters will 

apply to each BE verification test. Core and auxiliary parameters, sampling location, and 

sample/measurement approach are shown in Table 8. Core parameters are those that are required 

during each BE test cycle and are the minimum measurements required to verify treatment 

efficacy and the validity of the BE test cycle. Auxiliary parameters are: (1) useful indicators of 

core parameters, (2) required by the vendor or VO, or (3) otherwise advisable to assess test 

validity or treatment efficacy. Guidance on sampling methods, sample volumes, sample 

container type, preservation method, and maximum holding time for each parameter is shown in 

Table 9. Although the maximum holding times are listed, all analyses should be conducted as 

soon as possible, 

The TO, in conjunction with the TF and the vendor, will assess the use of continuous, in situ 

(inline) biological or other process measurements during verification testing. Any selected 

methods must be described and justified in the TQAP and approved for use by the VO. 

The TO shall present a detailed schedule for verification test sample collection and analytical 

methods in the TQAP. At a minimum, the TQAP shall contain the scheduled sample collection 

times (expressed as time from start of test), parameters for testing, number of replicates, and 

number of control samples. 

5.4.6.1 Water Quality Parameters & Analysis 

Water quality samples shall be collected as described in Section 5.3.2.1 and defined in the 

TQAP, with the volumes described in Table 9. Note that some analyses, following methods 

described in Table 10, must be performed within 6 hours of the sample collection. In cases where 

water quality samples can be stored for appropriate time periods, TO logistics may warrant 

outsourcing of water quality analyses to an independent, qualified laboratory, with agreement by 

the VO. Reliable, continuously recording in situ sensors are available for temperature, salinity, 

dissolved oxygen, chlorophyll a, and turbidity. Such sensors may, with VO approval, be used to 

measure water quality parameters during verification testing. Discrete analytical samples shall 

be collected to provide test-specific verification or calibration of the sensor data and to allow 

comparison of sensor data to vendor-supplied information as appropriate. Sensor maintenance 

and calibration shall be described in the test site operating procedures and the TQAP. Data 

quality objectives for quality control and quality assurance purposes are provided in Table 11. 

These data quality objectives and the related QA/QC measures should be discussed in the QAPP 

of the TQAP as described in Appendix A. 

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Protocol for the Verification of Ballast Water Treatment Technologies September 2010 

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Table 8. Core and Potential Auxiliary Parameter and Measurement Techniques 

Measurement Sample Location and Approach 1,2 

Parameter Class Challenge Water Post Treatment 

Temperature Core In situ, continuous In situ, continuous 

Salinity Core In situ or Discrete grab In situ or Discrete grab 

Total suspended solids Core Discrete grab Discrete grab 

Particulate organic Core Discrete grab Discrete grab 

matter 

Dissolved organic matter Core In situ, continuous, In situ, continuous, 

discrete discrete 

Dissolved oxygen Core In situ, discrete In situ, discrete 

pH Core In situ, continuous In situ, continuous 

Ambient Organism Core Discrete Discrete 

Concentration 

Ballast System Flow Core In situ, continuous In situ, continuous 

Rate 

Ballast System Pressure Core In situ, continuous In situ, continuous 

Sampling Flow Rates Core In situ, continuous In situ, continuous 

Chlorophyll a (biomass) Core In situ, continuous In situ, continuous 

Dissolved nutrients Auxiliary NA Discrete 

(N, P, Si) 

Turbidity Auxiliary In situ, continuous In situ, continuous 

ATP (living material) Auxiliary Continuous as available Continuous as available 

1	 In Situ = in-line or in-tank measurements; Discrete Grab = an acquired sample for analysis at a specific place and 

time; Continuous = measurement is continuous throughout the period of operation at some defined rate. 2	 The frequency and means for calibrating and validating performance of in situ monitoring devices must be 

addressed in the TQAP. 

Discrete samples for determination of total suspended solids, particulate organic matter (as 

carbon, POM), total dissolved organic matter (as carbon, DOC), and nutrient concentrations shall 

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be collected appropriate to the tests being conducted. The concentration of mineral matter may 

be determined as the difference between the total suspended solids and the particulate organic 

matter concentration (mass per liter basis). In addition, when appropriate, samples should be 

acquired or measurements made in situ to measure residual toxicity or the concentration of 

chemical residuals or disinfection by-products. Guidance for such measurements and sample 

collection are highly dependent on the chemicals of interest or in use; a qualified laboratory 

should be consulted for the appropriate handling and measurement methods. 

The analytical methods must be applied within defined holding times (Table ) after appropriate 

preservation, per industry standard procedures. Where available, US EPA, Standard Methods or 

other methods (i.e., ASTM) approved by the VO will be used to quantify each parameter. If 

standardized methods are not available, the sampling and analytical methods to be used shall be 

documented in the TQAP. These methods will follow accepted scientific practices and be 

accepted by the VO. 

5.4.6.2 Biological Parameters 

Biological samples will be collected using methods and techniques appropriate to the size class 

and anticipated concentration being measured. The samples for biological analyses will be 

acquired from each of the time integrated sample volumes acquired during the test cycle. The TO 

will ensure that the contents of the integrated sample collection tanks have been thoroughly 

mixed to ensure homogeneity prior to sub-sampling. 

The abundance of living ambient and test organisms (if used) will be quantified in (1) the uptake 

challenge water just prior to treatment and entry into control tank, (2) the discharge of the control 

tank after the appropriate hold time, (3) the discharge following an in-line BWTS and (4) the 

discharge from the holding tank (for both types of treatment) of treated water after the 

appropriate holding time. In the case of the control and treated discharge, biological samples 

will be retrieved from a point upstream of any pumps or significant components which could be 

expected to affect organism mortality or sample representativeness. 

37
 
== Parameter 

Table 9. 

Min. Sample 

Volume (mL) 1 

Sample Volumes, Containers and Processing 

Sample 

3 Processing/ Preservation Containers 3 

 CoreParameters 

Electronic in situ data 

(Temperature, pH, Salinity, 

etc.) 2 

Total suspended solids 

Dissolved organic carbon Particulate organic carbon 

Dissolved oxygen Phytoplankton Enumeration 

(Live/Dead Analysis) 4 

Zooplankton Enumeration 

(Live/Dead Analysis) Low Concentration/Discharge Bacteria 

NA 

100 

25 

500 300 

3 3 m

concentrated to 

1000 mL 

3 3 m

concentrated to 

1000 mL 

1000 

NA 

250 mL HDPE or 

glass 

40-mL glass vial 

Whatman GF/F in 

foil 

300 mL glass 

BOD bottle 

Dark 1000-mL 

HDPE bottle 

1 L flask 

1 L sterile HDPE 

Maintain digital archive. Process immediately or store at 4°C. 

Pass sample through a GF/F; freeze filtrate until analysis. 

Pass through a GF/F; freeze filter until analysis. 

Fix per Oudot et al. (1988); titrate 2-24 h later. 

No preservation; stain with Fluorescein Diacetate and CMFDA as 

described in the protocol 

No preservation; Sub-sample into well plate (20 1-mL wells 

observed). Observe with dissecting microscope and probe organisms to determine live/dead status. Fix with Lugol’s solution 

for total counts. 

Plate on appropriate media. 

Auxiliary

ParametersDissolved inorganic nutrients Chlorophyll a and phaeopigments 40 400 60-mL polyethylene bottle Whatman GF/F in foil Pass through a NucleporeTM (Whatman Inc., Piscataway, NJ) membrane filter; freeze filtrate until analysis. Pass through GF/F; fix with a saturated MgCO3 solution; freeze filter until analysis. 

GF/F: pre-ashed glass fiber filter.
 

1 Volume processed for analysis; volumes are quantitative.
 2 Conductivity, temperature, pressure, dissolved oxygen, chlorophyll a fluorescence, transmissometry.
 3 Name brand items (e.g., NucleporeTM, WhatmanTM) may be substituted with comparable items from a different manufacturer.
 4 Dinoflagellate methods are under development.
 Protocol for the Verification of Ballast Water Treatment Technologies September 2010 Version 5.1 

38 
Table 10. Recommendation for Water Quality Sample Analysis Methods 

Parameter Units Instrument Method/Reference 

Dissolved ammonium 

μM Autoanalyzer APHA Standard Method No. 4500-NH3 / 20th edition 

EPA Method No. 349.0 http://www.epa.gov/nerlcwww/m349

Dissolved inorganic nitrate and inorganic nitrite 

μM Autoanalyzer ESS Method No. 220.3 http://www.epa.gov/glnpo/lmmb/meth

APHA Standard Method Nos. 4500-NO2-B and 4500-NO3-F

EPA Method No. 353.4 http://www.epa.gov/nerlcwww/m353

Dissolved inorganic phosphate 

μM Autoanalyzer ESS Method No. 310.1 http://www.epa.gov/glnpo/lmmb/meth

EPA Method No. 365.5 http://www.epa.gov/nerlcwww/m365

Dissolved inorganic silicate 

μM Autoanalyzer EPA Method 366.0 http://www.epa.gov/nerlcwww/m366_0.p

Dissolved organic carbon 

μM Carbon Analyzer APHA Standard Method No. 5310-C, 20th edition 

ASTM Method Nos. D6317, D2579, D4129, D4839, D513-02 

LMMB Method No. 096 http://www.epa.gov/glnpo/lmmb/meLMMB Method No. 014 http://www.epa.gov/glnpo/lmmb/meEPA Method No. 440.0 http://www.epa.gov/nerlcwww/m440

Particulate organic matter μM Carbon analyzer 

CHN Analyzer 

or LMMB Method No. 097 http://www.epa.gov/glnpo/lmmb/me

APHA Standard Method No. 5310-C, 20th edition 

LMMB Method No. 014 http://www.epa.gov/glnpo/lmmb/meEPA Method No. 440.0 http://www.epa.gov/nerlcwww/m440

Chlorophyll 

a/phaeopigments 

μg/L Fluorometer EPA Method 445.0 http://www.epa.gov/nerlcwww/m445_0.p

EPA Method No. 446.0 http://www.epa.gov/nerlcwww/m446EPA Method 447.0 http://www.epa.gov/nerlcwww/m447_0.pASTM Method No. 3731-87 (1998) 

Total suspended solids Mg/L 5-place balance ESS Method No. 340.2 (LMMB Method No. 065) 

http://www.epa.gov/glnpo/lmmb/methods/methd340.pdf APHA Standard Method No. 2540D (1998) EPA Method 160.2 http://www.epa.gov/region09/qa/pdfs/160

Dissolved oxygen Mg/L Radiometer TitraLab EPA Method No. 360.1 (Probe Method) 

APHA Standard Method No. 4500-0G (Probe Method) 

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Table 11. Data Quality Objectives for Water Quality Samples 

Core 

Parameter 

Frequency of QC Sample 

Collection 

Method Detection 

Limit 

Data Quality Indicator 

Type/Acceptance 

Criteria 

Dissolved Procedural blank Ammonia and silica Procedural blank 

nutrients Two (2) per treatment cycle 0.02 μM <5 times MDL 1 

Sample replicates Nitrate, nitrite, Sample replicates 

Three (3) sample replicates phosphate ≤2% PD 2 

per treatment cycle 0.01 μM 

Total Procedural blank Procedural blank 

suspended Two (2) per treatment cycle 0.1 mg/L <5 times MDL 

solids (DI Sample replicates Sample replicates <10% 

water and Three (3) sample replicates RPD 3 

seawater) per treatment cycle 

DOC Procedural blank Procedural blank 

Two (2) per treatment cycle 20 μM ≤15% PD 

Sample replicates Sample replicates 

Three (3) sample replicates ≤10% RPD 

per treatment cycle 

POM Procedural blank Procedural blank 

Two (2) per treatment cycle 5.5 μM ≤15% PD 

Sample replicates Sample replicates 

Three (3) sample replicates ≤10% RPD 

per treatment cycle 

Chlorophyll Procedural blank Procedural blank 

a/phaeophytin Two (2) per treatment cycle 0.02 μg/L <5% PD 

Sample replicates Sample replicates 

Three (3) sample replicates <15% RPD 

per treatment cycle 

Dissolved Procedural blank Procedural blank 

oxygen NA NA 

Sample replicates Sample replicates 

Three (3) sample replicates <5% CV 4 

per treatment cycle 

1 MDL = method detection limit. 

2 Percent Difference (PD) = [(true concentration – measured concentration)/true concentration] × 100%. 3 Relative Percent Difference (RPD) = {[absolute value (replicate 1 – replicate 2)]/[(replicate 1 + replicate 2)/2]} 

×100. 4 Filter blanks used for QC purposes only. 

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5.4.6.3 Sample Volumes & Data Quality 

Samples from the discharges of successful treatments will likely have low concentrations of 

organisms. Enumeration of the organisms from these samples (determined from 20 one-mL 

subsamples from a concentrated whole water sample, as described below) is represented by the 

Poisson distribution, and therefore the cumulative or total count is the key test statistic (Lemieux 

et al., 2008b). Further, a chi-square transformation can be utilized to approximate the confidence 

intervals. 

Assuming, for organisms ≥ 50 μm, that the desired minimum precision is that the upper bound of 

the chi-square statistic should not exceed twice the observed mean (this corresponds to a 

coefficient of variation of 40%), a count of 6 organisms is required.3 

The volume required to successfully count 6 organisms is dependent on the whole water sample 

volume, concentration factor, number of sub-samples counted, and the target concentration. 

Table 12 provides the resultant upper bounds, based on the Poisson distribution for a 95% 

confidence interval from the chi-square transformation for a variety of sample volumes at a 

concentration factor of 3000 (3 m3 concentrated to 1 L) assuming 20 subsamples of 1 mL. Given 

these assumptions, 30 m3 must be sampled to enumerate 10 organisms/m3, with the desired level 

of precision given above. The total sample volume may be reduced accordingly if: the 

concentration factor is increased, the confidence limit is lowered (e.g., from 95% to 90%), 

or the volume of subsample analyzed is increased (e.g., from 20 mL to 40 mL). If the latter 

is done, the TF should conduct validation experiments to ensure counting accuracy is high (e.g., 

using microbeads as described in Lemieux et al. (2010), which is found in Appendix C). It is the 

responsibility of the Testing Organization to justify any changes to the volumes suggested in 

Table 12. As discussed previously, sample replication is unnecessary as the Poisson distribution 

pools the data to improve the measurement precision and assumes the organisms to be randomly 

distributed. Note that this approach would not be appropriate if samples are not 

continuously acquired on a time-averaged basis. 

In any case, sample size should be selected relative to the targeted concentration and to provide 

the level of precision required to supply a 95% upper confidence limit which is (1) no more than 

twice the observed mean and (2) does not exceed the targeted concentration or as otherwise 

defined by the TO. Examples are provided in Table 13 for standards for organisms larger than 

50 μm compared to standards that are currently proposed or considered domestically and 

internationally. The chart provides the volume of sample required assuming that the entire 

sample is concentrated to 1 L and 6 organisms are counted. N is the number of samples 

analyzed, with each sample dispensed into a well plate and having 20 one-mL subsamples 

observed. 

A similar approach for organisms ≥ 10 μm and < 50 μm may be applied; however, the targeted 

concentrations are considerably denser, and the anticipated total counts can be expected to be 

3 Mathematically, that relationship can be represented as follows: coefficient of variation = standard 

deviation/mean. For the Poisson distribution, the variance = (standard deviation)2 = mean, thus 

substituting the critical value of the mean, 6, gives a coefficient of variation = 60.5/6 ≈ 40%. 

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higher. The Poisson distribution assumption still applies, and a more stringent level of precision 

may be applied. Specifically, if the desired level of precision is set at a coefficient of variation of 

10% or the upper confidence limit is not more than approximately 20% of the estimated density, 

then the volumes given in Table 14 result. These volumes are the required whole water sample 

volume to be concentrated to 1 L as a function of the number of 1 mL sub-samples (N). 

5.4.6.4 Zooplankton Enumeration 

Time integrated in-line sample volumes should be concentrated at the time of sampling using 35 

μm mesh plankton nets (50 μm in the diagonal). The concentrated contents of the cod-end 

should be rinsed into a flask. The volume capacity of the flask will be dependent on the organism 

density of the sample but typically requires a range of 1 to 4 L capacity. Fresh, artificial 

seawater, filtered seawater, or freshwater, as appropriate, should be added to maintain oxygen 

levels for the living organisms to be counted. If the initial sample has a low concentration of 

zooplankton, the sample may need to be further concentrated before analysis. In this instance, 

the sample should be concentrated using 35 μm mesh. 

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1 

Protocol for the Verification of Ballast Water Treatment Technologies September 2010
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Table 12. Density Confidence Intervals for Poisson Distributions Using the Chi-Square Sta

2 Scaled Densities and Confidence Intervals
 

Count Data (Assumes whole water sample volume is concentrated to 1L,

with analysis of 20 one-mL subsamples from the concentrate

Whole Water Sample
 

Volume (V) = 

3 V = 1 m 3 3 3 V = 3 m V = 10 m V = 30 m 

Organism


Count


95%


Upper


Bound
 Upper Bound / Count 

95% Mean Upper Density Bound 

(m-3) (m-3) 

95% 95% 95% Mean Mean Mean Upper Upper Uppe

Density Density Density Bound Bound Boun

(m-3) (m-3) (m-3) (m-3) (m-3) (m-3

0 3.00 0 150 0 50 0 15 0 5 

1 4.74 4.74 50 237 17 79 5 24 2 8 

2 6.30 3.15 100 315 33 105 10 31 3 10 

3 7.75 2.58 150 388 50 129 15 39 5 13 4 9.15 2.29 200 458 67 153 20 46 7 15 

5 10.51 2.10 250 526 83 175 25 53 8 18 

6 11.84 1.97 300 592 100 197 30 59 10 20 

7 13.15 1.88 350 657 117 219 35 66 12 22 

8 14.43 1.80 400 722 133 241 40 72 13 24 

9 15.71 1.75 450 785 150 262 45 79 15 26 

10 16.96 1.70 500 848 167 283 50 85 17 28 

11 18.21 1.66 550 910 183 303 55 91 18 30 

12 19.44 1.62 600 972 200 324 60 97 20 32 

13 20.67 1.59 650 1033 217 344 65 103 22 34 

14 21.89 1.56 700 1094 233 365 70 109 23 36 

15 23.10 1.54 750 1155 250 385 75 115 25 38 

16 24.30 1.52 800 1215 267 405 80 122 27 41 

17 25.50 1.50 850 1275 283 425 85 127 28 42 

18 26.69 1.48 900 1335 300 445 90 133 30 44 

19 27.88 1.47 950 1394 317 465 95 139 32 46 

20 29.06 1.45 1000 1453 333 484 100 145 33 48 

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Table 13. Sample Volume Required Relative to Treatment Standards–Organisms ≥ 50 μm 

N = 1 3 5 

Concentration (individuals/m3) 

0.01 60,000 

3

) 1 Sample Volume Required (m 

20,000 12,000 

0.1 6000 2000 1200 

1 600 200 120 

10 60 20 12 

1 Assumes the entire volume is concentrated to 1 L, 20 1-mL subsamples are analyzed, and the desired precision is 

the 95% Confidence Interval of the Poisson distribution = 2 times the observed mean and not greater than the 

Standard Limit. 

Table 14. Sample Volume Required Relative to Treatment Standards Organisms ≥ 10 μm 

and < 50 μm 

N 1 = 2 3 4 

Concentration (individuals/mL) Sample Volume Required (L) 2 

0.01 6000 4000 3000 

0.1 600 400 300 

1 60 40 30 

10 6 4 3 

 1 The number of 1 mL sub-samples analyzed.
 

 2 Assumes the entire volume is concentrated to 1 L and the desired precision is that CV is not greater than 10%.
 

Subsamples should be analyzed immediately, and as analysis proceeds, the original sample 

should be held at ambient water temperature. Previous work has shown zooplankton die-off 

occurs in the sample after a hold time of 6 hours. The appropriate maximum hold time should be 

validated at each test facility so that the detectable zooplankton mortality over the hold time does 

not exceed 5%. 

Subsamples should be extracted using 5-mL serological, graduated pipettes with an Eppendorf 

pipette helper (or a similarly accurate instrument that can effectively capture swimming 

zooplankton). Subsamples should then be examined in multi-well plates, Bogorov chambers, 

Sedgewick Rafter Counting Chambers, or counting wheels. The subsample should be dispensed 

into the counting chamber while still allowing for the addition of a narcotizing agent. In 

addition, the counting chamber volume should be shallow enough to allow for adequate focusing 

on the organisms during analysis. All direct counts should be done using counting chambers 

placed under a stereo or compound microscope at magnifications ranging from 10× to 40×. 

Lugol’s iodine solution should be used as a euthanizing and preservation agent. It should be 

noted that this agent works particularly well on the standard test organism Artemia spp. And for 

ambient organisms that have chitinous exoskeletons. It has been documented, however, that 

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Lugol’s can have distorting effects on the preservation of some marine organisms, particularly if 

their bodies lack chitin or other types of hard body structure. Given the choices of preservation 

or euthanizing agents available, additional validation is advised when different zooplankton are 

present in samples, or when dealing with organisms found at specific TFs, to determine which 

fixative(s) work best in preserving the zooplankton concentrations for total direct counts. 

In samples from challenge water or a control tank, the zooplankton should first be examined to 

count the number of dead organisms, defined by a lack of visible movement during an 

observation time of at least ten seconds. Unmoving but intact zooplankton may be living, so 

they are gently touched with the point of a fine dissecting needle or probe to elicit movement. 

Given that each dead organism is monitored for at least 10 seconds for visible movement, 

viability measurements could be lengthy for samples with dense concentrations of dead 

organisms, thereby increasing the potential for sample bias due to sample degradation. 

Once the number of dead organisms has been tallied, the organisms within the wells should be 

killed and/or preserved (to eliminate motion of the live organisms) and total counts obtained. 

Live counts will then be calculated from the difference using the equation: Total # – # Dead = # 

Live. Because samples collected following treatment are expected to have few living organisms, 

the living organisms can be enumerated directly. 

Note that samples collected to verify challenge conditions are met may require taxonomic 

identification of dominant organisms. 

5.4.6.5 Organisms ≥ 10 μm and ≤ 50 μm (nominally protists) 

Laboratory concentration of this size of organisms in the whole water sample can be 

accomplished by gently passing the sample through a sieve with mesh ≤ 10 μm in the diagonal. 

Care should be taken to gently sieve organisms to ensure they are not killed in the process. 

Techniques and standardized methods for the enumeration and viability analyses of protists 

remain an active area of investigation. This protocol recommends use a combination of two vital 

stains: Fluorescein Diacetate (FDA, Molecular Probes-Invitrogen Carlsbad, CA) and 5­

chloromethylfluorescein diacetate (CMFDA, CellTrackerTM Green; Molecular Probes-Invitrogen 

Carlsbad, CA). When non-specific esterases in living cells cleave the stains, the resultant 

molecules fluoresce green when excited with a blue light (e.g., Selvin et al., 1988; 

www.invitrogen.com). 

This method utilizes manual epifluorescence microscopy to evaluate samples: FDA (final 

concentration 5 μM) and CMFDA (final concentration 2.5 μM) are added to a 1 mL sample that 

is incubated in the dark for 10 minutes, the sample is loaded into a Sedgewick Rafter Counting 

Chamber, and it is examined under epifluorescence using a Fluorescein Isothiocyanate (FITC) 

narrow pass filter cube (e.g., excitation 465-495 nm, dichroic mirror wavelength 505 nm, barrier 

filter 515-555 nm; Drake et al., 2010). Samples should be examined for a maximum of 20 

minutes because the signal fades as stain leaks from the cell. If a cell is labeled by either FDA or 

CMFDA (as exhibited by a characteristic fluorescent green color) or moves, or both, it is scored 

as viable. A photomicrograph should be taken of any such cells under fluorescent and brightfield 

(white light) illumination to create a visual record of viable cells. 

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Research on the dual staining method at four locations (including marine and estuarine sites) in 

the U.S. has shown the method to yield variable degrees of false positives (Type I error) from as 

little as 3% to nearly 40% (Steinberg et al., 2010). Thus, before Tos use the dual staining 

method or any other alternative method, it is necessary that it undergo on-site validation by 

preparing, examining, and analyzing ambient samples that are killed (i.e., negative controls).4 

From the perspective of environmental protection, this type of error is conservative, as it 

overestimates the number of viable organisms. In contrast, Type II errors (false negatives) 

underestimate the number of viable organisms. Encouragingly, the Type II error rate was 

uniformly low across all study sites: 0% in three locations and 1% at the remaining two 

locations (Steinberg et al., 2010). Nonetheless, the Type II error rate should also be determined 

during initial site validation of this method or alternative method validation on a seasonal basis, 

and as part of the on-going QA program.5 

The advantages of using manual microscopy with vital stains are: (1) the instrumentation 

required is available in most research laboratories, (2) the cost of materials is low, (3) sample 

incubation times are relatively short, (4) the protocol is straightforward, and (5) results can be 

generated fairly rapidly. The disadvantages of this method are that it takes several hours (4-5 

hours) to completely characterize the subsamples within a sample, and unless the microscope is 

equipped with a camera (which is recommended), there is no archive of the data collected. 

Additionally, manual counts are subject to errors from operator-specific biases as well as from 

fatigue effects during extended observation periods. 

Within this size class fall dormant cells or resting stages exhibited across a broad phylogenetic 

range of microalgae, heterotrophic protists, and metazoans (e.g., Marret and Zonneveld, 2003; 

Matsuoka and Fukuyo, 2000). To encompass this group, the term ‘cysts’ is used, which includes 

but is not limited to cysts of dinoflagellates, spores of diatoms, cysts of heterotrophic protists, 

and cysts of rotifers. Notably, spores of bacteria and fungi are not included; they are smaller in 

minimum dimension than the lower limit of the size class considered here (10 μm). 

With focus on dinoflagellates alone, many authors (e.g., Dobbs and Rogerson, 2005; Doblin and 

Dobbs, 2006; and references in both) have made the point that cysts in ships’ ballast water 

represent robust ecological hazards. Given their resistance to physiological stress, killing cysts 

may be the best, i.e., most stringent, test of ballast-water technologies. If cysts can be killed, 

then there is excellent reason to assume vegetative cells or non-resting stages will also be killed. 

But because of their low metabolic state and relative impermeability to stains, it may be difficult 

to assess the viability of cysts on an individual basis without painstaking, cultural analyses, 

which, if possible at all, may require weeks or months to complete. At present, no rapid, reliable 

4 For example, heat–killed, negative control samples are prepared by placing ambient water samples in a 50 oC water 

bath. Once the sample temperature reached 50 oC, it is held in the bath for an additional 10 minutes (Drake et al., 

2010; Steinberg et al., 2010). The sample is cooled to room temperature before being stained. Organisms should 

not show a green, fluorescent signal after heat killing; those that do fluoresce represent false positives and indicate 

the Type I error associated with the dual-stain method. 

5 One approach is to collect ambient protists and place them in one of four categories based on an organism’s 

fluorescence signal and movement: (1) fluorescent and moving, (2) fluorescent and non-moving, (3) non-fluorescent 

and moving, and (4) non-fluorescent and non-moving (Drake et al., 2010; Steinberg et al., 2010). Organisms binned 

as non-fluorescent and moving are obviously viable, but the combination of stains fails to indicate viability, 

representing the Type II (false negative) error. 

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method to determine cysts’ viability is in widespread use, and the FDA-CMFDA method has 

yielded variable results with dinoflagellates and cyst-like objects. This protocol allows for 

additional techniques for plankton assemblages to be developed (Section 5.4.8); should a method 

reliably indicating cyst viability become available, it is assumed that it would allow all viable 

organisms within the �rotest size class to be enumerated. 

5.4.6.7 Organisms <10 μm 

Bacteria samples should not need to be concentrated from the whole water sample prior to 

analysis. Sample analysis will be conducted according to standard microbial techniques. 

Multiple bacterial growth media will be used to assess the effectiveness of a treatment for 

bacteria6. Use of multiple types of media enables measurement of the response of different 

portions of the ambient bacterial community7

. The minimum number of media used will include 

two general-purpose (1 marine, 1 nutrient agar) media for culturable aerobic heterotrophic 

bacteria. Other media may be added during the development of the TQAP. The rationale and 

methods will be described in the TQAP. 

For culturable, aerobic, heterotrophic bacteria, 1 mL samples should be diluted in a 10-fold 

dilution series in sterile Phosphate Buffered Saline (PBS) or sterile ambient water. Next, 100 μl 

of each appropriate dilution should be spread onto the media recommended in the protocol, with 

triplicate plates for each dilution. Plates should be incubated at 25 °C and monitored during the 

incubation time to ensure overgrowth does not occur. Colonies should be monitored and counted 

after 5 days (or after 3-5 days, if colony overgrowth appears imminent on all plates) and 

recorded as colony forming units (CFUs) per 100 mL of sample water. 

For E. coli samples, USEPA Method 1603 should be used: 1mL, 10 mL and 100 mL water 

samples should be passed through 0.45 μm membranes, which should be placed on modified 

thermo-tolerant E. coli agar (mTEC) plates (Becton Dickson, Sparks, MD). Plates should be 

incubated at 35 ± 0.5oC for 2 hours to allow for cell wall repair. Next, plates should be incubated 

at 44.5oC in a waterbath for 22-24 hours. Total red and magenta colonies should be scored and 

data reported as E. coli colonies per 100 mL of sample water. Alternatively, an IDEXX Colilert 

kit (Westbrook, ME) can be used according to the manufacturer’s protocol. 

For Enterococci samples, a modified version of USEPA Method 1106.1 should be used: 10 mL 

and 100 mL water samples should be passed through 0.45 μm membranes, the membranes 

transferred onto mEnterococcus agar (mEA) plates, and the plates incubated at 35 ± 2oC for 24 

hours. Membranes with light and dark red colonies should be transferred to bile esculin agar 

(BEA) plates, which should be incubated for 4 hours at 35 ± 2oC. After incubation, colonies 

with black halos should be scored and data reported as Enterococci per 100 mL. Alternatively, 

an IDEXX Enterolert kit (Westbrook, ME) can be used according to the manufacturer’s protocol. 

Toxigenic Vibrio cholerae densities should determined by a DNA colony blot hybridization 

method that detects ctxA gene (Huq et al., 2006). Briefly, colonies are grown on TCBS agar, 

6 The suggested media for marine water include 2216 Marine Agar and salt-modified R2A agar; media for fresh 

water species may include Plate Count Agar and Nutrient broth (plus agar (15 g/L)`). 7 Note it is assumed that if all culturable bacteria are killed, all non-culturable bacteria are also killed. 

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purified, and inoculated with 2.5% yeast extract and nalidixic acid and fixed after incubation 

overnight. Viable V. cholerae O1 and O139 cells are enumerated using a direct-fluorescent 

antibody kit (New Horizons Diagnostics; Columbia, MD) for serogroups O1 and O139 using 

monoclonal antibodies tagged with fluorescein isothiocyanate (FITC) under an epifluorescence 

microscope. 

Appropriate controls (e.g., heat to remove vegetative cells for tests using resting stages or spores) 

for microbial plates will be used throughout the verification testing. Steps will also be taken to 

ensure the action of any treatment (e.g., a biocide) is stopped at the time of sample collection 

(i.e., treatment does not continue after sample collection). Any steps and controls used to verify 

the effectiveness of a neutralizer will be described and justified in the TQAP. 

5.4.6.8 Auxiliary Parameters 

Sampling and analysis of supplemental parameters may be required depending on vendorspecified information. For example, a vendor may define an additional treatment effectiveness 

based on removal of fecal coliform bacteria or other microorganisms of public health concern. 

In such cases, the TO, with VO acceptance, will determine the appropriate supplemental 

parameters, based on vendor-specific information, and shall determine sampling and analysis 

requirements for inclusion in the sample collection schedule in the TQAP. 

5.4.7 BE Validity Criteria 

At the conclusion of each BE verification test cycle, the TO should verify that all criteria 

necessary for a valid BE test were established and maintained, as appropriate. As a minimum, 

the test validity criteria should consist of: (1) operational parameters that demonstrate the 

requisite volumes were transferred and sampled and Vendor-specified flows, pressures, or other 

validation criteria were maintained, (2) water quality challenge conditions for uptake and 

discharge waters, including any toxicity sampling as required by the TQAP, were met, and (3) 

biological challenge conditions for ambient organism concentration and diversity in treatment 

and control samples were met. Note that the vendor-specified validation criteria should be 

limited to operational parameters; that is, the criteria should be employed to ensure the system 

was operated correctly and in accordance with the provided training and O&M manual. 

Parameters that document a system failure under proper usage do not invalidate a test. The types 

and locations for measurements in each category are summarized in Table 15, and requisite 

criteria are discussed by category. 

Each of the measurement criteria and its valid ranges are to be documented by the TO in the 

TQAP. The declared ranges should accommodate variability of ambient source water conditions 

as well as possible ranges for ambient organisms in challenge water. The declared range of valid 

conditions also indicates the degree to which the TO can control the test parameters. Following 

each individual test, the TO will produce a test validation matrix that summarizes valid ranges 

(from the TQAP) and corresponding mean values obtained during the test. Any significant 

deviations from the mean noted during the testing shall be discussed in the verification report. 

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5.4.7.1 Uptake Operations 

The ETV protocol provides minimum requirements for volume and flow in Section 5.2.3, with 

final ranges for volume, pressure and flow to be identified in the TQAP by the TO and the 

vendor. For all water transfers, the minimum volume is 200 m3, and the minimum sample 

collection volume associated with each transfer is 3 m3. Acceptable ranges for sample collection 

volumes, pressures, and flows are also to be identified in the TQAP. The test validation matrix 

should provide the valid ranges and the resulting mean values to verify if valid, in-range 

conditions were met over the duration of the test at the locations specified in Table 16. 

5.4.7.2 Water Quality Conditions 

Minimum water quality conditions for BE tests are provided in Table 3 for dissolved and 

particulate organics, mineral matter, total suspended solids, and temperature for the two salinity 

ranges. The TO, in conjunction with the vendor, will declare valid test ranges for these and other 

relevant water quality parameters of interest (e.g., pH, DO, etc.) in the TQAP and provide a list 

of the valid ranges and mean results for each test in the test validation matrix. If there is reason to 

measure these parameters in the discharge waters as well, these measurements should also be 

presented. 

5.4.7.3 Biological Diversity and Concentrations 

Table 3 presented requirements for biological challenge conditions to include a minimum of 5 

species from 3 separate phyla across the three requisite size classes. This specification refers to 

populations at the point of treatment and entry to the control tank. Valid population densities and 

diversity ranges for ambient organisms should be defined by the TO in the TQAP. These ranges 

are envisioned to be fairly broad to accommodate variations in ambient populations and 

dominant species over the duration of the ETV testing. The anticipated ranges and the measured 

mean values for the sampled populations are to be presented in the test validation matrix. 

Organism population densities and diversity are also to be measured in the discharge samples for 

both treated and control waters, where minimum living concentrations are required in the control 

discharge as noted in Table 7. These criteria are given by totals for each size class and should be 

shown in the test validation matrix with results broken out for the dominant 5 species present in 

each size class. 

5.4.7.4 Biological Treatment Efficacy Determination 

Treatment efficacy will be determined by the measurement of living ambient organism 

concentrations in the treatment discharge for the three size classes identified in Table 3. 

5.4.7.5 Toxicity Test for Biocide Treatments 

Toxicity tests conducted during the start-up for treatments involving biocides in marine and 

brackish waters will be selected from the following: 

Inland Silverside, Menidia beryllina, Larval Survival and Growth (EPA Method 1006.0):
 

http://www.epa.gov/OST/WET/disk1/ctm13.pdf)
 

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Table 15. Challenge Test Validation Criteria by Location 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Control Treatment Control Treatment Control Treatment Discharge Discharge Parameter Sample Sample Tank Tank Sample Sample Tank Tank Tank Tank 

Ballasting Operations 

Volume × × × × × × 

Pressure × × × × × × 

Flow × × × × × × 

Vendor-specified parameters × 

Water Quality Conditions 

Temperature, Salinity, pH, DO 

(can monitor source waters) × × 

Total Suspended Solids (TSS) × × × × 

Dissolved Organic Carbon 

(DOC) × × × × 

Particulate Organic Material 

(POM) × × × × 

Mineral Matter (MM) × × × × 

Environmental Contaminants × 

Biological Diversity and Concentrations 

Ambient organisms/m3

; ≥ 50μm 

(live/dead) × × × × 

Ambient organisms/mL; 

≥ 10 and < 50μm (live/dead) × × × × 

Ambient organisms/mL; 

< 10 μm (live/dead) × × × × 

Sea Urchin, Arbacia punctulata, Fertilization Test (EPA METHOD 1008.0:
 

http://www.epa.gov/OST/WET/disk1/ctm15.pdf)
 

Mysid Acute Toxicity Test (EPA OPPTS Method 850.1035: 

http://www.epa.gov/opptsfrs/OPPTS_Harmonized/850_Ecological_Effects_Test_Guidelines/D 

rafts/850-1035.pdf) 

Additional guidance can be found in ASTM (1996a, 1996b) and Klemm, et al. (1994). Tests and 

species selected for toxicity testing during commissioning will be specified in the TQAP in 

accordance with the salinity ranges identified for testing. 

5.4.8 Alternative and Emerging Methods 

New methods for analysis and enumeration of living plankton communities are being developed 

to meet the relatively complex and demanding needs of ballast water treatment system testing. 

These methods include, but are not limited to, rapid analytical measurements, vital stains and 

dyes, and molecular probes. The inclusion or substitution of these techniques to those described 

above is acceptable. However, at a minimum the method(s) selected for any given size class 

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should provide a quantitative measurement of the concentration of living organisms. If nonstandard methods are selected, they should be validated by the TO to the satisfaction of the VO. 

5.4.9 Operation and Maintenance Verification Factor 

The operation and maintenance (O&M) of the ballast water treatment system will be verified. 

The verification has been designed as a minimum volume requirement, allowing sufficient time 

to verify operation and maintenance of the ballast water treatment system. It is anticipated that 

O&M testing commences in between BE test cycles to ensure some equipment run-time occurs 

prior to each BE verification test. In this manner, any changes in treatment efficacy due to 

equipment operation over time may be observed. 

The TO is responsible for monitoring and maintaining the system, in accordance with the 

Vendor’s O&M manual, throughout the testing to ensure stable operating conditions (as mutually 

agreed to by the vendor) and proper operating effectiveness. All system components will be 

monitored for proper operation throughout the test period. All maintenance activity completed 

during the verification testing shall be documented for inclusion in the verification report. 

All required monitoring and maintenance activities should be coordinated with the TO in 

advance of verification testing, and detailed in a monitoring and maintenance plan included in 

the TQAP. The monitoring and maintenance plan shall address the following requirements, as 

applicable: 

� A monitoring and maintenance schedule for the testing period (as shipboard systems are 

generally designed to require minimal regular maintenance, visual inspections by the 

operator may be all that is required); 

� Equipment and component calibration methods and frequencies; 

� Monitoring and maintenance activities and procedures shall be described and 

documentation forms provided – maintenance documentation forms must identify the TF, 

date and time, describe the work performed, observations of the treatment system, and 

results of the work; and 

� Operating characteristics and vendor-specified ranges required for proper operating 

conditions shall be described (e.g., system temperature, flows entering and exiting the 

system, power levels). 

Other information that must be addressed in the TQAP includes: 

� Monitoring requirements to ensure a proper operating environment; 

� Continuous on-line O&M monitoring requirements, as specified by the vendor; and 

� Credentials of all personnel involved in operating, monitoring and maintaining the 

treatment system. 

All monitoring and maintenance documentation must be maintained in a written record at the TF 

and will be included in the verification report. 

To help address predictability and reliability verification factors, qualitative and quantitative 

O&M performance indictors will be evaluated. The means and methods to evaluate or quantify 

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O&M performance indicators shall be included in the TQAP and described in a schedule for 

collecting this information. 

5.4.9.1 Qualitative O&M Performance Indicators 

Qualitative O&M performance indicators will include, but are not limited to: 

5.4.9.2 Visual Observations 

Visual inspections of the treated ballast water quality (e.g., turbidity, color) and treatment system 

conditions (e.g., foaming, floating material, settled solids) will be performed at each maintenance 

or monitoring event. Visual observations will also include the inspection of the treatment system 

prior to, during and following each test cycle for equipment and process failures, corrosion, 

leaks, impediments of flow (entering or exiting the system) and any other system issues that 

could impact performance. Specific visual indicators shall be defined in the TQAP. 

5.4.9.3 Operability 

Observations regarding the ease of start-up and operation during testing and the ease of 

monitoring system performance shall be noted and recorded. 

5.4.9.4 O&M Manual 

The TO shall evaluate the usefulness and quality of the O&M manual, and a written report on the 

evaluation shall be prepared. 

5.4.9.5 Operator Skills 

The level of operator expertise required to operate and maintain the treatment system shall be 

noted and compared with that indicated by the vendor. 

5.4.9.6 System Accessibility 

The ease of access and required clearances for system operation and required maintenance shall 

be noted. 

5.4.9.7 Quantitative O&M Performance Indicators 

Quantitative O&M performance indicators shall include, but are not limited to: 

5.4.9.8 Time demand 

Personnel time required to start-up, shutdown, operate, and maintain the treatment system shall 

be recorded in the monitoring and maintenance log. 

5.4.9.9 Residual 

Volumes of residual materials, (e.g., solids removed via filtration systems, etc.), mass generation 

rates, and concentrations shall be determined during verification testing. Results will be 

recorded in m3

, gallons or pounds per m3, or gallons of water treated, as appropriate. Factors 

related to the disposal of residuals (such as storage requirements and handling hazards) shall also 

be addressed. 

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5.4.9.10 Chemical Use 

Usage rates and concentrations of any chemicals (e.g., biocides) used as part of the treatment 

system and its operation during verification testing (per test cycle) will be measured and 

recorded. Results shall be reported for residuals and possible by-products. 

5.4.9.11 Power consumption 

The power consumed per test cycle by the treatment system will be monitored and recorded (e.g., 

kWh per m3 of water treated shall be calculated for use in cost factors below). The peak 

electrical load at system start-up will also be monitored and recorded as will fluctuations in 

consumption during test cycles. 

5.4.9.12 Other Consumables 

The use of any other consumables, such as filter cartridges, shall be monitored, documented, and 

reported. 

5.4.9.13 Supplemental Parameters 

Depending on vendor claims, supplemental monitoring, maintenance, and O&M performance 

indictors may be required. These will be described, along with requirements for performance 

monitoring, in the TQAP. 

5.4.9.14 Upset Conditions 

Upset conditions are those events or occurrences outside the operating parameters defined in the 

TQAP that result in either malfunctioning of the equipment, exception from normal operating 

conditions, or conditions causing alarms that indicate the system is producing or discharging 

treated water that exceeds the stated set points or limits for effective treatment. The cause of 

upset conditions may be due to conditions at the TF or the technology. These events may 

include both events in which the system is operating within the manufacturer’s specifications and 

those that are within specification but do not result in adequate treatment. The TO shall notify 

the vendor and the VO immediately when an upset condition is identified. The TO shall correct 

the upset condition as soon as possible to bring the treatment system back on line. For unusual 

upset conditions, the TO will work with the vendor to identify and correct the problem. The 

occurrence of all upset conditions, the causes, the results, and the means to correct the upset shall 

be documented at the time of the occurrence and shall be described in the verification report. 

As sampling is continuous over the course of the test, any upset conditions during the test need to 

be noted and a post-test review conducted to determine their cause and assess the impact on test 

results. (This task will be done by the TO and approved by the VO.) This review will determine 

where inclusion of these data is appropriate for performance assessment and the statistical 

analysis presented in the verification report. If the cause of an upset condition cannot be 

determined or the condition cannot be qualified as a true upset, then the sampling results shall be 

used in the statistical analysis for the verification report. 

5.4.9.15 Reliability 

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The mechanical reliability of the technology will be determined by comparing the Vendor 

projected mean-time between failure (MTBF) with the maintenance events observed during 

testing. The comparison will be reported in the verification report. 

The reliability of the treatment system to achieve treatment will be determined by (1) the number 

of instances where the treatment system or technology does not achieve the stated performance 

goal per the total number of test cycles, and (2) the standard deviation of the mean for biological 

performance data (e.g., percent removal). 

Reliability performance measures will take into consideration any vendor provided information 

that assists in the projection of the performance such as CT (concentration-time) disinfection 

information or power/energy curves. Any adjustments made to the system, outside of the 

vendor-specified operation and maintenance claims, to achieve the performance goals will be 

noted in the maintenance log and specified in the verification report. 

Specific performance reliability indicators along with the planned methods for evaluating and 

reporting them will be identified in the TQAP. 

5.4.9.16 Cost Factors 

Verified cost factors will include the following as applicable: 

5.4.9.17 Power consumption 

Power consumption will be reported as total kWh necessary to operate all equipment to achieve 

desired biological treatment performance. 

5.4.9.18 Consumable or expendable materials 

Amounts of all consumables or expendables, including chemicals or other items required for 

treatment, shall be itemized and reported. 

5.4.9.19 Replacement parts used during normal maintenance 

The number of replacement parts will be itemized and reported. Any unanticipated replacement 

of parts will be specified separately. 

5.4.9.20 Labor time to start-up, operate, and maintain the treatment system 

The total number of hours for each activity will be recorded and reported. 

5.4.9.21 By-product or waste materials produced 

By-products that require treatment or disposal will be reported as an expression of total volume 

treated or disposed. 

5.4.9.22 Environmental Acceptability 

Two performance indicators will determine the environmental acceptability of a treatment 

system: water quality and treatment residuals. 

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The data used to evaluate the environmental acceptability of a system will be taken from the 

water quality data collected at the point of discharge as detailed in Section 5.3.2. These data will 

include but may not be limited to the following parameters: 

� Temperature 

� pH 

� Salinity 

� Total suspended solids 

� Particulate organic matter 

� Dissolved organic matter 

� Dissolved oxygen 

� Dissolved nutrients 

� Biochemical oxygen demand 

� Biological efficacy 

The results of these tests at the point of discharge will be compared to the range of expected 

natural conditions and reported in the verification report. 

Additional analytical parameters will be included as necessary for reporting on any residual 

material that may result from treatment; for example residual biocides and disinfection 

byproducts. The additional parameters, the potential impact to the environment, and the 

analytical methods will be detailed in the TQAP 

It will be the responsibility of the TF to obtain NPDES discharge permits and to ensure that 

discharge is within permitted limits. Additionally, toxicity testing of any biocide treatment will 

be conducted, as discussed under Section 5.4.4. Verification testing will not begin unless the 

results of the toxicity tests are acceptable. 

5.4.9.23 Safety 

Safety is of concern during the operation of any equipment or machinery and during the use of 

potential hazardous materials, but it is of particular concern while on board ship, where staff is 

limited and access to land based emergency infrastructure is unavailable. Therefore, the safety 

of the treatment system will be evaluated during verification testing. 

The performance indicators for this verification factor will be technology specific, but, to the 

extent possible, required indicators shall include: 

� Listing of all dangerous or hazardous materials, including submittal of Material Safety 

Data Sheets (MSDS); 

� Potential to compromise the normal ship ballasting or deballasting cycle (i.e., impediment 

of flow); 

� Visual indicators of potential threats to shipboard operations, such as exposed or 

improper housing of power cables, structural stability of the system, external 

temperatures of the treatment system, and any other treatment-specific factors that may 

pose a threat to the operator or compromise the safety of ship operations; and 

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� Review of the vendor provided O&M manual for adequacy of cautions and guidance on 

ways to minimize the potential for, and directives to mitigate, a hazardous situation. 

The method for evaluating these and other items identified by the TO in reviewing the 

technology documentation shall be described in the TQAP. 

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Chapter 6 

Reporting Verification Testing Results 

Deviations from this protocol or any TQAP prepared for BWTS testing shall be described in the 

verification report, which shall include supporting documentation that provided the basis for 

acceptance of the deviations. All testing results will be presented in the report, including all data 

regarding challenge conditions, results of verification testing for all verification factors, and any 

vendor supplied data or information. A summary verification statement will also be prepared. 

The outline for the report shall include: 

� Verification Statement 

� Executive Summary 

� Introduction and Background 

� Description of the Treatment System or System 

� Experimental Design (including a description of all deviations from the protocol and the 

basis for accepting the deviations) 

� Description of Challenge Conditions 

� Methods and Procedures 

� Results and Discussion 

� Verification Testing Operation and Monitoring QA/QC 

Appendices: 

� TQAP 

� BE Test Validation Matrix 

� Vendor-supplied Operation and Maintenance Manual 

� Data Generated During Testing 

� QA/QC Records 

� Maintenance Logs 

� Any other records maintained during testing, such as chain of custody forms 

� Any other information provided by the Vendor, which may be of use to the stakeholder 

community 

Upon completion of the draft report the VO, the vendor, and the TF QA manager will review the 

document and supporting data, and provide comment. The comments will be addressed or 

stricken with approval of all parties and the final report will be submitted to NSF International 

(ETV Water Quality Protection Center partner) and EPA for QA and technical review. The final 

verification report and statement will be processed for clearance and posted on the ETV 

Program’s web site. 

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Chapter 7 

Quality Assurance/Quality Control (QA/QC) 

To ensure the quality and integrity of data gathered during testing activities, a Quality Assurance 

Project Plan (QAPP) will be prepared by the TO and included as part of the TQAP. The QAPP 

will describe the project scope, management, procedures for measurements and data acquisition, 

project assessment and oversight, and data validation and usability assessments necessary to 

meet the project goals. The written document will communicate all decisions related to project 

design and completion to the project team so work is performed according to written 

specifications. The generic format for a QAPP is included in Appendix A. EPA also provides 

guidance in preparing quality management plans, QAPPs and other quality management 

documents on their web site: http://www.epa.gov/quality. 

7.1 Project Management 

The QAPP will list all project participants and clearly define their roles and responsibilities. In 

addition, this section will describe project scheduling, data quality objectives, training and 

certification requirements (as applicable), and required documentation. The information 

included in this section will ensure that all participants understand the scope of the study and 

their explicit roles. Due to the complexity of testing in accordance with these protocols, it is 

advisable that each test cycle be preceded by a briefing or meeting in which the TF personnel 

critically review the plan of action, test operation, and conduct so they are familiar with the 

TQAP and their responsibilities. It is further recommended that this briefing or review be 

accompanied by a standardized test form that identifies the specific, quantitative set points and 

objectives that may be actively used throughout the test cycle to identify or record specific 

events, measurements or alarms. The consolidated and completed test form from each cycle 

should be included in an Appendix of the test report. 

7.2 Measurement and Data Acquision 

A detailed description of the experimental design and its components will be included in the 

QAPP. Specific requirements with regard to use, maintenance, and calibration of equipment, 

analytical procedures, chain-of-custody procedures, sample collection, data management and 

documentation, records management, project scheduling, experimental design assumptions, and 

disclosure of non-standard techniques or equipment will be discussed. 

7.3 Verification of Test Data 

The data quality objective process will be used to develop the QAPP and establish the locations, 

types and numbers of samples to be collected, the quality control samples (duplicates, blanks, 

spikes, etc.) required for both field and laboratory samples, and will establish the data quality 

criteria and measures of acceptability that are appropriate for the project. The TQAP will also 

detail a corrective action plan to describe actions to be taken if acceptance criteria for accuracy, 

precision and completeness are not met. 

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7.4 Assessment 

The effectiveness of QA/QC will be monitored through assessments of general and projectspecific activities. The QAPP will include detailed information on the types of assessments to be 

utilized (e.g., management, technical, and/or quality assurance assessments), appropriate 

response actions, reporting requirements, and assessment and reporting authority. To increase 

facility-to-facility and test-to-test comparisons, and TF internal QA/QC, standardized spiked and 

blank samples shall be incorporated into the sample analysis procedures. Spikes may be 

accomplished using inert objects such as stained, killed organisms or microbeads of appropriate 

size for the specific analyses. The methods to be used for spiked and blank samples shall be 

described in the QAPP. 

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Chapter 8 

Data Management, Analysis and Presentation 

8.1 Data Management 

Any data collected during testing activities must be capable of withstanding challenges to its 

validity, accuracy, and legibility. Data will be recorded in standardized formats and in 

accordance with the following minimum requirements: 

� Data are entered directly, promptly, and legibly; 

� Hand-entered data are recorded legibly in ink; all original data records include, as 

appropriate, a description of the data collected, the unit, the unique sample identification, 

the name of the person collecting the data, and the date and time of data collection; 

� Any changes to the original entry do not obscure the original entry, document the reason 

for the change, and are initialed and dated by the person making the change; 

� All deviations from the QAPP must be documented in writing, and approved by the TO; 

documentation and communication include an assessment of the impact the deviation has 

on data quality; and 

� Data in electronic format shall be included in a commercially available program for word 

processing, spreadsheet calculations, database processing, or commercial software 

developed especially for the data collection and processing on a specific hardware 

instrument or piece of equipment; backup of computer databases should be performed on 

a daily basis, if possible. 

Project-specific data management requirements, including the types of data to be collected and 

managed and how they will subsequently be reported, shall be defined in the data handling 

section of the TQAP. QA/QC activities for data management will be described in the QAPP and 

included in the TQAP. 

8.2 Data Analysis and Presentation 

Hand-recorded data gathered during verification testing will be entered into electronic format (a 

spreadsheet or other database product capable of performing graphical and simple statistical 

analyses). Following reduction, data will be presented in a graphical, tabular, or other logical 

format and accompanied by a detailed discussion to be included in the verification report. 

Treatment effectiveness will be calculated for each size class of ambient organisms as 

concentration per unit volume in the discharge and may be related to relevant standards as 

identified by the vendor or the vendor’s claims. In addition, viability data will be reported for 

STOs used in bench-scale experiments. Additional measures or comparisons may also be used 

to assess treatment efficacy, including percent organism removal by size class, or as a 

comparison of treated discharge to the control tank discharge. All methods will be described in 

the TQAP. The treatment effectiveness will be discussed in the verification test report with raw 

data included as an appendix. 

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Chapter 9 

Environmental, Health, and Safety Plan 

The TO shall develop an Environmental, Health, and Safety (EHS) Plan to be included in the 

TQAP. The EHS Plan shall identify all environmental concerns and potential hazards associated 

with the verification testing process and the TF, as well as the required measures to prevent 

exposure to the identified hazards. The TO shall be responsible for informing all personnel at 

the test site, including employees, contractors, and visitors, of the potential hazards and safety 

measures to be employed at the test site. The EHS plan shall address the following issues, as 

applicable: 

� Permitting requirements for equipment operation, effluent discharge, and waste disposal; 

� Biological, chemical, mechanical, electrical, and other hazards; 

� Environmental hazards will be defined in accordance with local, state and federal 

regulations; 

� Handling, storage, and disposal of all biological material and chemicals associated with 

the testing; 

� Safeguards and protocols to prevent the accidental release to the environment of any nonambient organisms if used in the test process; protocols of the form supplied in Part II 

ANS Task Force ANS Research Evaluation Protocol are recommended 

(http://www.anstaskforce.gov/Documents/Research_Evaluation_Protocol_ANSTF.pdf); 

� Material Safety Data Sheets (MSDS); 

� Conformance with the local electrical code; 

� Conformance with the local plumbing code; 

� Ventilation of equipment, trailers, or buildings housing equipment, if gases generated by 

the equipment could present a safety hazard; 

� Confined space entry hazards; 

� Fire safety; and 

� Emergency contacts for 911, the nearest hospital (provide directions), local fire 

department, the site manager, and all other important contacts. 

Any other environmental, health, or safety issues specific to the test location or ballast water 

treatment system to be tested must be addressed. A copy of the EHS plan, including all MSDS, 

shall be maintained and readily accessible at the test site. A one-page summary of emergency 

contacts shall be placed inside a clear plastic cover and kept at the verification-testing unit. 

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References
 

Anderson, A., J. Cordell, F. Dobbs, R. Herwig, and A. Rogerson. 2008. Screening of Surrogate 

Species for Ballast Water Treatment. Final Report to NSF International, Ann Arbor, MI. 

ASTM. 1996a. Standard Guide for Conducting Acute Toxicity Tests Starting with Embryos of 

Four Species of Saltwater Bivalve Mollusks. In Annual Book of ASTM Standards, 

Section 11, Water and Environmental Technology, Volume 11.05 (724-94), West 

Conshohocken, PA. 

ASTM. 1996b. Standard Guide for Conducting Acute Toxicity Tests with Echinoid Larvae, In 

Annual Book of ASTM Standards, Section 11, Water and Environmental Technology, 

Volume 11.05 (E1563-95). West Conshohocken, PA. 

Dobbs, F.C., and A. Rogerson. 2005. Ridding ships’ ballast water of microorganisms. Environ. 

Sci. Technol. 39: 259A-264A. 

Doblin, M.A., and F.C. Dobbs. 2006. Setting a size-exclusion limit to remove toxic 

dinoflagellate cysts in ships’ ballast water. Mar. Pollut. Bull. 52: 259-263. 

Drake, L.A., M.K. Steinberg, S.H. Robbins, S.C. Riley, B.N. Nelson, and E.J. Lemieux. 2010. 

Development of a method to determine the viability of organisms ≥ 10 μm and < 50 μm 

(nominally protists) in ships’ ballast water: a combination of two vital, fluorescent stains. 

NRL Letter Report 6130/1011 , Washington, D.C. 

Garthright, W.E. 1993. Bias in the logarithm of microbial density estimates from serial 

dilutions. Biometrical Journal 35: 299-314. 

Huq, A., C.A. Grim, R.R. Colwell, G.B. Nair. 2006. Detection, Isolation, and Identification of 

Vibrio cholerae from the Environment. Current Protocols in Microbiology: 6A.5 

Klemm, D.J., G.E. Morrison, T.J. Norberg-King, W.H. Peltier, and M.A. Heber. 1994. ShortTerm Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to 

Marine and Estuarine Organisms (Second Edition) EPA/600/4-91/003, Washington, DC. 

Lemieux, E.J., T.P. Wier, M.K. Steinberg, S.H. Robbins, S.C. Riley, E.C. Schrack, W.B. Hyland, J.F. 

Grant, C.S. Moser, and L.A. Drake. 2010. Design and preliminary use of a commercial filter skid to 

capture organisms ≥ 50 μm in minimum dimension (nominally zooplankton) for evaluating ships’ 

ballast water management systems at land-based test facilities. NRL Letter Report 6130/1029, 

Washington, DC. 

Lemieux, E.J., J. Grant, T. Wier, S. Robbins, S. Riley, W. Hyland, L. Davis, T. Donowick, B. 

Stringham, W.J. Kinee, B. Brown, and R. Everett. 2008a. Pilot Environmental 

Technology Verification (ETV) Test Report of the Severn Trent DeNora BalPureTM 

Ballast Water Treatment System, NRL Letter Report 6130/6098, Washington, DC. 

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Protocol for the Verification of Ballast Water Treatment Technologies September 2010 

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Lemieux, E.J., S. Robbins, K. Burns, S. Ratcliff, and P. Herring. 2008b. Evaluation of 

Representative Sampling for Rare Populations using Microbeads. Report No. CG-D-03­

09, United States Coast Guard Research and Development Center, Groton, CT. 

Marret, F., and K.A.F. Zonneveld. 2003. Atlas of modern organic-walled dinoflagellate cyst 

distribution. Rev. Palaeobot. Palynol. 125: 1-200. 

Matsuoka, K., and Y. Fukuyo. 2000. Technical Guide for Modern Dinoflagellate Cyst Study. 

http://dinos.anesc.u-tokyo.ac.jp/technical_guide/main.pdf. 

Oudot C., R. Gerard, P. Morin, and I. Gningue. 1988. Precise shipboard determination of 

dissolved oxygen (Winkler procedure) for productivity studies with a commercial system. 

Limnol. Oceanogr. 33: 146–150. 

Richard, R.V., J.F. Grant, and E.J. Lemieux. 2008. Analysis of Ballast Water Sampling Port 

Designs Using Computational Fluid Dynamics. Report No. CG-D-01-08. US Coast 

Guard Research and Development Center, Groton, CT. 

Selvin, R., B. Reguera, I. Bravo, and C.M. Yentsch. 1988. Use of Fluorescein Diacetate (FDA) 

as a single-cell probe of metabolic activity in dinoflagellates cultures. Biol. Oceanogr. 6: 

505-511. 

Steinberg, M.K., S.C. Riley, E.J. Lemieux, and L.A. Drake. 2010. Mult-site Validation of a 

Method to Determine the Viability of Organisms ≥ 10 μm and < 50 μm (Nominally 

Protists) in Ships’ Ballast Water Using Two Vital, Fluorescent Stains. Letter Report No. 

6130/1016, Published by the US Naval Research laboratory, Washington, D.C. 

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Appendix A: Quality Assurance Project Plan Outline
 

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Quality Assurance Project Plan
 

A Quality Assurance Project Plan (QAPP) shall be prepared as part of the TQAP for evaluating 

the performance of ballast water treatment technologies. Information on preparing QAPPs is 

provided on the EPA web site: http://www.epa.gov/quality. The generic format for QAPPs 

include: 

A.1 Project Descriptions, Objectives and Organization 

•	 The purpose of the study shall be clearly stated. 

•	 The processes to be evaluated will be described. 

•	 The TF, apparatus and technology set-up will be fully described. 

•	 Project objectives shall be clearly stated and identified as being primary or non-primary. 

•	 Responsibilities of all project participants shall be identified. Key personnel and their 

organizations shall be identified, along with the designation of responsibilities for planning, 

coordination, sample collection, measurements (i.e., analytical, physical, and process), data 

reduction, data validation (independent of data generation), data analysis, report preparation, 

and quality assurance. 

A.2 Experimental Approach 

•	 Technology installation and shakedown procedures will be identified. 

•	 Technology startup procedures will be identified. Startup will comprise a number of tasks to 

implement and check operating and sampling protocols. Tasks will include establishing feed 

makeup (including and procedures for challenge water preparation) and performing 

calibration checks on monitoring systems, identifying sampling and monitoring points and 

identifying the types of samples to be collected. 

•	 Physical, analytical or chemical measurements to be taken during the study will be provided. 

Examples include flow rates, pH, salinity, total suspended solids, particulate organic matter, 

dissolved organic matter, dissolved oxygen, dissolved nutrients, biochemical oxygen 

demand, biological organisms, O&M performance indicators, etc. 

•	 Sampling and monitoring points for each test unit and the type of sample to be collected 

(grab or composite) will be identified. 

•	 The frequency of sampling and monitoring as well as the number of samples required will be 

provided. This includes the number of samples needed to meet QA/QC objectives. 

•	 Planned approach for evaluation objectives (data analysis). This will include formulas, units, 

and definition of terms and statistical analyses to be performed in the analysis of the data. 

Example graphical relationships will be provided. 

•	 Demobilization of the technology, including scheduling and site restoration requirements, 

will be described. 

A.3 Sampling Procedures

•	 Whenever applicable or necessary to achieve project objectives, the method used to establish 

steady-state conditions shall be described. 

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•	 Each sampling/monitoring procedure to be used shall be described in detail or referenced. If 

compositing or splitting samples is required, those procedures shall be described. 

•	 Sampling or monitoring procedures shall be appropriate for the matrix or analyte being 

tested. 

•	 If sampling/monitoring equipment is used to collect critical measurement data (e.g., used to 

calculate the final concentration of a critical parameter), the QAPP shall describe how and at 

what frequency the sampling equipment is calibrated. 

•	 If sampling/monitoring equipment is used to collect critical measurement data, the QAPP 

shall describe how cross-contamination between samples is avoided. 

•	 When representativeness is essential for meeting a primary project objective, the QAPP shall 

include a discussion of the procedures to be used to assure that representative samples are 

collected. 

•	 A list of sample quantities to be collected, and the sample amount required for each analysis, 

including QC sample analysis, shall be specified in the QAPP. 

•	 Containers used for sample collection for each sample type shall be described in the QAPP. 

•	 Sample preservation methods (e.g., refrigeration, acidification, etc.) and holding times shall 

be described in the QAPP. 

•	 A sample of the chain of custody form to be used during testing shall be provided, including 

records of times and other critical parameters such as storage temperatures, light condition, 

etc. 

A.4 Testing and Measurement Protocols 

•	 Each measurement method to be used shall be described in detail or referenced in the QAPP. 

Modifications to EPA-approved or similarly validated methods shall be specified. 

•	 For unproven methods, the QAPP shall provide evidence that the proposed method is capable 

of achieving the desired performance. 

•	 For measurements that require a calibrated system, the QAPP shall include specific 

calibration procedures, and the procedures for verifying both initial and continuing 

calibrations (including frequency and acceptance criteria, and corrective actions to be 

performed if acceptance criteria are not met). 

A.5 QA/QC Checks 

A.5.1 Data Quality Indicators 

•	 Statistical analyses shall be carried out on data obtained for all performance measurements. 

As part of the assessment of data quality, six data quality indicators (DQIs) can be used to 

interpret the degree of acceptability or utility of the data. At a minimum, the QAPP shall 

include a protocol for assessing the following DQIs, and acceptable limits and criteria for 

each of these indicators: representativeness, accuracy, precision, bias, comparability, and 

completeness. 

•	 The TO shall determine acceptable values or qualitative descriptors for all DQIs in advance 

of verification testing as part of the experimental design. The assessment of data quality will 

require specific field and laboratory procedures to determine the data quality indicators. All 

details of DQI selection and values shall be documented in the QAPP. 

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A.5.1.1 Representativeness 

Representativeness refers to the degree to which the data accurately and precisely represent the 

conditions or characteristics of the parameter represented by the data. In this testing, 

representativeness will be ensured by executing consistent verification procedures. 

Representativeness will also be ensured by using each method at its optimum capability to 

provide results that represent the most accurate and precise measurement it is capable of 

achieving. For equipment operating data, representativeness entails collecting a sufficient 

quantity of data during operation to be able to detect a change in operations. 

A.5.1.2 Accuracy 

For water quality analyses, accuracy refers to the difference between a sample result and the 

reference or true value for the sample. Loss of accuracy can be caused by such processes as 

errors in standards preparation, equipment calibrations, loss of target analyte in the extraction 

process, interferences, and systematic or carryover contamination from one sample to the next. 

Loss of accuracy for microbial species can be caused by such factors as error in dilution or 

concentration of microbiological organisms, systematic or carryover contamination from one 

sample to the next, improper enumeration techniques, etc. The TO shall discuss the applicable 

ways of determining the accuracy of the chemical and microbiological sampling and analytical 

techniques in the TQAP. 

For equipment operating parameters, accuracy refers to the difference between the reported 

operating condition and the actual operating condition. For water flow, accuracy may be the 

difference between the reported flow indicated by a flow meter and the flow as actually 

measured on the basis of known volumes of water and carefully defined times. Meters and 

gauges must be checked periodically for accuracy, and when proven dependable over time, the 

time interval between accuracy checks can be increased. In the TQAP, the TO shall discuss the 

applicable ways of determining the accuracy of the operational conditions and procedures. 

From an analytical perspective, accuracy represents the deviation of the analytical value from the 

known value. Since true values are never known in the field, accuracy measurements are made 

on the analysis of QC samples analyzed with field samples. QC samples for analysis shall be 

prepared with laboratory control samples, matrix spikes, and spike duplicates. It is 

recommended for verification testing that the TQAP includes laboratory performance of one 

matrix spike for determination of sample recoveries. Recoveries for spiked samples are 

calculated in the following manner: 

 

 

 

 

100(SSR − SR ) % Recovery = (A-1) SA 

where: SSR = spiked sample result 

SR = sample result 

SA = spike amount added 

Recoveries for laboratory control samples are calculated as follows: 

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 100( foundconce ntration ) % Recovery =	 (A-2) trueconcen tration 

 

 

 

 

 

 

 

 

 

For acceptable analytical accuracy under the verification testing program, the recoveries reported 

during analysis of the verification testing samples must be within control limits, where control 

limits are defined as the mean recovery plus or minus three times the standard deviation. 

A.5.1.3 Precision 

Precision refers to the degree of mutual agreement among individual measurements and provides 

an estimate of random error. Analytical precision is a measure of how far an individual 

measurement may be from the mean of replicate measurements. The standard deviation and the 

relative standard deviation recorded from sample analyses may be reported as a means to 

quantify sample precision. The coefficient of variation (CV) may be calculated in the following 

manner: 

 

 

 

S (100) % CV =	 (A-3) X average 

where: S = standard deviation
 

Xaverage = the arithmetic mean of the recovery values
 

 

 

 

 

 

 

 

 

 

 

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Standard Deviation is calculated as follows: 

 

 

 

 

(Xi − X )

2 

Standard Deviation = (A-4) 

n −1 

where: Xi = the individual recovery values 

X = the arithmetic mean of the recovery values 

n = the number of determinations 

The QAPP shall list and define all other QC checks and/or procedures (e.g., detection limits 

determination, blanks, spikes, surrogates, controls, etc.) used for the project. 

For each specified QC check or procedure, required frequencies, associated acceptance criteria, 

and corrective actions to be performed if acceptance criteria are not met shall be included in the 

QAPP. 

A.6 Data Reporting, Data Reduction, and Data Validation 

•	 The reporting requirements (e.g., units) for each measurement and matrix shall be identified 

in the QAPP. 

•	 Data reduction procedures specific to the project shall be described, including calculations 

and equations. 

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•	 The data validation procedures used to ensure the reporting of accurate project data to 

internal and external clients should be described. 

•	 The expected product document that will be prepared shall be specified. 

A.7 Assessments 

Whenever applicable, the QAPP shall identify all audits (i.e., both technical system audits 

[TSAs] and performance evaluations [PEs]) to be performed, who will perform these audits, and 

who will receive the audit reports. 

A.8 References 

References shall be provided in the QAPP in the body of the text as appropriate. 

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Appendix B: Anderson, et al. Approach for Evaluation of Standard
 

Test Organisms
 

Taken From:
 

Final Report
 

To NSF International
 

For
 

Contract No 03/06/394
 

Woods Hole Oceanographic Institute
 

Woods Hole, MA 02543
 

“Screening of Surrogate Species for 

Ballast Water Treatment” 

3 July, 2008 

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MATERIALS AND METHODS 

Common Methods 

Synthetic Water Preparation 

The freshwater and seawater used in these experiments were prepared in each PI’s lab using 

synthetic solutions for uniform consistency and replication purposes. The seawater for these 

experiments was prepared by dissolving Instant Ocean® salts in Milli-Q or equivalent deionized 

water to achieve the desired salinity. Freshwater was prepared either as WC medium (Guillard, 

1975) (for heterotrophic protists and phytoplankton) or according to the EPA’s recommended 

aquatic toxicology testing protocol for freshwater organisms 

(http://www.epa.gov/waterscience/WET/disk2/) (for bacteria and zooplankton). Freshwater and 

seawater were prepared using Milli-Q water or equivalent water (bacteria and zooplankton only) 

and was filtered through glass fiber filters (marine zooplankton) or filter-sterilized (all other taxa 

and water combinations) before use. Additional details are found in the taxon-specific methods 

sections. 

Treatments 

The treatments, their associated vendors, dose, and literature citation are summarized in Table 1. 

Phase I tests separately involved thermal, glutaraldehyde, and hypochlorite treatments. All other 

treatments were administered in Phase II tests. The methods common to all taxa tested are 

provided in the text following the table. Please note there occasionally were deviations from the 

specific concentrations or times listed here, and those deviations, as well as other specific 

methods, are given in the taxon-specific section of the Materials and Methods. In particular, the 

data sets for the zooplankton species are more extensive than for the other three taxa. 

Thermal treatment: Regardless of the organisms or the volume of the experimental container in 

which they were held, the thermal treatment was begun by immersing the container into a water 

bath (accurate to ± 0.5o C) heated to the test temperatures (35°, 40°, 45°, and 50° C). Timing of 

the treatment began once the water in the container reached the criterion temperature. After 4 

hours (bacteria and zooplankton) or 8 hours (heterotrophic protists and phytoplankton) of 

treatment, viability was assessed. Controls consisted of vessels containing organisms held for 

the same time at room temperature. 

Chlorine (sodium hypochlorite): Household bleach (UltraClorox® Regular Bleach) has a 

concentration of 6.0% sodium hypochlorite. Appropriate volumes were added to generate four 

experimental concentrations of sodium hypochlorite (0.25, 0.5, 1.0, and 2.0 mg/L). Additional 

concentrations were tested in the zooplankton study; see details in the taxon-specific section. 

After 24 hours of exposure, viability was assessed. Control vessels containing organisms were 

not treated with hypochlorite, but were held for the same time at room temperature. 

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Table 1. Summary of treatment and experimental conditions. 

Treatment Vendor or 

source 

Concentration or 

Intensity 

Exposure 

time References 

Thermal 

treatment 

Water bath 35 ̊, 40 ̊, 45 ̊, 

50 ̊ C 

4 hours or 8 

hours 

Hallegraeff et al. 1997; Rigby 

et al. 1999 

Chlorine 

(sodium 

hypochlorite) 

Chlorox bleach Aqueous solution of 

sodium hypochlorite. 

Final concs. of 0.25, 

0.5, 1.0, 2.0 mg/L 

24 hours Sano et al. 2004; Bolch and 

Hallegraeff 1993 

Chlorine 

dioxide 

(EcochlorTM) 

Ecochlor, Inc. Final concs. of 1, 2, 

4, 6 ppm 

24 hours T. Perlich, Echochlor, Inc. 

(pers. comm., 19 & 20 Oct. 

2004) 

Glutaraldehyde Fisher Scientific Final concs. of 50, 

100, 500 and 1000 

mg/L 

24 hours Sano et al. 2003 

UV light UV collimator 

designed and built 

by Dr. E. “Chip” 

Blatchley, Purdue 

University 

UV light (256 nm) at 

10, 25, 50, 100 

mJ/cm2 

Dose independent of 

exposure time 

between c. 30 

sec. to 2 min. 

Azanza et al. 2001; Montani 

et al. 1995; Sutherland et al. 

2001; Sutherland et al. 2003 

Ozone Enaly OZX-300U 

or Clear Water 

Tech UV-275 

Total initial residual 

oxidant (TRO) level 

of 0.25, 0.5, 1.0, and 

2.0 mg Br2/L in 

seawater 

24 hours after 

achieving initial 

level of TRO 

Hoigné 1998; Langlais et al. 

1991; Cooper et al. 2002 

Hydrogen 

peroxide 

Local drugstore Final concs. of 0.5, 

1, 10 and 20 ppm 

24 hours after 

achieving intial 

conc. 

Kuzirian et al. 2001 

Deoxygenation BBL GasPak 

SystemTM 

Anoxia (0 mg/L 

oxygen) 

24, 48, 72 hours Tamburri et al. 2002; P. D. 

McNulty, NEI Treatment 

Systems, Inc. (pers. comm.. 

27 Oct. 2004) 

SeaKleen® Vitamar, Inc. 0.25, 0.5, 1.0, 2.0 

mg/L active 

ingredient 

24 hours Cutler et al. 2003; Sano et al. 

2004 

PeraClean® 

Ocean 

Degussa AG Final concs. of 50, 

100, 200 and 400 

ppm 

24 hours http://dmses.dot.gov/docimag 

es/pdf81/175321_web.pdf 

Note:	 NB SeaKleen® was tested only with zooplankton species and Geobacillus stearothermophilus, as it was not 

provided by its manufacturer for general distribution to the PIs. 

Chlorine dioxide (EcochlorTM): Species were tested against four concentrations (1.0, 2.0, 4.0, 

and 6.0 ppm) of chlorine dioxide (EcochlorTM). Reagents were provided by Ecochlor, Inc. 

(Acton, MA). A concentrated stock solution (3,000 ppm) was prepared as per manufacturer 

directions by dissolving the two-part reagent into one liter of distilled water. This stock was 

maintained in the refrigerator in a glass bottle until required. To make working solutions, 

appropriate volumes were added to either WC or sterile artificial seawater to generate the four 

experimental concentrations listed above. Additional concentrations were tested in the 

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zooplankton study; see details in the taxon-specific section. Treatment times were 24 hours in all 

cases, during which time experimental and control organisms were incubated at room 

temperature. After 24 hours, viability was assessed. 

Glutaraldehyde: Glutaraldehyde (Fisher Scientific, Cat. No. G151-1 or for the zooplankton and 

bacteria studies, 25% solution from Sigma Chemical Company) was used at concentrations of 

50, 100, 500, and 1000 mg/L in either artificial seawater or WC medium. Additional 

concentrations were tested in the zooplankton study; see details in the taxon-specific section. 

Controls consisted of organisms incubated in all ways the same, save the addition of 

glutaraldehyde. After 24 hours of treatment at room temperature, the viability of the organisms 

was determined. 

Ultraviolet light: This treatment utilized a collimator built by Dr. E. “Chip” Blatchley, Purdue 

University, which has a 256 nm UV bulb to provide controlled dosages of UV light onto the test 

surface. Different dosages were delivered by varying the length of time organisms were 

exposed. The protocols employed were developed by Drs. Russell Herwig and Adelaide Rhodes 

(University of Washington). The test organisms and medium were transferred into disposable 50 

mm diameter petri dishes, which were positioned ca. 1 cm beneath the end of the collimator. 

Dosages used for zooplankton, protists, and phytoplankton were: 10 mJ/cm2 (72 seconds); 25 

mJ/cm2 (180 seconds); 50 mJ/cm2 (360 seconds); and 100 mJ/cm2 (720 seconds). Similar 

dosages were used for bacteria but since these experiments were performed later in the contract 

period and following extensive usage of the same UV lamp, the exposure time needed to be 

slightly increased to achieve the similar doses. The doses (time of exposure) for the bacteria 

were: 10 mJ/cm2 (80 seconds); 25 mJ/cm2 (200 seconds); 50 mJ/cm2 (400 seconds); and 100 

mJ/cm2 (800 seconds). Additional higher dosages were tested in the zooplankton study since 

some of the species were tolerant of 100 mJ/cm2

; see details in the taxon-specific section. 

During the UV exposure, organisms suspended in freshwater or seawater in the petri dishes were 

stirred with a small magnetic stir bar to ensure equal exposure of all organisms placed in the 

dish. The control exposures were conducted in the same way, but the UV light was not turned 

on. 

Ozone: Ozone (O3) is a gas that is also an oxidizing biocide. It is commonly used for the 

disinfection of freshwater and seawater, such as in drinking water, freshwater and seawater 

aquaria. Ozone can be generated in a corona-discharge tube by passing an electrical current 

through an atmosphere that is enriched in oxygen. A variety of commercial ozone generators are 

available, but we decided to use a small inexpensive unit that could generate ozone for benchscale experiments. We chose the Enaly (http://www.ozone.enaly.com/, Shanghai, China) OZX-300 U 

unit with air dryer and built-in aerator. 

We calibrated the unit using synthetic seawater and freshwater (R. Herwig and A Rhodes, 

University of Washington). To calibrate the instrument, ozone was bubbled into test water for 

various times, and Total Residual Oxidant (TRO) levels were measured for seawater and the 

ozone concentrations were measured for freshwater. Ozone in freshwater is toxic by direct 

contact with the organism. In seawater, ozone quickly reacts with bromide ions (Br-

) to form 

bromines, compounds that are long lasting disinfectants. In freshwater, this transformation does 

not occur. The concentration of bromines was measured using the DPD (N,N-diethyl-p73
 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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phenylenediamine) colorimetric test (Hach DREL/2010 spectrophotometer with the DPD Total 

Chlorine Powder Pillows (US EPA Method 8016), Hach Company, Loveland, CO). Using a 

calibration curve (Figure 2), the times required to achieve the desired concentration of ozone 

were calculated. A similar procedure was used to calibrate freshwater ozone concentrations. We 

directly measured ozone concentrations in freshwater at various times using a Hach DREL/ 2010 

spectrophotometer with Ozone Accuvac ampoules that measure the formation of indigo (US 

EPA Method 8311). The freshwater ozone test was difficult to perform because of the rapid 

disappearance of ozone, so we repeated the calibration three times (Figure 3). 

To expose the zooplankton, phytoplankton, and protists to ozone, we placed the test organisms in 

500 ml of seawater or freshwater, inserted the airstone provided by the manufacturer (Enaly) into 

the liquid, and let the generator run for the required time. Ozone testing with bacteria followed a 

slightly different protocol because small volumes of liquid were treated. For freshwater testing, 

ozone was produced using the Enaly ozone generator. The UV-275 (ClearWater Tech, LLC, San 

Luis Obispo, California), which produces ozone using UV light, was used for testing seawater 

conditions, because the Enaly generator produced ozone too rapidly. 

TRO Concentration for Marine Treatments vs. Time 

y = 1.1803x + 0.203 

R2 = 0.9981 

0 

1 

2 

3 

4 

5 

6 

7 

8 

9 

0 1 2 3 4 5 6 7 

Time 

Concentration (mg CL2/L)

Trial 1 

Trial 2 

Trial 3 

Average of All Trials 

Linear (Average of All Trials) 

Figure 2. TRO concentrations (measured as mg Cl2/L) for synthetic seawater treated with 

ozone versus time using the Enaly ozone generator. 

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Protocol for the Verification of Ballast Water Treatment Technologies September 2010 

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Ozone Concentrations for Freshwater 

Treatments vs. Time 

y = 0.4923Ln(x) - 0.4348 

R2 = 0.9763 

0 

0.2 

0.4 

0.6 

0.8 

1 

1.2 

0 5 10 15 20 25 

Time 

Concentration (mg/L) 

Figure 3. Ozone concentrations for freshwater versus time using the Enaly ozone 

generator. 

Exposure times for each experimental run were based on a standard curve of ozone 

concentrations versus time (Figure 4) (Russell Herwig and Jake Perrins, University of 

Washington). Containers holding test bacteria were injected with ozone to four experimental 

TRO concentrations (0.25, 0.50, 1.0, and 2.0 mg Br2/L). Following exposure, organisms were 

held for 24 hours at room temperature, after which their viability was determined. Controls were 

similarly treated, but ozone was not injected into the test containers. Additional ozone 

concentrations were tested in the zooplankton study; see details in the taxon-specific section. 

Hydrogen Peroxide: Hydrogen peroxide (3%) was purchased locally at drug stores by the PIs. 

Only unopened bottles were used to make treatment solutions. Appropriate volumes were added 

to either WC or sterile artificial seawater to generate four experimental concentrations of 

hydrogen peroxide (0.5, 1.0, 10.0, and 20.0 ppm). Additional concentrations were tested in the 

bacteria and zooplankton studies; see details in the taxon-specific section. All treatments lasted 

24 hours at room temperature. Viability was assessed thereafter. 

Deoxygenation: The deoxygenation tests used a BBLTM GasPakTM anaerobic system (Becton 

Dickinson, Becton Drive, Franklin Lakes, NJ 07417). The GasPakTM system is self-contained 

and produces anaerobic conditions when used with hydrogen and CO2 generating GasPakTM 

envelopes (BD# 271040). Test organisms and media were placed in Petri dishes and the dishes 

placed in the chamber, together with GasPakTM dry anaerobic test strips (BD # 271051). The 

strips were monitored for color change when conditions became anaerobic, approximately 2 

hours after activation. The containers were sealed to prevent infiltration of any oxygen over the 

time course of the experiment, and treatments lasted 24, 48, and 72 hours at room temperature. 

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2.5 

Ozone diffusion in saltwater 

y = 0.5033x - 0.2673 

R2 = 0.9914 

2 3 4 5 6 

Time (min) 

TRO mg/L as Br2 O3 mg/L 

2 

1.5 

1 

0.5 

0 

0 1 

Ozone diffusion in freshwater 

1 

0.75 

0.5 

0.25 

0 

y = 0.1943x + 0.0173 

R2 = 0.9517 

0 1 2 3 4 5 6 

Time (min) 

 

 

 

 

 

 

 

 

 

 

 

 

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Figure 4. Ozone standard curves used to test bacteria in seawater and freshwater. (For 

seawater, bromine was measured as TRO. For freshwater, ozone was directly measured.) 

Experimental controls consisted of containers not exposed to anaerobic conditions for same time 

periods. After treatment, the viability of the organisms was determined. 

SeaKleen® (zooplankton and Geobacillus stearothermophilus only): As mentioned earlier, 

Vitamar, Inc. chose not to distribute SeaKleen® to the PIs. The zooplankton and bacteria 

investigators, however, had some material remaining from previous work and were able to 

perform a series of experiments. See the taxon-specific methods for details. 

PeraClean® Ocean: The manufacturer of PeraClean® (Degussa Corporation, 379 Interpace 

Parkway Parsippany, NJ 07054) supplied the product, certified to contain 15.4% peracetic acid 

and 14.2% hydrogen peroxide. PeraClean® volumes were added either to WC or sterile artificial 

seawater to generate four experimental concentrations (50, 100, 200, and 400 ppm). Treatment 

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times were 24 hours in all cases and tubes were incubated at room temperature. Controls 

consisted of containers identical in all ways save the addition of PeraClean® Ocean. 

Experimental Replication and Controls 

Each combination of treatment level and species tested were run in quadruplicate, as were the 

controls, for the bacteria, heterotrophic protists, and phytoplankton. Zooplankton experimental 

combinations were replicated at least three times. In any case where high mortality (defined as 

>20%) was determined for the controls, the experiment was repeated until controls achieved an 

acceptable level of survival. 

Viability Determinations 

For the potential surrogate species studied here, viability was determined either though an 

assessment of their growth potential or in the case of zooplankton species, their ability to move 

or respond to mechanical stimulation. Thus, two different approaches were followed, one 

culture-based and the other behavioral. 

Bacterial viability was determined based on the ability of the species to grow on agar plates 

comprised of a standard general or selective bacteriological medium. This classic technique 

yields so-called “colony-forming units” (CFUs) that can be pointed to and counted. 

Effectiveness of a treatment was expressed as the log-scale mortality in the treatment relative to 

the control. 

Viability of both heterotrophic protists and phytoplankton was assessed using the “most probable 

number” (MPN) method (Throndsen 1978), also known as the “extinction dilution method” of 

Imai et al. (1984). The MPN data yield an estimate of the abundance of cells (or cysts) capable 

of dividing (or germinating and dividing). It is important to understand the dormancy and 

excystment characteristics of the species being investigated if this method is to be used, since 

cysts sometimes will not germinate, but are nevertheless viable in the long term. 

Briefly, the MPN counting method begins with pipetting medium into a set of tubes, one set for 

each of the four replicate tubes of organisms (experimental or control). The inoculum volume 

added to each MPN tube differs by a factor of 10. After mixing, samples of the MPN series are 

then transferred to multiwell plates and following an appropriate incubation period, wells are 

examined by inverted microscopy to determine cell viability. The number of positive scores 

(i.e., the presence of viable cells in a well) is entered into a Most Probable Number program 

(Blodgett, 2003) to determine the number of organisms per ml. Percent survival is calculated by 

comparing the MPN from the treated samples with the MPN of the control after incubation. We 

emphasize this MPN technique is suitable for pure cultures of heterotrophic protists or 

phytoplankton, but is not a useful tool for mixed cultures. 

To assess viability of zooplankton following treatment, animals were placed in a counting wheel 

and examined under at least 10× magnification to determine their survival (indicated by motion) 

or mortality (lack of movement after a few seconds of observation or prodding with a wire 

pointer). 

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Bacteria Methods 

General 

The Phase I assessment utilized two Gram negative pathogen-indicator organisms, Enterococcus 

avium (ATCC 14025) and Vibrio cholerae (ATCC 14033), and two Gram positive spore-forming 

organisms, Geobacillus stearothermophilus (ATCC 7953), and Bacillus subtilis (ATCC 6633). 

E. avium and V. cholerae were purchased from the American Type Culture Collection (ATCC) 

(www.atcc.org) as freeze dried pellets of cells. G. stearothermophilus and B. subtilis were 

purchased as spores from SGM Biotech, Inc. (www.sgmbiotech.com). Spores of these species are 

also available from other commercial sources. Spore suspensions from these and other bacterial 

species are commonly used for disinfection experiments and the testing of disinfectants, so the 

suspensions are routinely available. Phase II experiments were conducted solely with G. 

stearothermophilus, based on its superior performance (as a potential surrogate species) in Phase 

I of the investigation. G. stearothermophilus is a thermophilic bacteria that has a recommended 

incubation temperature of 55° C. 

At the beginning of an experiment, sterile containers (test tubes or petri dishes) were inoculated 

with a live bacterial culture or spore suspension to achieve a final concentration of 104 to 105 

cells/mL. Two methods were used to provide this concentration. In the first, freeze-dried pellets 

of E. avium and V. cholerae were rehydrated and streaked onto agar media, as suggested by 

ATCC, and incubated for 24 hours at 37o C. Colonies were harvested with a sterile inoculating 

loop, and plated onto general purpose agar media, Trypticase Soy Broth Agar for E. avium, and 

Nutrient Agar for V. cholerae. To collect cells for the surrogate challenge tests following a 24 hr 

incubation, colonies on the agar media were harvested with a sterile inoculating loop and 

suspended in Phosphate Buffered Saline (PBS). The cell suspensions were placed in a 

spectrophotometer and the cell concentration was determined by measuring the absorbance at 

450 nm. An absorbance of 0.05 was equivalent to a concentration of 106 cells/mL. The bacteria 

suspension was then distributed into sample containers to yield a final concentration of 104 to 105 

cells/mL. 

In the second method, G. stearothermophilus and B. subtilis spore suspensions were purchased at 

the vendor-reported spore concentrations of 107 and 108 spores/mL. Spores were diluted to an 

estimated 106 spores/mL in PBS. An aliquot of the PBS spore suspension was then added to the 

sample containers to have a final concentration of 104 spores/mL. 

In either case, to ensure uniform bacteria distribution among replicate treatments and controls, all 

bacterial cell and spore aliquots were removed from the same intermediate PBS suspension for 

use in a specific surrogate test. The stressor was added or applied after bacteria were diluted into 

the synthetic freshwater or seawater in the sample containers. The total experimental volume 

(after bacterial cell or spore, and stressor addition) was 10.0 mL for experiments conducted in 16 

× 125 mm screw-capped test tubes, and 5.0 mL for the two experiments (UV and anaerobic 

treatment) conducted in 50 mm diameter sterile plastic petri dishes. 

After treatment, culturable bacteria were enumerated as colony forming units (CFU) using the 

agar spread-plate method. Petri dishes (100 mm diameter) containing agar medium were 

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inoculated directly with a 0.1 mL (10-1 dilution) aliquot from the treatment or control sample, 

which was spread evenly over the agar surface using a sterile, bent-glass rod. Nutrient Agar was 

used to enumerate G. stearothermophilus, B. subtilis, and V. cholerae cells, and Trypticase Soy 

Broth Agar (TSBA) was used to enumerate E. avium. Two serial dilutions (10-2 and 10-3 

dilutions) were prepared as necessary to ensure an accurate count when the number of culturable 

bacteria was greater than 200 to 300 CFU per inoculated petri dish on the 10-1 dilution. The 

spread-plate samples were inoculated in triplicate (three plates per dilution) and incubated in the 

dark for 24 hours. Petri plates for E. avium, V. cholerae, and B. subtilis were incubated at 37o C, 

and G. stearothermophilus plates were incubated at 55o C in a dry incubator. After incubation, 

bacterial colonies were counted and the data were reported as CFU/mL. 

Thermal Treatment (Phase I) 

As described in “Common Methods”. Following treatment, bacteria were enumerated (as above) 

after the incubation period. 

Biocide Treatments (Phase I and II) 

Biocides (sodium hypochlorite, chlorine, chlorine dioxide (EcochlorTM), glutaraldehyde, 

hydrogen peroxide, Seakleen®, and PeraClean® Ocean—all except ozone, see below) were 

added to test tubes as aqueous solutions, and were prepared from liquid or solid stocks using 

deionized water. Biocide stocks were prepared at ten times their final concentrations. A volume 

of 1.0-mL of the stock solution was added to treatment test tubes containing 9.0 mL of the 

bacteria or spore suspension in synthetic freshwater or seawater, resulting in the desired final 

biocide concentration. Control test tubes received 1.0 mL of deionized water in place of the 

treatment solution. After addition of the biocide, treatment and control test samples were mixed 

with a vortexer and held in the dark at room temperature for 24 hours. Bacteria were enumerated 

(as above) following this incubation period. 

Ultraviolet Light Treatment (Phase II) 

The UV treatment tests were performed as described in “Common Methods”. Specifically for 

bacteria, UV treatment was conducted by putting 5.0-mL samples containing 104 bacterial cells 

or spores/mL in 50 mm sterile plastic petri dishes, and placing the dish, with the lid removed, 

under the collimated UV beam. Bacteria in the dish were gently stirred with a small clean 

magnetic stir bar during treatment. After the treatment, the petri dishes were covered with the 

plastic lid, and held in the dark at room temperature for 24 hours before enumerating culturable 

bacteria by counting colonies on the inoculated media. 

Ozone Treatment (Phase II) 

Prior to ozone treatment, 1.00-L volumes of synthetic seawater and freshwater were filter 

sterilized and amended with bacteria or spores to achieve a final concentration of 104 cells/mL. 

Fifty (50.0) mL of these bacterial suspensions were placed into sterile, 500-mL graduated glass 

cylinders using a sterile pipet, and ozonated for the required time to achieve the target ozone 

dose or Total Residual Oxidant (TRO) level. The graduated cylinder reduced the water volume 

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necessary to submerge the air stone diffuser and allowed for a tall water column, which increased 

ozone absorption efficiency. After treatment, a 10-mL aliquot of ozonated water was transferred 

from the graduated cylinder into a screw-cap test tube and held in the dark at room temperature 

for 24 hours. Control samples (also 10 mL) were taken from the original 1.00-L volume of the 

bacteria-amended water, and held in the dark at room temperature for 24 hours. Viable bacteria 

remaining in both control and treatment test tubes after 24 hours were plated on agar media. 

Viable organisms were reported as CFUs. 

Deoxygenation Treatment (Phase II) 

The deoxygenation treatment was generally performed as described in “Common Methods”. 

Specifically for bacteria, 5.0-mL samples containing 104 bacterial cells or spores/mL of a given 

species were placed in 50 mm sterile plastic petri dishes. The combination of the small sample 

volume and its placement in a petri dish yielded a large surface area for the sample, reducing the 

time required to achieve anoxic conditions. Petri dishes were placed inside a BD BBL GasPak® 

Jar and held for 48 hours in the dark at room temperature. The 5.0 mL control samples 

containing 104 bacteria cells/mL also were placed in 50 mm plastic petri dishes and held in the 

dark, not under anaerobic conditions, at room temperature for 48 hours. After 48 hours, petri 

dishes were removed from the dark and the GasPak® jars. Plates were agitated before removing 

an aliquot for plating, incubation, and enumeration of culturable bacteria. 

Heterotrophic Protist Methods 

Sources of Organisms 

Acanthamoeba sp. was obtained from stocks maintained by the Oceanographic Center (OC) of 

Nova Southeastern University (NSU). This strain was originally isolated from soil adjacent to 

Hollywood beach, Florida. Chilomonas sp. was purchased from Carolina Biological Supply. 

Rhynchomonas sp. was isolated from mangrove water in the vicinity of the OC of NSU. Axenic 

Tetrahymena pyriformis were purchased from the American Type Culture Collection (ATCC, 

Maryland, USA) and Vannella anglica and Vanella platypodia from the Culture Collection of 

Algae and Protozoa (CCAP, Dunstaffnage, UK). Uronema sp. was isolated from a beach in 

Oregon by A. Hartz. 

Culture Methods 

The seven heterotrophic protists tested (Figure 5) were cultured and maintained in various media 

formulations as summarized in Table 2. The freshwater amoebae, Acanthamoeba sp. and 

Vannella platypodia, were cultured on non-nutrient agar plates (NNAS) streaked with E. coli as a 

food source. In the case of the acanthamoebae, when food was depleted cyst formation was 

induced; a typical Petri dish generated ca. 10 million cysts within one week. The freshwater 

flagellate Chilomonas sp. was cultured in amoeba saline (AS) with an added sterile rice grain. 

Nutrients leaching from the rice stimulated attendant bacteria in the culture to grow and provided 

prey for this bactiverous protist. The freshwater ciliate Tetrahymena pyriformis was maintained 

in axenic culture in tubes of proteose peptone yeast (PPY) medium. The marine flagellate 

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Rhynchomonas sp. and the ciliate Uronema sp. were cultured in filtered sterile seawater seeded 

with rice grains (again to stimulate the growth of bacterial prey). The saltwater amoeba Vannella 

anglica was cultured on malt yeast agar plates made with 75% seawater (MY75S). All cultures 

were incubated at 21°C and subcultured weekly to maintain healthy, exponentially growing 

stocks. The one exception was Acanthamoeba sp., which survived well in encysted form. This 

amoeba was cultured every three weeks. 

NNAS was made by dissolving 15 g non-nutrient agar in 1 L amoeba saline. AS was prepared 

from five stock solutions: NaCl, 1.20 g/100 mL; CaCl2, 0.04 g/100 mL; MgSO4, 0.04 g/100 mL; 

Na2HPO4 , 1.42 g/100 mL; KH2PO4, 1.36 g/100 mL. Ten (10) mL of each stock solution was 

added to 950 mL dH2O. PPY was made with proteose peptone (20.0 g), yeast extract (2.5 g), 

and dH2O (1 L). MY75S comprised sterile filtered natural seawater (750 mL), dH2O (250 mL), 

malt extract (0.1 g), yeast extract (0.1 g), and bacteriological agar (15.0g). 

Table 3. Growth media used for the routine cultivation of surrogate protists. 

Surrogate Growth Conditions

1 Acanthamoeba sp. cysts NNAS agar seeded with E. coli 

2 Chilomonas sp. Amoeba saline enriched with rice grains 

3 Rhynchomonas sp. Filtered sterile seawater enriched with rice grains 

4 Tetrahymena pyriformis PPY 

5 Uronema sp. Filtered sterile seawater enriched with rice grains 

6 Vannella anglica MY75S 

7 Vannella platypodia NNAS agar seeded with E. coli 

Figure 5. Photomicrographs of heterotrophic protists used for Phase I testing. (Top row, 

left to right: Acanthamoeba sp. cysts, freshwater, 15-18 μm; Chilomonas sp., freshwater, 13-15 μm; 

Rhynchomonas sp., marine, 4-7 μm; Tetrahymena pyriformis, freshwater, 60 μm; Bottom row, left to 

right: Uronema sp., marine, 24 μm; Vannella anglica, marine, 21-24 μm; Vannella platypodia, 

freshwater, 16-21 μm. For Phase II testing, only Acanthamoeba sp. cysts, T. pyriformis, and Uronema sp. 

were used.) 

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Thermal Treatment (Phase I) 

Six protozoan species plus Acanthamoeba sp. cysts were tested against four temperature 

stressors, 35°, 40°, 45°, and 50° C. Three of the trophic protists were freshwater and the others 

were marine. The acanthamoebae cysts were from a freshwater source, however these cysts are 

unaffected by marine conditions and previous work has shown that trophs even replicate in 

seawater (Booton et al., 2004). In all cases, the treatment time was 8 hours at the experimental 

temperature. Each experimental run was tested in quadruplicate. The organisms were 

transferred to a sterile tube (16 mL) with either 10 mL artificial seawater (Instant Ocean®, 

Aquarium Systems, Mentor, OH), in the case of the marine protists, or 10 ml WC medium (Table 

3). To concentrate organisms, cells were either washed off agar plates into the respective 

medium or rinsed from the base of Petri dishes after pouring off excess medium. Tetrahymena 

pyriformis were maintained in axenic PPY medium. Prior to experimentation, cells were 

pipetted into amoeba saline and centrifuged three times (3 min at 3000 rpm) to concentrate and 

wash cells free of this organically rich medium. Acanthamoeba sp. cysts were tested in both 

artificial seawater (SW Acanthamoeba) and WC medium (FW Acanthamoeba). In all cases, 

densities of cells in the experimental tubes was sufficient to allow reliable enumeration but not 

so excessive as to promote interference between cells. The tubes were immersed in a water bath 

to maintain the correct incubation temperature (+/- 0.1°C). After the 8 hours incubation, the 

number of viable organisms was determined using an MPN method (see “Common Methods” 

above and next paragraph as well). For each experimental run, a control set of 4 tubes was set up 

to ensure that cells remained viable over the experimental period. The controls were kept with 

the minimum of bacteria (to limit cell replication) in either artificial seawater or WC medium. 

Incubation was at 21° C, a temperature chosen to optimize the survival of the protozoa. 

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Table 3. Preparation of inorganic medium WC. 

1. Prepare 7 Stock Solutions: 

a. NaNO3: 85.01 g/L 

b. CaCl2· 2H 2O:36.76 g/L 

c. K2HPO4: 8.71 g/L 

d. MgSO4· 7H 2O36.97 g/L 

e. Na2O3Si· 9H 2O14.21 g/L 

f. NaHCO3 12.6 g/L 

g. Trace Metal Mix
 

MnCl2· 4H 2O0.18 g/L
 

ZnSO4· 7H 2O0.022 g/L
 

(NH4)6Mo7O24· 4 H 2O0.0046 g/L
 

CoCl2· 6H 2O0.012 g/L
 

CvSO4· 5H 2O0.01 g/L
 

H3BO3 0.006 g/L
 

2. Add 1 mL of each of the 7 stock solutions to 993 mL diH 2O
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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The most probable number (MPN) counting method was used to count the number of protozoa 

after treatment (in the experimental tubes and the control tubes). For each tube, medium was 

pipetted into a set of MPN tubes containing either amoeba saline or filtered, sterilized seawater 

(depending on whether the cells were freshwater or marine). The inoculum volume added to 

each MPN tube differed by a factor of 10. One set of MPN tubes were inoculated with 10 mL of 

water, 1 mL of water, 0.1 mL of water and 0.01 mL of water. A separate MPN series was set up 

for each of the 4 replicate experimental or control tubes. The MPN tubes were sub-sampled after 

briefly vortexing (5 sec) to ensure thorough mixing of cells. Nine replicate aliquots of each tube 

(20 μl) were added to 1 ml of amoeba saline (or sterile filtered seawater, as appropriate) held in 

nine wells of a 24 well tissue culture plate and incubated at 21°C. To promote excystment and 

growth of surviving cysts, nutrients were added to stimulate bacterial prey. Two 2 μL Bacto 

Casitone medium (Difco) was added to each well. After 1, 3, and 5 days, wells were examined 

by inverted microscopy to detect the presence or absence of a population of growing protozoa. 

The number of positive scores were entered into a Most Probable Number (MPN) program 

(Blodgett, 2003) to determine the number of organisms per ml. Percent survival was calculated 

by comparing the MPN from the treated samples with the MPN of the control after incubation. 

Statistical analyses were performed on the MPN data using single-factor ANOVA and the 

Tukey-Kramer procedure for determining difference in mean. Data was analyzed using the 

PHStat2 add-in for Microsoft Excel (Version 10, Prentice Hall, 2001). 

Chlorine (sodium hypochlorite) Treatment (Phase I) 

Household bleach was added to either amoeba saline or sterile artificial seawater to generate the 

four experimental concentrations of sodium hypochlorite. The organisms were transferred to a 

sterile tube (16 mL) containing either 10 mL artificial seawater, in the case of the marine protists, 

or 10 mL WC medium for freshwater protists. After 24 hours at room temperature, the number 

of surviving organisms was determined using the MPN method. 

Glutaraldehyde Treatment (Phase I) 

As described in “Common Methods”. After 24 hours of treatment, the viability of the organisms 

was determined using the MPN method. 

Chlorine Dioxide (EcochlorTM) Treatment (Phase II) 

Three heterotrophic protists (Acanthamoeba sp. cysts, Tetrahymena pyriformis, and Uronema 

sp.) were selected for Phase II testing based on the results of the Phase I tests. These three 

species were easy to culture in large numbers, were robust during harvesting, and grew out 

rapidly in the MPN counting protocols. Moreover, they were easy to observe under the 

microscope when scoring positive or negative growth. 

The three species were tested against four concentrations of chlorine dioxide (EcochlorTM). The 

organisms were transferred to a sterile tube (16 mL) containing either 10 mL artificial seawater 

32 ppt (Instant Ocean®, Aquarium Systems, Mentor, OH), in the case of the marine protists, or 

10 mL WC medium for freshwater protists. 

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To concentrate Acanthamoeba sp. cysts, cells were harvested off agar plates by scraping the 

surface with FalconTM Cell Scrapers. Cysts were decanting into 100 mL of WC medium. 

Tetrahymena pyriformis was maintained in axenic protease peptone and yeast (PPY) medium. 

Prior to experimentation, cells were pipetted into WC medium and centrifuged three times for 

three minutes at 3000 rpm to concentrate and wash. Uronema sp. was harvested from Petri 

dishes by washing cells into 100 mL of artificial seawater. In all cases, densities of cells in the 

experimental tubes was sufficient to allow reliable enumeration but not so excessive to promote 

interference between cells. 

After 24 hours incubation, the number of viable organisms was determined using the MPN 

method described above. For each experimental run, a control set of 4 tubes was set up to ensure 

that cells remained viable over the experimental period. Like the experimental runs, the controls 

were kept with the minimum of bacteria (to limit cell replication) in either artificial seawater or 

WC medium. Incubation was 24 hours at 21°C, a temperature chosen to optimize the survival of 

the protozoa. 

The enrichment of surviving cells was similar to protocols used in Phase I with a few minor 

modifications. For the ciliates Tetrahymena pyriformis and Uronema sp., 20 μL aliquots were 

inoculated into nine wells of a 24 well tissue culture plate containing 1 ml of appropriate media 

enriched with the prey bacterium E. coli. Plates were incubated at 21°C for 7 days. 

Acanthamoeba sp. cysts were inoculated (20 μL aliquots) onto non-nutrient agar plates (NNAS) 

streaked with E. coli prey. After 7 days, agar plates were examined by dissecting microscopy 

with transmitted light to detect the presence or absence of a population of growing amoebae. 

The number of positive scores were entered into a Most Probable Number (MPN) program 

(Blodgett, 2003) and percent survival was calculated by comparing the MPN from the treated 

samples with the MPN of the controls after incubation. 

PeraClean® Ocean Treatment (Phase II) 

As described in “Common Methods”. PeraClean® Ocean volumes were added to either WC or 

sterile artificial seawater to generate four experimental concentrations. The organisms were 

transferred to sterile tubes (16 mL), each containing 10 mL of the experimental (or control) 

media. After 24 hours the number of surviving organisms was determined using the MPN 

method. 

Hydrogen Peroxide Treatment (Phase II) 

As described in “Common Methods”. After 24 hours of treatment, the viability of the organisms 

was determined using the MPN method. 

Ultraviolet Light Treatment (Phase II) 

The test organisms were transferred into sterile 60x15 mm polystyrene Petri dishes along with 10 

mL of appropriate media. The dish was positioned 1 cm beneath the end of the collimator box 

and dosages were delivered as indicated in “Common Methods”. After exposure, medium and 

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organisms were transferred to sterile 16 mL glass tubes for 24 hours incubation at room 

temperature, after which viability of the organisms was determined using the MPN method. 

Ozone Treatment (Phase II) 

Generally as described in “Common Methods.” The test organisms contained in 500 mL of 

appropriate medium were placed in 500 mL glass media bottles. Holes were drilled in bottle lids 

to accommodate tubing leading to an airstone that was used to deliver the ozone. Ozone was 

injected into marine media for 9, 18, 35, and 90 secs to generate four experimental 

concentrations of ozone (0.25, 0.50, 1.0, and 2.0 mg Br2/L). Since ozone reaction chemistry 

differs in freshwater and seawater (see “Common Methods”), only three experimental 

concentrations could be achieved in the case of the freshwater treatments. Here, ozone was 

injected into WC media for 4, 8, and 16 minutes to generate the different experimental 

concentrations of ozone (0.25, 0.50, and 1.0 mg Br2/L). 

After treatment exposure, suspensions were transferred to sterile 16 mL glass tubes for 24 hours 

incubation at room temperature. Each treatment, including controls, was replicated 4 times. 

After 24 hours of treatment, the viability of the organisms was determined using the MPN 

method. 

Deoxygenation Treatment (Phase II) 

Generally as described in “Common Methods.” Test organisms were transferred to sterile 100 × 

15 mm polystyrene Petri dishes along with 10 mL of appropriate media and dishes were placed 

in the GasPak® chamber. Each treatment, including controls, was replicated 4 times. After 24 

hours of treatment, the viability of the organisms was determined using the MPN method. 

Phytoplankton Methods 

Phase I 

Phase I experiments testing the impact of hypochlorite, glutaraldehyde, and thermal stressors 

were conducted on eight phytoplankton species of which five were marine forms [Chlorella sp. 

(CCMP256), Chaetoceros affinis (CCMP158), Skeletonema costatum (CCMP780), Scrippsiella 

trochoidea (CCMP1599), Scrippsiella lachrymosa (both vegetative and cyst forms, clone: B10­

1)] and four were freshwater forms [Prymnesium parvum (CMS204), Microcystis aeruginosa 

(UTEX B 2672) and Fragilaria crotensis (UTCC269)]. The marine species were cultured in 

modified f/2 medium with or without silicate (Guillard and Ryther 1962), where Na2SeO3 has 

been added to a final concentration of 10-8 M and the concentration of CuSO4

.

5H2O has been 

reduced to the same level. For culture upkeep, the medium was prepared in autoclaved, 0.2 μm 

filtered natural seawater. The freshwater species were grown in WC medium (Guillard, 1975) 

made with Milli-Q water. All cultures were maintained at 20o

C on a 14:10h light: dark cycle (ca. 

200 μmol photons·m-2·sec-1 irradiance provided by cool white fluorescent bulbs). All marine 

culture experiments were conducted in f/2 medium made with sterile filtered Instant Ocean® 

water adjusted to a salinity of 32 as the base. 

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Sterile, disposable polystyrene tubes (17 × 100 mm) filled with 7 mL of mid-exponential growth 

phase culture were used for all experiments except for the UV light and deoxygenation trials, for 

which sterile, polystyrene Petri dishes (50 × 11 mm) containing 5 mL of culture were used. 

The thermal trial tubes were supported on floating racks and incubated in separate water baths 

adjusted to the criterion temperatures. After 8 hours, the cultures were transferred to room 

temperature and individually pipetted into 96 well tissue culture plates for most probable number 

(MPN) determinations. Phytoplankton species were exposed to the other Phase I treatments, 

glutaraldehyde and hypochlorite, for 24 hours at room temperature, then pipetted into culture 

plates preparatory to MPN determinations. 

For MPN estimates, each well of the tissue culture plate was filled with 270 μL of f/2 medium, 

with the exception of wells in row H, which were loaded with 300 μL of treatment culture (6 

replicate wells per replicate treatment tube). 30 μL of the culture from row H wells were then 

serially diluted into the wells above using a 12 position multi-channel pipette so that a final 

dilution series ranging from 10-0 to 10-7 was achieved. The plates were sealed with tape around 

the perimeter to minimize evaporative loss, incubated at 20oC and monitored after 21 days with 

an inverted microscope at 100× to determine cell viability. A gridded tally sheet was scored with 

the results and the data were entered into a most probable number calculator Excel spreadsheet 

(Dr. R. Blodgett, Division of Mathematics, FDA/CFSAN) to derive the MPN for the experiment. 

Phase II 

Based on the results of the Phase I trials, 3 species, S. lachrymosa cysts (B10-1), Chaetoceros 

affinis (CCMP158), and Chlorella sp. (CCMP256) were advanced for Phase II testing. 

Experiments with chlorine dioxide (EcochlorTM), hydrogen peroxide, and Peraclean® Ocean 

were conducted in the same manner described for the glutaraldehyde and hypochlorite Phase I 

trials. 

For the UV light exposures, 5 mL of culture was loaded into a 50 mm diameter polystyrene Petri 

dish and exposed, uncovered, to the collimator UV light. Samples were then transferred into 

disposable 15 mL centrifuge tubes so that the samples could be mixed well prior to MPN 

dilutions. 

For the deoxygenation treatment, 5 mL samples were pipetted into Petri dishes, which were 

placed on a perforated plastic support within a glass desiccator. Four desiccators in total were 

used – 1 each for the 12, 24, 48, and 72 hour incubations. Two BD BBLTM GasPak sachets, 

clipped onto the support in an upright position, were then activated within each desiccator per the 

manufacturer’s protocol. The desiccators were covered with the airtight lid and were closely 

monitored until duplicate indicator strips revealed that anaerobic conditions were achieved. This 

event marked the “time 0” for the experiments. At the termination of the incubation time, the 

indicator strips were again checked to ensure that anaerobic conditions had persisted through the 

course of the incubation. The Petri dish culture contents were then transferred into disposable 15 

mL centrifuge tubes as above for MPN dilutions. 

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Zooplankton Methods 

Species Selection 

We obtained and isolated representative species from the list of organisms provided by the ETV 

Technical Panel and suggested in the “Request for Proposals” (Table 4). This list was developed 

with the understanding that in some cases, species might not be obtainable and the final list 

would substitute equivalent taxa more readily available in the Pacific Northwest. For example, 

the European harpacticoid copepod species, Tisbe battagliai, was replaced with a common and 

abundant Tisbe species of the Pacific Northwest, Tisbe cf. furcata. 

After collecting or purchasing the organisms, cultures were established using a standardized 

protocol based on EPA recommendations and refined to accommodate the large number of 

organisms being screened. Many of the suggested organisms were not easy to obtain or to 

culture to the numbers required for bench-top testing, so the list was augmented with organisms 

either easily obtained from the field in the Pacific Northwest region or ordered from a reputable 

provider of live organisms (Table 5a,b). We verified species identifications of all purchased 

species. In the course of the project, we screened 35 species distributed among 12 major taxa as 

potential surrogate species: copepods (17 species); cladocerans (7); rotifers (4); amphipod (1); 

barnacle (1); isopod (1); branchiopod (1); annelid (1); abalone (1); and insect (1). 

Three criteria were used in the Phase I testing to reduce the list of surrogates for Phase II tests: 

performance in the preliminary trials; ability to culture the organisms to high enough densities 

for future full-scale testing in experimental ballast water tanks; and ability to determine the 

efficacy of the treatments, i.e., the organisms’ viability, using a microscope. Application of these 

three criteria allowed us to narrow the list of potential surrogates to four marine and three 

freshwater zooplankton species representing three marine classes (one branchiopod, two 

copepods, and one annelid) and three freshwater classes (one cladoceran, one copepod, and one 

ostracod) (Table 6). These seven species were used in the Phase II trials. 

Table 4. Potential surrogate zooplankton species identified by the ETV program and 

related studies. 

Fresh Water Marine Water 

Daphnia pulex (resting) 

Daphnia magna 

Brachionus calyciflorus (resting) 

Culex (insect larvae) 

Diaptomus pallidus (adult) 

Ceriodaphnia lacustris (adult) 

Acartia hudsonica (warm – resting) 

Acartia tonsa (cold – resting) 

Brachionus calyciflorus (adult) 

Tisbe battagliai (adult) 

Nitokra lacustris 

Artemia 

Mussel larvae 

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Table 5a. Potential surrogate zooplankton species ordered from commercial sources or 

provided by other researchers. 

Organism and Type Acquisition Procedure Experimental Status 

Harpacticoid copepods 

Tisbe sp. cf. furcata (CA) Essential Live Feeds, WA Phase I 

Nitokra lacustris Essential Live Feeds, WA Phase I & II 

Calanoid copepods 

Acartia tonsa Algagen, LLC, in Vero Beach, FL Phase I 

Cyclopoid copepods 

Acanthocyclops robustus Carolina Biological Supply, NC Phase I 

Macrocyclops albidus Essential Live Feeds, WA Phase I & II 

Cladocerans 

Daphnia pulex Carolina Biological Supply, NC Phase I 

Daphnia magna Frieda Taub’s Lab, UW Phase I 

Ceriodaphnia dubia Carolina Biological Supply, NC Phase I 

Chydorus sphaericus* Carolina Biological Supply, NC Phase I & II 

Moina sp. Florida Aquafarms, FL Rejected for experimentation; very 

low densities in culture. 

Mollusks 

Red Abalone Carolyn Friedman’s Lab, UW Phase I 

Rotifers 

Brachionus plicatilis Carolina Biological Supply, NC Phase I 

Brachionus calyciflorus Florida Aquafarms, FL Phase I 

Philodina citrine Carolina Biological Supply, NC Rejected for experimentation 

because it is not easy to filter intact 

for observation. 

Lecane monostyla Carolina Biological Supply, NC Rejected for experimentation 

because it is not easy to filter intact 

for observation. 

Branchiopods 

Artemia salina Brine Shrimp Direct Phase I & II 

Ostracods 

Ostracod sp.* Carolina Biological Supply, NC Phase I & II 

Annelids 

Nereis virens Sea Bait Ltd., Maine Phase I & II 

Mosquito larvae 

Culex sp. Florida Aquafarms, FL Phase I 

*While these organisms were started from cultures from Carolina Biological Supply, these were not the 

organisms ordered. They were contaminants in with the D. pulex. 

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Table 5b. Potential surrogate zooplankton species collected by the University of 

Washington ballast water team. 

Organism and Type Acquisition Procedure Experimental Status 

Harpacticoid copepods 

Tigriopus californicus Deception Pass State Park, WA. 

April, 2005 

Phase I & II 

Tisbe sp. Washington Field sampling at NOAA 

Fisheries Manchester Research 

Station, WA. April, 2005 

Rejected for experimentation because 

needs to be kept at 10 degrees C. 

Harpacticus uniremis NOAA Fisheries Manchester 

Research Station, WA. April, 

2005 

Rejected for experimentation due to 

length of life cycle and univoltine 

reproduction limit the culture of this 

species throughout the year 

Mesochra sp. Jakle’s Lagoon, San Juan Island, 

June, 2005 

Rejected for experimentation due to 

low densities in culture, and because 

three harpacticoid copepods were 

already available for testing. 

Calanoid copepods 

Eurytemora affinis Columbia River sampling. April, 

2005 

Rejected for experimentation due to 

extremely low densities in culture. 

Eurytemora americana Jakle’s Lagoon on San Juan 

Island, June, 2005 

Phase I 

Calanus pacificus NOAA Fisheries Manchester 

Research Station, WA. April, 

2005 

Rejected for experimentation because 

it Could not be kept alive on the 

variety of diets available 

Acartia hudsonica Jakle’s Lagoon on San Juan 

Island, June, 2005 

Rejected for experimentation due to 

extremely low densities in culture. 

Diaptomus nevadensis Soap Lake, WA. August, 2005. Rejected for experimentation because 

it Could not be kept alive on the 

variety of diets available 

Diaptomus sp. Collected from Lake Washington, 

October 2005 

Phase I 

Cyclopoid copepods 

Corycaeus anglicus NOAA Fisheries Manchester 

Research Station, WA. April, 

2005 

Rejected for experimentation because 

it did not adapt easily to the laboratory 

conditions 

Cladoceran 

Bosmina longirostris Lake Washington sampling, 

October, 2005 

Phase I 

Daphnia villosa Soap Lake, WA, August 2005 Rejected for experimentation because 

it could not be kept acclimated to full 

strength salinity or freshwater. 

Cirripedia 

Cirripedia larvae Columbia River sampling. April, 

2005 

Rejected because it is not easy to 

culture nor is it available commercially 

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Organism and Type Acquisition Procedure Experimental Status 

Amphipods 

Eogammarus 

confervicolus 

Duwamish River Estuary, WA. 

April, 2005 

Rejected for experimentation due to 

extremely low densities in culture. 

Isopods 

Gnorimosphaeroma sp. Duwamish River Estuary, WA. 

April, 2005 

Rejected for experimentation due to 

extremely low densities in culture. 

Table 6. Zooplankton species used in Phase II treatments. 

Fresh Water Marine Water 

Macrocyclops albidus (copepod) 

Chydorus sphaericalis (cladocerans) 

ostracod sp. 

Artemia salina (branchiopod) 

Tigriopus californicus (copepod) 

Nitokra lacustris (copepod) 

Nereis virens (annelid) 

Culture Conditions 

Organisms were acclimated to standard laboratory conditions by placing them in an incubator 

held at 20oC with a 12 hours light-12 hours dark photoperiod. Depending on the densities 

achievable, organisms were kept either in 1 L of media in a half-gallon glass jar, or in a 150 mL 

vented tissue culture flask. For regular maintenance, animals were screened and placed in fresh 

culture media biweekly. (Twenty-four hours before experiments, animals were placed in fresh 

media and allowed to evacuate their guts.) All cultures were kept in batch conditions, meaning 

that no aeration was provided and all animals were manually filtered when the culture media was 

changed. 

Marine organisms were fed a combination of the live algae Tetraselmis suecica, freeze-dried 

phytoplankton (PhytoplanTM, Two Little Fishies, FL), and powdered fish food (Boyd’s Vita-diet, 

Boyd Enterprises, FL) on a weekly basis. Freshwater animals were provided with the live alga 

Scenedesmus sp., freeze-dried phytoplankton (PhytoplanTM, Two Little Fishies, FL), and 

powdered fish food (Boyd’s Vita-diet, Boyd Enterprises, FL) on the same schedule. The same 

media was used for the experiments and the maintenance conditions, with the exception two 

commercial products added to the freshwater maintenance media: Kent Marine R/O Right 

powdered formula (a mixture of major salts of sodium, magnesium, calcium, and potassium; 

Kent Marine, WI) and Kent Freshwater Essential liquid (a mixture of essential trace minerals to 

reproduce natural freshwater; Kent Marine, WI). Organisms were held up to one year using 

these techniques. 

Experimental Conditions and Methods 

The experiments for zooplankton differed from those for phytoplankton, where the dilution 

media required the presence of nutrients to determine whether the organisms were affected by 

the treatments and not starvation. An elevated level of nutrients was not required to keep the 

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animals alive in the zooplankton trials. The seawater comprised filter-sterilized artificial 

seawater (Instant Ocean®) without nutrient supplementation. Salts were dissolved in glass fiberfiltered reverse osmosis water to a salinity of 30. 

For freshwater organisms, the test medium consisted of filter-sterilized (glass fiber-filtered) 

reverse osmosis water with a testing medium based on the EPA’s recommended aquatic 

toxicology testing dilution protocols for freshwater organisms. Depending on the sensitivity of 

the organisms, one of two EPA protocols was utilized to prepare the dilution water for testing 

(see http://www.epa.gov/waterscience/WET/disk2/). In the toxicity-test methods, synthetic water is 

referred to as diluted mineral water (DMW). 

Whether seawater or fresh, the pH of test water was adjusted to match the culture conditions of 

the organism to be tested. Water was used within 7 days of preparation. 

Freshwater and marine experiments were conducted at room temperature in containers of 

volumes appropriate to the size of the organisms being tested. Except for the thermal treatments, 

which were terminated directly after 4 hours of exposure to the target temperature, and the 

deoxygenation treatments, which were terminated after 48 hours in the degassing units, percent 

survivals were determined after 24 hours in the dark. Biocides were treated as instantaneous 

stressors, but the time of exposure to the treatment stressors for the ozone and UV treatments 

varied based on desired exposure levels. Table 7 provides detailed information on the source, 

concentration, and exposure time. 

Because so many zooplankton species were resistant to the levels originally proposed for this 

project, many of them were tested at higher levels or concentrations to provide a basis for 

comparison with the performance of other zooplankton. 

Phase I testing identified the seven most promising organisms among the different classes to be 

tested (Table 7). Only the organisms listed in Table 6 were carried forward into Phase II testing. 

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Table 7. Zooplankton treatments (proposed and realized) and experimental conditions. 

Treatment 

stressor 

Vendor or 

source 

Concentration 

or Intensity Test volume Actual Conditions and Exposure Times 

Thermal 

treatment 

Water bath 35°, 40°, 45°, 

50 ̊C 

150 mL or 1 L 35 ̊, 40 ̊, 45 ̊ for 4 hours 

Chlorine (sodium 

hypochlorite) 

Chlorox bleach Aqueous 

solution of 

sodium hypochlorite. Final 

concs. of 0.25, 

0.5, 1.0, 2.0 

mg/L 

150 mL or 1 L Final conc. of 1, 2, 4, 8 mg/L for 

24 hours 

Chlorine dioxide 

(EcochlorTM) 

Ecochlor, Inc. Final concs. of 1, 

2, 4, 6 ppm 

150 mL Final conc. of 1, 2, 4, 8 and 16 

ppm for 24 hours 

Glutaraldehyde Fisher Scientific Final concs. of 

50, 100, 500 and 

1000 mg/L 

150 mL Final conc. of 50, 100, 200, 400, 

800, 1200 and 1600 mg/L for 24 

hours 

UV light UV collimator 

designed and 

built by Dr. E. 

“Chip” 

Blatchley, 

Purdue Univ. 

UV light (256 

nm) at 10, 25, 

50, 100 mJ/cm2 

50 mL Final exposures of 50, 100, 200, 

and 400 mJ/cm2 – dose depends on 

exposure time 

Ozone Aquatic EcoSystems, UVtype, 2.4 g with 

air. Connected 

to tubing and 

an air stone. 

Total initial 

residual oxidant 

(TRO) level of 

0.25, 0.5, 1.0, 

and 2.0 mg Br2/L 

0.5 L Final exposure of 0.5 and 1 mg Br 

L-1 for FW and 1,2, 4, and 8 mg Br 

L-1 residual oxidant level for 

Marine for 24 hours after 

achieving initial level of TRO 

Hydrogen 

peroxide 

Fisher Scientific Final concs. Of 

0.5, 1, 10, and 20 

ppm 

150 mL Final conc. of 10, 20, 40, 80, 160, 

and 320 ppm for 24 hours after 

achieving initial concentration 

Deoxygenation Sparge with N2 

(95%) and CO2 

(5%) mixture, 

at levels to 

reduce pH to 

5.5, then seal 

container. 

Anoxia (0 mg/L 

oxygen) 

50 mL 48 hours after achieving anoxia 

SeaKleen® Vitamar, Inc. 0.25, 0.5, 1.0, 2.0 

mg/L active 

ingredient 

150 mL Final conc. of 1, 2, 4, 8, and 16 

mg/L for 24 hours 

PeraClean® 

Ocean 

Degussa AG Final concs. of 

50, 100, 200, and 

400 ppm 

150 mL Final concs. of 50, 100, 200, and 

400 ppm 

Note:	 Entries in bold indicate alterations from the originally proposed protocol (see Table 2 of this 

report). 

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Thermal Treatment (Phase I) 

For zooplankton, only three temperatures were tested, 35o, 40o, and 45oC. Regardless of 

organism and volume of culture container (150 mL of medium in 200 mL beakers or 1 L of 

medium in half-gallon glass jars), the test container was filled either with artificial seawater or 

freshwater medium and the test organisms were screened and added. After 4 hours of treatment 

in the dark, the viability of organisms was immediately assessed. Controls were incubated 

simultaneously at room temperature in the dark. 

In the case of 1 L treatments, animals were recollected by passing the test medium twice through 

a 20μm Nitex filter screen. As this method proved to be inefficient in recollecting soft-bodied 

organisms such as rotifers or animals which disintegrated due to the treatment, we switched to 

150 mL treatments in 200 mL beakers, which enabled us to count organisms without having to 

recollect them on a filter screen. Animals were placed in a counting wheel and examined under 

at least 10x magnification to determine survival (indicated by motion) or mortality (lack of 

movement after a few seconds of observation or prodding with a wire pointer). 

Biocide Treatments (Phase I and II) 

Biocide (sodium hypochlorite, glutaraldehyde, chlorine dioxide (EcochlorTM), SeaKleen®, 

PeraClean® Ocean, hydrogen peroxide) experiments with zooplankton were conducted in 

microcosms of non-reactive materials, incubated at natural ambient temperatures in the dark for 

24 hours. At least 20 animals were placed in the microcosms and tested at various doses of the 

treatments with a no-treatment control. Since variability was low in biocide treatments, three 

replicates at each dosage level were adequate for comparison. Experimental controls consisted 

of organisms held either in artificial seawater or freshwater medium for 24 hours in the dark. All 

treatments were analyzed for survival and mortality after 24 hours. 

When the originally proposed doses of biocides were not sufficient to kill more than 50% of 

treatment organisms, dosages were increased until 50% mortality (or more) was obtained or until 

the dose of biocide reached more than 100 times the maximum dose originally proposed. Details 

about individual biocides follow. 

Chlorine (sodium hypochlorite) 

As described in “Common Methods”. At the start of the experiment and after 24 h, the level of 

total residual oxidant (TRO) was measured using a Hach Kit 2010. 

Glutaraldehyde 

As described in “Common Methods”, except that organisms were exposed to a wider range of 

glutaraldehyde concentrations: 50, 100, 200, 400, 800, 1200 and 1600 mg/L (Table 7). 

93
 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Protocol for the Verification of Ballast Water Treatment Technologies September 2010 

Version 5.1 

Chlorine dioxide (EcochlorTM) 

As described in “Common Methods”, except that organisms were exposed to a wider range of 

chlorine dioxide concentrations: 1, 2, 4, 8, and 16 ppm (Table 7). 

SeaKleen® 

An organic biocide, SeaKleen® is available in several formulations and concentrations from the 

manufacturer. We examined the sensitivity of prospective surrogate organisms to a wettable 

powder that contains 85% active ingredient. Fresh stock solutions were prepared from the 

powder before each series of trials. SeaKleen® was tested at 1, 2, 4, 8, and 16 mg/L of the active 

ingredient (Table 7). 

PeraClean® Ocean 

As described in “Common Methods”. 

Hydrogen peroxide 

As described in “Common Methods”, except that organisms were exposed to a wider range of 

hydrogen peroxide concentrations: 10, 20, 40, 80, 160, and 320 ppm (Table 7). 

Ultraviolet Light Treatment (Phase II) 

Generally as described in “Common Methods”, except that organisms were exposed to a greater 

range of exposures: 50, 100, 200, and 400 mJ/cm2 (Table 7). A small polystyrene petri dish (60 

× 15 mm, Falcon) containing the test organisms and less than 0.5 cm of medium was placed 

beneath the collimator light beam. We determined there was no significant production of heat 

generated by the lamp. Organisms were generally checked for mortality only once, 24 hours 

after exposure. When no mortality was observed, we occasionally held onto samples for further 

analysis at 48 and 72 hours. Four replicates were conducted for each combination of treatment 

level and species. 

Ozone Treatment (Phase II) 

Generally as described in “Common Methods”. To expose zooplankton to ozone, we placed 

them in one half liter of media, placed the air stone in the media, and let the generator run for the 

predetermined periods. In the case of the marine treatments, times were adjusted if exposure 

level was less than anticipated. We were not able to do this for the freshwater experiments. A 

lid through which the ozone delivery hose was fitted was placed on freshwater containers to 

ensure maximum contact between the organisms and ozone for each 24 hour experiment. After 

treatment, we replaced the hose-fitted lid with a sealed lid. All treatment jars were placed in the 

dark for 24 hours before analyzing organism survival. 

94
 
 

 

 

 

 

 

Protocol for the Verification of Ballast Water Treatment Technologies September 2010 

Version 5.1 

Deoxygenation Treatment (Phase II) 

Generally as described in “Common Methods”, except that only a 48 hours treatment was run. 

At least 20 organisms from each taxon being tested were placed in a small polystyrene petri dish 

(60 × 15 mm, Falcon), and the dish itself was placed inside the BD BBL GasPak® Jar System. 

95
 
 

 

 

 

 

 

Protocol for the Verification of Ballast Water Treatment Technologies September 2010 

Version 5.1 

Appendix C: Design and Preliminary Use of a Commercial Filter 

Skid to Capture Organisms ≥ 50 μm in Minimum Dimension 

(Nominally Zooplankton) for Evaluating Ships’ Ballast Water 

Management Systems at Land-based Test Facilities 

96
 
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22-06-2010 NRL Letter Report March 2009-April 2010

Design and Preliminary Use of a Commercial Filter Skid to Capture

Organisms Ż 50 μm in Minimum Dimension (Nominally Zooplankton) for

Evaluating Ships’ Ballast Water Management Systems at Land-Based Test

Facilities

Edward J. Lemieux, Code 6130, NRL, DC, Lisa A. Drake, SAIC, Key West,

FL, Stephanie H. Robbins, SAIC, Key West, FL, Mia K. Steinberg, Code

6130, NRL, DC, Scott C. Riley, SAIC, Key West, FL, Elizabeth C. Schrack,

University of Virginia, Charlottesville, VA, Wayne B. Hyland, Azimuth

Technical Consultants, Key West, FL, Jonathan F. Grant, Battenkill

Technologies, Inc., Manchester Center, VT, Cameron S. Moser, EXCET, Inc.,

Key West, FL, Tim P. Wier, EXCET, Inc., Seattle, WA

US Naval Research Laboratory

4555 Overlook Ave., SW

Washington, DC 20375

6130/1029

United States Coast Guard

Environmental Standards Division (CG-5224)

2100 2nd Street, S.W.

Washington, DC 20593

USCG

Distribution Statement A: Approved for public release, distribution is unlimited.

International and national standards dictating the number of living organisms discharged in ships’ ballast water are in the process of

being enacted. Developing a method to accurately determine whether how many organisms are present in treated water (e.g., with

sparse concentrations of organisms) with statistical confidence requires large volume (e.g., > 10 m^3) are concentrated and

examined. Here, we describe efforts to (1) develop and validate a procedure for retaining organisms > 50 μm in minimum

dimension (nominally zooplankton) in large volumes of water used for testing ballast water management systems, (2) design and

build the equipment to do so, and (3) validate the equipment. Importantly, zooplankton needed to be kept alive and minimally

affected by filtering and handling, since ballast water discharge standards prescribe the number of viable (living) organisms.

U U U Unclassified 46

Edward Lemieux

202-404-2123

 

Science and Engineering

Design and Preliminary Use of a Commercial Filter Skid to 

Capture Organisms ≥ 50 μm in Minimum Dimension 

(Nominally Zooplankton) for Evaluating Ships’ Ballast 

Water Management Systems at Land-Based Test Facilities

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Table of Contents 

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3 !-^)%+*)H:$; =^^%0$9E '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' T

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3'T Q+9%0D)$"A ''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' 3

3'T'2 Q+9%0D)$" 7+A+H:)L%$:+0H ''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' V

3'T'T Q):E0" 0J Q+9%0D)$" /0,H:+HL'''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' V

3'T'3 >H:)%9$;+D%$:+0H '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' W

3'3 B+;:)% M$LA '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' 1

3'3'2 B+;:)% M$LA B+);" O%+$;A_@$:)% B;0# 6$:) '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' Y

3'3'T B+;:)% M$LA_($D0%$:0%< $H" B+);" K$;+"$:+0H #+:E Q+9%0D)$"A '''''''''''''''''''''''''''''''''' Y

3'3'3 B+;:)% M$LA_8)$*A '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' `

3'3'V B+;:)% M$LA_O0-+9+:< 0J 8)$;$H: 0H :E) 8)$*A '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' `

3'V B+;:)% 8F+"''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' U

3'V'2 B+;:)% 8F+" 6)a,+%)*)H:A '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' U

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3'V'3 /0*^$%+A0H 0J b00^;$HF:0H +H :E) M$;;$A: O$HF IA' :E) B+;:)% 8F+" '''''''''''''''''''''''''' 24

3'V'V !JJ)9: 0J /%0#"+HL 0H =*D+)H: b00^;$HF:0H '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' 22

V 6)A,;:A ''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' 22

V'2 Q+9%0D)$"A ''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' 22

V'2'2 Q+9%0D)$" 7+A+H:)L%$:+0H ''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' 22

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V'2'3 >H:)%9$;+D%$:+0H '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' 2T

V'T B+;:)% M$LA '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' 2T

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1 =9FH0#;)"L)*)H:A ''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' T1

Y 6)J)%)H9)A ''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' T1

=^^)H"+- 2 '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' T`

=^^)H"+- T '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' TU

=^^)H"+- 3 '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' 34

=^^)H"+- V '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' 32

=^^)H"+- W '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' 3T

=^^)H"+- 1 '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' 3`

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Table of Figures 

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1 Introduction 

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Z3 3

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3

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$H" )I$;,$:)"' 

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T 

2 Objectives 

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H,*D)% 0J viable [;+I+HL\ 0%L$H+A*A [>QX. T44W\' 

3 Experimental Approach 

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^;$HF:0H H): $H" J+;:)% D$LA. %)A^)9:+I);<\' 

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3.1 Ballast Water Treatment Test Facility (BWTTF) Description 

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3 

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$H" ")J)HA+D;) %)A,;:A $%) L)H)%$:)"' O0 :E$: )H". :E) M@OOB +A ")A+LH)" :0 90H",9: 

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J,;;< +HA:%,*)H:)" A)$#$:)% A:0%$L) $H" :%$HAJ)% A<A:)* :E$: %)^;+9$:)A :E) I0;,*)A. J;0#A. $H" 

^%)AA,%)A :<^+9$; 0J D$;;$A: #$:)% A<A:)*A 0H *$%+H) I)AA);A' OE) M@OOB ^,*^A 9$H ^%0I+") 

Z2 3

J;0# %$:)A ,^ :0 W224 ; *+H [23W4 L^*\. $H" 0HZA+:) :$HFA +H9;,") $ 3`T * [242.444 L$;\ 

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"+L+:$; 90HI)%:)%A 90HH)9:)" :0 $ %)",H"$H: 90*^,:)% A)%I)% A<A:)* :0 ;0L $;; $9:+0HA $H" "$:$' = 

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^$%$*):)%A "+A^;$<)"' 

3.2 Microbeads 

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2Z*; 8)"L)#+9F 6$J:)% 90,H:+HL 9E$*D)%A ,A+HL $H X;<*^,A 8bN24 "+AA)9:+HL *+9%0A90^). $ 

5+F0H =b244 Q,;:+C00* *+9%0A90^). 0% $ 5+F0H !9;+^A) O8244 +HI)%:)" *+9%0A90^)' 

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3.2.1 Microbead Disintegration 

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#)%) "+A^)HA)" +H:0 W *; 0J J+;:)%)" A)$#$:)% [4'TT g*\. $H" :)H *+9%0D)$"A 0J )$9E A+C) 9;$AA 

#)%) "+A^)HA)" +H:0 W *; 0J O<^) >> #$:)%' OE) J0,% #);;A #)%) 90I)%)" $H" ^;$9)" 0H $ 

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3.2.2 Method of Microbead Counting 

OE) *+9%0D)$" %)90I)%< )JJ+9+)H9< #$A ;0# +H +H+:+$; :%+$;A ,A+HL W4 g* "+$*):)% *+9%0D)$"A +H 

J+);" $H" ;$D0%$:0%< )-^)%+*)H:A. #E+9E #$A A,%^%+A+HL. $A :E) *+9%0D)$"A E$" D))H $990,H:)" 

J0% +H $;; ^0AA+D;) ^;$9)An :E) J+;:%$:). J+;:%$H: [*$:)%+$; %):$+H)" +H :E) J+;:)% D$L\. %)A+",) +H :E) 

D)$F)% :E$: +H+:+$;;< E);" :E) *+9%0D)$"A. $H" %)A+",) +H :E) ^+^)::) ,A)" :0 "+A^)HA) A$*^;)A' 

P+I)H :E+A ,HD$;$H9). +: A))*)" ^0AA+D;) :E) *):E0" J0% +H+:+$;;< 90,H:+HL *+9%0D)$"A :0 $"" :0 

J+;:)% D$LA. A+)I)A. 0% ^;$HF:0H H):A #$A A0*)E0# ,H")%90,H:+HL :E)*' GH:+; :E+A ^0+H:. :E) 

*+9%0D)$"A E$" D))H 90,H:)" s"%<s. :E$: +A. $ A:)%+;) ^+^)::) :+^ #$A "+^^)" +H:0 :E) D0::;) 

90H:$+H+HL :E) *+9%0D)$"A :0 ");+I)% $ ^+;) 0J *+9%0D)$"A :0 $ L%+"")" 8)"L)#+9F 6$J:)% [86\ 

90,H:+HL 9E$*D)%. #E+9E "+" H0: E$I) #$:)% 0H +:' =J:)% :E) *+9%0D)$"A #)%) 90,H:)". :E) 86 

#$A L)H:;< %+HA)" #+:E O<^) >> #$:)% J%0* $ Aa,+%: D0::;) +H:0 $ D)$F)%. $H" :E) D)$F)%fA 90H:)H:A 

#)%) ^0,%)" +H:0 $ J+;:)% D$L 0% A+)I) J0% $H )-^)%+*)H:' O0 )HA,%) :E) 86 "+" H0: E$I) $H< 

%)*$+H+HL *+9%0D)$"A. +: #$A ^;$9)" 0H $ ?+* #+^) $H" )-$*+H)" ,H")% :E) 

*+9%0A90^)' c)%E$^A %+HA+HL :E) "%< *+9%0D)$"A +H:0 $ D)$F)% 9$,A)" :E)* :0 D) "+A^)%A)" +H:0 

:E) $+% %$:E)% :E$H :E) D)$F)%' 
W 

 

=""%)AA+HL :E+A E<^0:E)A+A. %)90I)%< )JJ+9+)H9< 0J 244 W4Zg* *+9%0D)$"A #$A "):)%*+H)" +H W 

:%+$;A #+:E e"%<f 90,H:+HL $H" V :%+$;A #+:E e#):f 90,H:+HL' >H :E) ;$::)% *):E0". $ ^+^)::) :+^ #$A 

"+^^)" +H:0 :E) D0::;) 90H:$+H+HL :E) *+9%0D)$"A $H" :$^^)" +H:0 $ "%0^ 0J O<^) >> #$:)% 0H $ 86 

A;+") :0 %)*0I) :E) *+9%0D)$"A J%0* :E) ^+^)::) :+^' OE) #$:)% "+" H0: :0,9E :E) )"L) 0J :E) 

A;+"). H0% #$A $ 90I)% A;+^ ,A)"' =J:)% :E) *+9%0D)$"A #)%) "+A^)HA)" ["%< 0% #):\ +H:0 :E) 86. 

:E)< #)%) 90,H:)" $H" %+HA)" J%0* :E) 86 #+:E $ Aa,+%: D0::;) 90H:$+H+HL O<^) >> #$:)% +H:0 $ 

D)$F)%' O0 "):)%*+H) :E$: 244 *+9%0D)$"A #)%). +H J$9:. ");+I)%)" :0 :E) D)$F)%. 

+:A 90H:)H:A #)%) ^+^)::)" +H:0 M0L0%0I 9E$*D)%A $H" 90,H:)"' @E)H :E) )H:+%) A$*^;) #$A 

90,H:)". :E) ^+^)::) $H" D)$F)% #)%) )-$*+H)" J0% $H< %)A+",$; *+9%0D)$"A' B0% )$9E :%+$;. :E) 

H$*) 0J :E) A9+)H:+A: 90,H:+HL :E) *+9%0D)$"A #$A %)90%")" :0 "):)%*+H) +J :E) ^00% %)90I)%< 

#$A $::%+D,:$D;) :0 $ L+I)H %)A)$%9E)%' 

3.2.3 Intercalibration 

O0 )HA,%) $;; *+9%0D)$"A #)%) 90,H:)". $H +H:)%9$;+D%$:+0H )-)%9+A) #$A 90H",9:)" +H #E+9E :#0 

0DA)%I)%A 90,H:)" :E) A$*) 2W *; A$*^;) "+A^)HA)" +H $ M0L0%0I 90,H:+HL :%$<' B0,% :%$<A 

[)$9E #+:E $ "+JJ)%)H: A$*^;) J%0* :E) J+;:)% D$L\ #)%) 90,H:)" D< )$9E 0DA)%I)%. $H" :E)H )$9E 

:%$< #$A %)90,H:)" D< )$9E 0DA)%I)%' =H $""+:+0H$; 24 :%$<A [)$9E #+:E $ "+JJ)%)H: A$*^;) J%0* 

:E) J+;:)% D$L\ #)%) 90,H:)" D< D0:E 0DA)%I)%A. J0% $ :0:$; 0J 2`' =;:E0,LE :E) A)$#$:)% ,A)" J0% 

:E) )-^)%+*)H: E$" D))H J+;:)%)". ")D%+A [A)"+*)H: $H" ^;$HF:0H\ #$A ^%)A)H: +H :E) A$*^;)A' 

OE,A. )-:%$ 9$%) #$A %)a,+%)" :0 J+H" :E) *+9%0D)$"A. $H" )$9E :%$< :00F $^^%0-+*$:);< VW *+H :0 

)-$*+H)' 

O0 "):)%*+H) :E) "+JJ)%)H9) D):#))H 0DA)%I)%A :E) *)$H 0J :E) 0DA)%I)%Af 90,H:A J0% $ A+HL;) 

:%$< #$A "):)%*+H)". $H" :E) ^)%9)H: "+JJ)%)H9) J0% )$9E 0DA)%I)%fA 90,H: #$A 9$;9,;$:)" $A 

J0;;0#An 

!a,$:+0H 2' /$;9,;$:+0H :0 "):)%*+H) ^)%9)H: "+JJ)%)H9) D):#))H *+9%0D)$" 90,H:A' 

/0,H:A 0JQ)$H 244

/0,H: 2XDA)%I)% Z /0,H:A 0JQ)$H 

24

M)9$,A) :E)%) #)%) :#0 0DA)%I)%A. :E) ^)%9)H: "+JJ)%)H9) #$A :E) A$*) J0% )$9E 0DA)%I)% J0% 

)$9E :%$< 90,H:)"' 

>H $ A)^$%$:) :%+$;. 0H) 0DA)%I)% "+A^)HA)" 2W *; 0J A$*^;) J%0* :E) J+%A: J+;:)% D$L $H" 90,H:)" +: 

J+I) :+*)A' !$9E :+*). AE) AE00F :E) :%$< D)J0%) 90,H:+HL :0 "):)%*+H) +J :E) *0I)*)H: 0J ")D%+A 

$H" *+9%0D)$"A %)A,;:)" +H $ "+JJ)%)H: *+9%0D)$" 90,H:' 
1 

3.3 Filter Bags 

>H ;$D0%$:0%< :%+$;A ,A+HL *+9%0D)$"A. H<;0H J+;:)% D$LA [2` 9* tY”u +H "+$*):)% $: :E) :0^ $H" V2 

9* t16”u ;0HL\ #+:E I$%+0,A *)AE A+C)A [244 g*. W4 g*. TW g*. 0% 24 g*\ #)%) ,A)" A+HL;< 

[GH+I)%A$; B+;:)%A. >H9'. =AD,%< c$%F. 5&\' >H A0*) 9$A)A. A+)I)A [T4 9* t8”u +H "+$*):)%\ #+:E 

TW g* $H" 24 g* H<;0H *)AE #)%) ,A)" +H ;+), 0J J+;:)% D$LA :0 :)A: %)90I)%< +H $ I)%< 

A:%$+LE:J0%#$%" #$< [+')'. J%0* $ J;$: A,%J$9) J%)) 0J A)$*A $H" "+*^;)A J0,H" +H J+;:)% D$LA\' 

>H J+);" :%+$;A. J+;:)% D$LA 2` 9* [Y”\ +H "+$*):)% $H" V2 9* [16”\ ;0HL #)%) ^;$9)" +H J+;:)% 

E0,A+HLA $%%$HL)" +H A)%+)A' = I$%+):< 0J *)AE A+C)A #$A ,A)"n TW g*. 3W g*. W4 g*. 244 g*' 

!$9E J+;:)% D$L #$A )H9;0A)" +H $ J+D)%L;$AA J+;:)% E0,A+HL [N$<#$%" B;0# /0H:%0; 8<A:)*A. 

/;)**0HA. 5/\ 90H:$+H+HL $ ^0;<^%0^<;)H) J+;:)% D$AF): [B+L,%) 2\' B;0# $H" ^%)AA,%) #)%) 

*0H+:0%)" ,A+HL $ ^$"";) #E)); J;0# A)HA0% "0#HA:%)$* 0J :E) A)90H" E0,A+HL $H" #+:E :E%)) 

^%)AA,%) A)HA0%A [0H) D)J0%) $H" $J:)% :E) J+%A: E0,A+HL $H" 0H) $J:)% :E) A)90H" E0,A+HLd $;; 

A)HA0%A #)%) *$H,J$9:,%)" D< PB 8+LH):. !; Q0H:). /=\' B;0# #$A 90H:%0;;)" *$H,$;;< ,A+HL $ 

W 9m (2”\ "+$^E%$L* I$;I) $: :E) D0::0* 0J :E) %+LE:ZE$H" J+;:)% E0,A+HL [B+L,%) 2. D;$9F E$H";)\' 

=*D+)H: A)$#$:)% #$A :$F)H ,^ D< $ 34ZE^. E0%+C0H:$; 9)H:%+J,L$; ^,*^. ^$AA)" :E%0,LE $ 2W 9* 

[6”\. ^0;<I+H<; 9E;0%+") [cK/\ ;+H). ^$AA)" :E%0,LE $ *$H+J0;". $H" ");+I)%)" :0 :E) N$<#$%" 

,H+:A D< $ W 9* [T”\. %)+HJ0%9)" cK/ ^;$A:+9 E0A)' B%)AE#$:)% #$A $"")" :0 $ 3Z*3 A:0%$L) :$HF 

,A+HL $ E0A) $H" ^,*^)" :0 :E) N$<#$%" ,H+:A :E%0,LE $ W 9m (2”\. %)+HJ0%9)" cK/ ^;$A:+9 E0A)' 

B+L,%) 2' O#0 V2 9* ;0HL J+;:)% D$LA +H N$<#$%" E0,A+HL $%%$HL)" +H A)%+)Ad #$:)% )H:)%)" :E) 

A<A:)* I+$ :E) L%))H E0A) $: :E) :0^ 0J :E) ;)J:ZE$H" E0,A+HL' 

!$%;< J+);" :%+$;A )*^;0<)" J+;:)% D$LA #+:E TWZg* *)AE' ($:)% :%+$;A ,A)" J+;:)% D$LA #+:E 3W g* 

*)AE. D)9$,A) +: D)::)% $^^%0-+*$:)" :E) *)AE A+C) ,A)" D< %)A)$%9E)%A $: :)A: J$9+;+:+)A +H :E) G8 

$H" $D%0$" [3Y g*\ $H" #$A $I$+;$D;) +H 90**)%9+$;;< *$H,J$9:,%)" J+;:)% D$LA' O0 9$^:,%) 

0%L$H+A*A +H :E) v W4 g* A+C) 9;$AA. :E) >QX /0HI)H:+0H [T44V\ A:$:)A :E) E<^0:)H,A) 0J :E) 
Y 

*)AE *,A: D) H0 ;0HL)% :E$H W4 g*d :E$: %)a,+%)*)H: +A *): ,A+HL 3W g* *)AE [E<^0:)H,A) i 

VU'W g*\' 

@E)H *+9%0D)$"A #)%) ,A)" $A ^%0-+)A J0% C00^;$HF:0H. +**)"+$:);< ^%+0% :0 :E) :%+$;. :E)< #)%) 

%+HA)" +H:0 $ J+;:)% D$L ^;$9)" +H :E) A)90H" J+;:)% E0,A+HL +H A)%+)A ["0#HA:%)$* 0J $ E0,A+HL 

#+:E $ J+;:)% D$L ,A)" $A $ ^%)J+;:)%\' OE) 90I)% 0J :E) A)90H" J+;:)% E0,A+HL #$A %)*0I)". $H" :E) 

E0,A+HL E$" D))H J+;;)" #+:E J%)AE#$:)%' =J:)% :E) 90I)% #$A A)9,%)" 0H :E) J+;:)% E0,A+HL $H" 

:E) $+% D;)" J%0* :E) E0,A+HL ,A+HL $ I$;I) 0H +:A 90I)%. #$:)% #$A ^,*^)" :E%0,LE :E) A<A:)*' 

OE) J+;:)% D$LA #)%) %):%+)I)" $H" :E) *+9%0D)$"A 90,H:)" J%0* :E) J+;:)% D$L :0 #E+9E :E) 

*+9%0D)$"A E$" D))H $"")" $H" J%0* :E) J+;:)% D$L "0#HA:%)$* 0J +:' XJ:)H. $ J+;:)% D$L #$A 

,A)" $A $ ^%)J+;:)% ,^A:%)$* 0J :E) J+;:)% D$L 90H:$+H+HL :E) *+9%0D)$"A :0 %)*0I) ^;$HF:0H. 

A)"+*)H:. $H" "):%+:,A' 

3.3.1 Filter Bags Field Trials—Water Flow Rate 

!" #$%$&'()$ %*$ +,,&",&(+%$ '$-* -(.$ /"& /(0%$& 1+2- +&&+)2$# () -$&($- +)# %*$ +,,&",&(+%$ 

3+%$& /0"3 &+%$ %*&"42* %*$'5 /(6$ %&(+0- 3$&$ &4)7 !*$ /0"3 &+%$- 6+&($# 89: 2,'5 ;9: 2,'5 ;:< 

2,'=5 +- #(# %*$ /(0%$& 1+2-> '$-* -(.$- 89: ?'5 :< ?'5 ;<< ?'=7 !*$ #(//$&$)%(+0 ,&$--4&$ 

1$%3$$) /(0%$& *"4-()2- 3+- '")(%"&$#5 +)# 3*$%*$& %*$ /(0%$& 1+2- @0"22$# 3+- )"%$#7 

3.3.2 Filter Bags—Laboratory and Field Validation with Microbeads 

A) 0+1"&+%"&B %&(+0-5 '(@&"1$+#- 3$&$ @"4)%$# 4-()2 + C$#2$3(@D E+/%$& @"4)%()2 @*+'1$& () %*$ 

3$% "& #&B '+))$&5 &()-$# ()%" + 1$+D$& 3(%* !B,$ AA 3+%$&5 +)# %*$ 1$+D$&>- @")%$)%- 2$)%0B 

&()-$# ()%" + /(0%$& 1+2 "& ")%" + -($6$7 !*$ '+%$&(+0 &$%+()$# ") %*$ /(0%$& 1+2 "& -($6$ 3+- &()-$# 

3(%* !B,$ AA 3+%$& ()%" + 1$+D$&5 +)# (%- @")%$)%- 3$&$ %&+)-/$&&$# 3(%* + -$&"0"2(@+0 ,(,$% ()%" 

F"2"&"6 @"4)%()2 @*+'1$&-7 !*$ &$@"6$&B $//(@($)@B "/ :< ?' '(@&"1$+#- 3+- 0"3 () %*$ /(&-% 

0+1"&+%"&B 8+)# /($0#= %&(+0-5 -" ;:< ?' +)# ;<< ?' '(@&"1$+#- 3$&$ +##$# %" $G,$&('$)%- 

1$@+4-$ %*$(& 0+&2$ -(.$ &$)#$&$# %*$' $+-($& %" /()#5 %*4- %*$B -$&6$# +- H4+-(I,"-(%(6$ @")%&"0-7 

!*$ /"00"3()2 ,+&+'$%$&- 3$&$ &$@"&#$# %" +@@"4)% /"& +)B 1(+-$-J %B,$ "/ /(0%$& 1+2 8,0+-%(@I

%",,$# 6-7 /$0%I%",,$#=5 %*$ 204$ 4-$# %" -$+0 %*$ -$+'-5 *"3 %*$ -$+'- 3$&$ 204$# 8$7275 ")@$ ") 

%*$ ()-(#$ +)# ")@$ ") %*$ "4%-(#$=5 %*$ '$%*"# "/ @"4)%()25 +)# %*$ /(0%$& 1+2 )4'1$& 8%" &$@"&# 

%*$ )4'1$& "/ 4-$-=7 

K/%$& ()(%(+0 /($0# %&(+0- 3(%* +'1($)% -$+3+%$& B($0#$# 0"3 '(@&"1$+# &$@"6$&B $//(@($)@($-5 

/&$-*3+%$& 3+- 4-$# $G@04-(6$0B () %*$ /($0#7 L)$ 9:I?' /(0%$& 1+2 3+- ,0+@$# () $+@* "/ %*&$$ 

M+B3+&# /(0%$& *"4-()2- +&&+)2$# () -$&($- 83(%* %*$ ,&$--4&$ +)# /0"3 -$)-"&- #$-@&(1$# +1"6$=7 

K /(0%$& 1+2 -$&6$# +- + ,&$/(0%$& %" &$'"6$ #$1&(- /&"' %*$ 3+%$&N + D)"3) )4'1$& "/ 

'(@&"1$+#- () + 1$+D$& 3+- &()-$# 3(%* !B,$ AA 3+%$& ()%" %*$ -$@")# /(0%$& 1+2 () -$&($-7 K/%$& 

+,,&"G('+%$0B OPQ: 0(%$&- 8;<<< 2+0= 3+- ,4',$# %*&"42* %*$ -B-%$' +% + /0"3 &+%$ "/ 9: 2,'5 

%*$ -$@")# +)# %*(&# /(0%$& 1+2- 3$&$ &$'"6$#5 &()-$# 3(%* !B,$ AA 3+%$&5 +)# %*$ '+%$&(+0 

&$%+()$# () %*$ 1+2- 3+- &()-$# ()%" + 1$+D$&5 ,(,$%%$ ()%" F"2"&"6 @"4)%()2 @*+'1$&-5 +)# 

$G+'()$# /"& '(@&"1$+#-7 !*$ /(0%$& 1+2-> -$+'- 4-$# () /($0# %&(+0- 3$&$ -$+0$# 3(%* ORS 

:9<<J 9: ?' /(0%$& 1+2- 3$&$ -$+0$# %3(@$ ") %*$ ()-(#$7 !" &$#4@$ *+)#0()2 %('$5 %*$ O: ?' 
Q 

/(0%$& 1+2- 3$&$ -$+0$# ")@$ ") %*$ ()-(#$ 3(%* %3(@$ +- '4@* -$+0+)% %B,(@+00B 4-$# /"& %*$ 9: 

?' /(0%$& 1+2-N + 0+1"&+%"&B %&(+0 -*"3$# ;<<T &$@"6$&B "/ :< ?' '(@&"1$+#-7 

A) 0+1"&+%"&B +)# /($0# %&(+0-5 %*$ /(0%$& 1+2-> -$+'-5 %*$ 1$+D$&-5 +)# ,(,$%%$- 3$&$ $G+'()$# /"& 

&$-(#4+0 '(@&"1$+#-7 

3.3.3 Filter Bags—Seams 

K/%$& ()(%(+0 $G,$&('$)%- -*"3$# + 0"3 &$@"6$&B $//(@($)@B "/ '(@&"1$+#-5 (% 3+- #(-@"6$&$# %*+% 

%*$ *"0$- +% %*$ -$+'- 3$&$ '4@* 2&$+%$& %*+) %*$ )"'()+0 '$-* -(.$5 +)# (% 3+- *B,"%*$-(.$# 

%*$ '(@&"1$+#- ,+--$# %*&"42* %*$ /(0%$& 1+2- 6(+ %*$ *"0$-7 K -4&6$B "/ /(0%$& 1+2 '+)4/+@%4&$&- 

-*"3$# 1+2- 3(%* *$+%I3$0#$# -$+'- 3$&$ +6+(0+10$5 3*(@* 3"40# +'$0("&+%$ %*$ ,&"10$'5 14% 

1+2- 3$&$ +6+(0+10$ ")0B () '(@&"/(0+'$)% "& ,"0B,&",B0$)$ /$0% '+%$&(+07 U$(%*$& 3+- 

+,,&",&(+%$5 +- "&2+)(-'- @"40# )"% 1$ &$%&($6$# /&"' %*$-$ -4&/+@$-7 

V0"-()2 "// %*$ 1+2-> -$+'- 3(%* + '+&()$ -$+0+)% 3+- + 6(+10$ -"04%(")5 +- 0")2 +- %*$ @4&$# 

-$+0+)% 3+- '+00$+10$5 )"% %+@DB 8-" (% 3"40# )"% %&+, ."",0+)D%")=5 +)# )")I%"G(@ %" '+&()$ 

"&2+)(-'- "6$& %*$ -*"&% %('$ %*$B 3"40# 1$ -$H4$-%$&$# () %*$ /(0%$& 1+2- #4&()2 -+',0()2 

8+,,&"G('+%$0B 9 *=7 C$6$) -$+0+)%- 3$&$ $6+04+%$#J R+&()$ W"",S 8X@0$@%(@ Y&"#4@%-5 A)@75 

X42$)$5 LE=5 AUC!KU! K#*$-(6$ 8WVZ X0$@%&")(@-5 E"@D/" A[=5 \$0#]L) Y^V P;PZ 

Y0+-%(@ Y(,$ V$'$)% 8AYC V"&,"&+%(")5 W+&#$)+5 VK=5 9I,+&% $,"GB 8_"*) V7 `"0,* V"',+)B5 

R")'"4%* _4)@%(")5 U_=5 3*(%$ ORZ R+&()$ a+-% V4&$ :9<< K#*$-(6$ C$+0+)% 8OR5 C%7 Y+405 

RU=5 +)# X0'$&>-S U"I\&()D$ E411$& V$'$)% 8X0'$&>- Y&"#4@%-5 A)@75 V"04'14-5 LM=7 

b-()2 +Ic; @' 8;d”= 0")2 /(0%$& 1+2 3(%* ;< ?' '$-*5 $+@* +#*$-(6$ 3+- +,,0($# () 

+,,&"G('+%$0B :I@' 0")2 -$@%(")- %*$ "4%-(#$ +)# ()-(#$ "/ %*$ 1+2-5 +)# %*$ +#*$-(6$- @4&$# /"& 

Od * 80")2$& %*+) +)B "/ %*$ &$@"''$)#$# @4&()2 %('$-=7 

3.3.4 Filter Bags—Toxicity of Sealant on the Seams 

!" %$-% (/ ."",0+)D%") 3$&$ D(00$# 1B 1&($/ $G,"-4&$ %" %*$ /(0%$& 1+2-> -$+0+)% 8ORZ :9<<5 

@*"-$) +/%$& %*$ %&(+0 #$-@&(1$# () -$@%(") O7O7O=5 1("+--+B $G,$&('$)%- 3$&$ @")#4@%$# 3(%* 1&()$ 

-*&(', Artemia franciscana 8,&$6("4-0B 4-$# +% UE[e\ +- + -%+)#+&# %$-% "&2+)(-'= +)# 

-41-$H4$)%0B 3(%* +'1($)% ."",0+)D%") 8@",$,"#-=7 VB-%- "/ A. franciscana 3$&$ ,4&@*+-$# 

/&"' + 6$)#"& 8F&()$ C*&(', `(&$@%5 L2#$)5 b!= +)# ()@41+%$# () :I?'I/(0%$&$#5 +$&+%$# 

+&%(/(@(+0 -$+3+%$& 8-+0()(%B f Od= /"& 9c * () 9:gV 3(%* + ;9J;9 0(2*%J#+&D @B@0$ 4)#$& /04"&$-@$)% 

1401- 8P9 ?R X()-%$()- 'I9 -

I;=7 M+%@*$# )+4,0(( +,,&"G('+%$0B ;9 * "0# +)# O<< I c<< ?' 0")2 

3$&$ &$'"6$# /&"' %*$ @40%4&$ () ; '0 +0(H4"%- +)# #(-,$)-$# ()%" $+@* "/ $(2*% Y$%&( #(-*$- 8cP 

'' #(+'$%$&= 3(%* ;< '0 "/ <799 ?'I/(0%$&$# -$+3+%$&7 U$G%5 %*$ #(-*$- 3$&$ $G+'()$# 4-()2 + 

#(--$@%()2 '(@&"-@",$ %" $)-4&$ +00 A. franciscana 3$&$ 0(6()2 8#$%$&'()$# 1B '"6$'$)%=5 3*(@* 

3+- %*$ @+-$7 a"4& Y$%&( #(-*$- -$&6$# +- @")%&"0-5 +)# %" %*$ &$'+()()2 /"4&5 + -%&(, 

8+,,&"G('+%$0B c @' G ; @'= "/ 9: ?'5 )B0") /(0%$& 1+2 3(%* + 1$+# "/ -$+0+)% 8+,,&"G('+%$0B O 

@' G <7: @'= %*+% *+# 1$$) @4&$# /"& 9c * 3+- +##$#7 
h 

Y$%&( #(-*$- 3$&$ +&&+)2$# *+,*+.+�B ") + -*$0/ () %*$ ()@41+%"& 8#$-@&(1$# +1"6$= /"& 9 *5 %*$ 

$-%('+%$# +'"4)% "/ %('$ ."",0+)D%") 3"40# 1$ -$H4$-%$&$# () + /(0%$& 1+2 #4&()2 @"00$@%(")7 

K/%$& %*$ ()@41+%(")5 %*$ )4'1$& "/ #$+# A. franciscana 3+- @"4)%$#7 A/ +) "&2+)(-' 3+- )"% 

'"6()25 (% 3+- 2$)%0B ,&"##$# 3(%* + ,&"1$5 +)# (/ (% #(# )"% '"6$ 3(%*() ;< -5 (% 3+- -@"&$# +- 

#$+#7 a"00"3()2 %*$ %+00B5 +00 -+',0$- 3$&$ /(G$# 3(%* [42"0>- ("#()$ -"04%(")5 %*$ %"%+0 )4'1$& 

"/ A. franciscana @"4)%$#5 +)# %*$ )4'1$& "/ 0(6()2 "&2+)(-'- #$%$&'()$# 1B -41%&+@%(")7 

!*$ %&(+0 3+- &$,$+%$# 3(%* +'1($)% @",$,"#-5 3*(@* 3$&$ @")@$)%&+%$# /&"' + -$+3+%$& *"-$ 

")%" + O;I?' -($6$7 V",$,"#- "/ +00I0(/$ *(-%"&B -%+2$- 8i :< ?' () '()('4' #('$)-(")= 3$&$ 

&$'"6$# /&"' %*$ -+',0$ ()#(6(#4+00B 4-()2 + ,(,$%7 !" $+@* "/ $(2*% Y$%&( #(-*$- 8cP '' 

#(+'$%$&= @")%+()()2 ;< '0 "/ <799 ?' /(0%$&$# -$+3+%$&5 9< @",$,"#- 3$&$ +##$#7 a"4& #(-*$- 

-$&6$# +- @")%&"0-5 +)# -%&(,- "/ '$-* 3(%* -$+0+)% 3$&$ +##$# %" %*$ "%*$& /"4& Y$%&( #(-*$- 

8%&$+%'$)%=7 `(-*$- 3$&$ $G+'()$# %" $)-4&$ +00 @",$,"#- 3$&$ 0(6()2 8%*$B 3$&$=5 +)# #(-*$- 

3$&$ +&&+)2$# *+,*+.+�B ") + -*$0/ () %*$ ()@41+%"& +)# ()@41+%$# /"& 9 * +% %*$ -$%%()2- /&"' 

%*$ ,&$6("4- %&(+07 a"00"3()2 ()@41+%(")5 %*$ )4'1$& "/ #$+# @",$,"#- 3+- @"4)%$# +)# 

,&$-$&6$# +- +1"6$7 C%+%(-%(@- 3$&$ @+0@40+%$# 3(%* C(2'+Y0"%S 67 ;; 8CB-%+% C"/%3+&$ A)@75 C+) 

_"-$5 VK=7 

3.4 Filter Skid 

3.4.1 Filter Skid Requirements 

!*$ /(0%$& -D(# 3+- #$-(2)$# 3(%* -$6$&+0 @")-(#$&+%(")- () '()#J /(&-%5 (% 3+- #$-(&+10$ %" 

@")-%&4@% +) +,,+&+%4- 4-()2 @"''")5 @"''$&@(+00B +6+(0+10$ ,&"#4@%- -" %*$ -D(# @"40# 1$ 

&$,0(@+%$# $+-(0B +% "%*$& %$-% /+@(0(%($- +)# #(# )"% &$H4(&$ -,$@(+0 /+1&(@+%(") %$@*)(H4$-7 

C$@")#5 %*$ ,&"%"%B,$ 3+- #$6$0",$# %" $0('()+%$ 0+&2$5 @4'1$&-"'$ ,0+)D%") )$%- @4&&$)%0B 

4-$# 1B %$-% /+@(0(%($-5 -" (% )$$#$# %" *+6$ )$%- () -$0/I@")%+()$# /(0%$& *"4-()2-7 !*( %*$ 

+,,+&+%4- 3+- #$-(2)$# -" (% '(2*% 1$ +@@"''"#+%$# () /4%4&$ -*(,1"+&# -+',0()2 ,&"2&+'-5 

$7275 + -'+00 /""%,&()% 3+- &$H4(&$#7 F$@+4-$ %*$ -D(# 3"40# 1$ 4-$# 3(%* -$+3+%$&5 %*$ '+%$&(+0- 

)$$#$# %" 3(%*-%+)# + @"&&"-(6$ $)6(&")'$)% 8$7275 -%+()0$-- -%$$0 "& Y^V=7 F"%* %*$ @"-% +)# 

+6+(0+1(0(%B "/ @"&&"-(")I&$-(-%+)% '$%+0- 3$&$ @")-(#$&$#7 W&+#$ O;d -%+()0$-- -%$$0 3+- @*"-$) 

/"& %*$ /(0%$& *"4-()2-5 +)# Y^V 3+- @*"-$) /"& %*$ ,(,()27 

3.4.2 Toxic Effects of Stainless Steel Filter Housings 

K 1("+--+B %$-% 3+- @")#4@%$# %" #$%$&'()$ (/ %*$ 1&()$ -*&(', Artemia franciscana "& +'1($)% 

."",0+)D%") 3"40# #($ /&"' $G,"-4&$ %" -%+()0$-- -%$$0 /(0%$& *"4-()27 VB-%- "/ A. franciscana 

3$&$ *+%@*$# +- () -$@%(") O7O7c5 +)# O ;I'0 -41-+',0$- "/ ;9I* "0# U+4,0(( 8O<< I c<< ?' 0")2= 

3$&$ &$'"6$# /&"' %*$ @40%4&$ +)# $6+04+%$# H4+0(%+%(6$0B 4-()2 + #(--$@%()2 '(@&"-@",$7 A/ + 

)+4,0(4- 3+- )"% '"6()25 (% 3+- 2$)%0B ,&"##$# 3(%* + ,&"1$5 +)# (/ (% #(# )"% '"6$ 3(%*() ;< -5 

(% 3+- -@"&$# +- #$+#7 K00 )+4,0(( 8P< I PP ,$& -41-+',0$= 3$&$ 0(6()27 !3" '0 +0(H4"%- "/ %*$ 

@40%4&$ 3$&$ #(-,$)-$# ()%" $+@* "/ /(6$ ;<<< '0 1$+D$&- @")%+()()2 Q<< '0 "/ <7c: ?'I/(0%$&$# 

-$+3+%$&7 
;< 

K'1($)% ."",0+)D%") 8,&('+&(0B @",$,"#-= 3$&$ @")@$)%&+%$# /&"' + -$+3+%$& *"-$ ")%" + OPI

?' -($6$7 !*$ /(0%&+)# 3+- -4-,$)#$# () <7c: ?'I/(0%$&$# -$+3+%$&7 K ; '0 -+',0$ 3+- 

&$'"6$# +)# ."",0+)D%") 8i :< ?' () '()('4' #('$)-(")= 3$&$ H4+0(%+%(6$0B +--$--$#J ; 3+- 

#$+#5 dI;< 3$&$ 0(6()27 a"4&I'0 +0(H4"%- "/ %*$ -+',0$ 3$&$ ,0+@$# ()%" $+@* "/ %*$ /(6$ 1$+D$&-7 

!3" 1$+D$&- -$&6$# +- @")%&"0-5 3(%* ")0B -$+3+%$&5 A. franciscana5 +)# +'1($)% ."",0+)D%")7 

!" %*$ &$'+()()2 %*&$$ 1$+D$&-5 ;< 3+-*$&- 82&+#$ O;d -%+()0$-- -%$$05 ;7h '' %*(@D5 +)# OQ7; 

'' () #(+'$%$& 3(%* + *"0$ () %*$ @$)%$& ;:7Q '' () #(+'$%$&= 3$&$ +##$#7 !*$(& -4&/+@$ +&$+ 

8CK= () %*$ 1$+D$& 3+- @+0@40+%$# %" $H4+0 %*$ CKJ6"04'$ &+%(" "/ 3+%$& () + /(0%$& -D(# *"4-()27 

F$+D$&- 3$&$ +&&+)2$# *+,*+.+�B ") + -*$0/ +)# @"6$&$# 3(%* + 10+@D %&+B () %*$ ()@41+%"& /"& 

97: *5 %*$ $-%('+%$# +'"4)% "/ %('$ ."",0+)D%") 3"40# 1$ -$H4$-%$&$# () + -%+()0$-- -%$$0 /(0%$& 

*"4-()2 #4&()2 @"00$@%(")7 K/%$& %*$ ()@41+%(")5 %*$ 3+-*$&- 3$&$ &$'"6$# /&"' %*$ %&$+%'$)% 

1$+D$&- 4-()2 /"&@$,-5 +)# %*$ @")%$)%- "/ $+@* 1$+D$& 3$&$ 2$)%0B ,"4&$# %*&"42* + OP ?' 

-($6$5 %*$ /(0%&+)# &()-$# 3(%* /(0%$&$# -$+3+%$& ()%" + 1$+D$&5 +)# %*$ $)%(&$ 6"04'$ %&+)-/$&&$# %" 

+ Y$%&( #(-*7 !*$ )4'1$& "/ #$+# A. franciscana +)# ."",0+)D%") 3$&$ @"4)%$#7 a"00"3()2 %*$ 

%+00B5 +00 -+',0$- 3$&$ /(G$# 3(%* [42"0>- ("#()$ -"04%(")5 %*$ %"%+0 )4'1$& "/ "&2+)(-'- 

@"4)%$#5 +)# %*$ )4'1$& "/ 0(6()2 "&2+)(-'- #$%$&'()$# 1B -41%&+@%(")7 

 

3.4.3 Comparison of Zooplankton in the Ballast Tank vs. the Filter Skid 

A) + ,&$0('()+&B $G,$&('$)% %" #$%$&'()$ (/ @"00$@%()2 ."",0+)D%") 8@",$,"#-= 4-()2 %*$ /(0%$& 

-D(# D(00$# %*$'5 %*$ ,$&@$)%+2$ "/ 0(6()25 +'1($)% @",$,"#- 3+- H4+)%(/($# +/%$& %*$B 3$&$ 

,4',$# ()%" + 1+00+-% %+)D +% UE[e\5 +)# %*+% )4'1$& 3+- @"',+&$# %" %*$ ,$&@$)%+2$ "/ 

0(6()2 ."",0+)D%") @"00$@%$# () %*$ /(0%$& 1+2- () %*$ -D(# +- %*$ %+)D 3+- #&+()$#7 !*$ 1+00+-% 

%+)D 3+- /(00$# 3(%* 9;h 'O 8:P5PP: 2+0= "/ +'1($)% -$+3+%$& +% + /0"3 &+%$ "/ OQOc 0 '()I; 8;<;O 

2,'=5 +)# (''$#(+%$0B +/%$&3+&#-5 + 6$&%(@+0 ,0+)D%") %"3 3+- %+D$) () %*$ %+)D 4-()2 + 9: ?' 

'$-*5 <7P: ' '"4%* #(+'$%$&5 )B0") ,0+)D%") )$%7 L&(2()+00B5 + H4+)%(%+%(6$ -+',0$ 3+- %" 1$ 

@"00$@%$#5 14% 3*$) %*$ U(-D() 1"%%0$ 4-$# %" @"00$@% %*$ -+',0$ 1&"D$5 + H4+0(%+%(6$ ,0+)D%") %"3 

3+- %+D$) ()-%$+#7 !*$ )$% 3+- &()-$# 3(%* +'1($)% -$+3+%$& +)# (%- @")%$)%- -4-,$)#$# () 

/(0%$&$# -$+3+%$& 8<799 ?'= +)# #(04%$# ;<G 3(%* /(0%$&$# -$+3+%$&7 K/%$& %*$ @")%+()$& 3+- 

-$+0$# +)# 2$)%0B ()6$&%$# OG5 9 -41-+',0$- 3$&$ &$'"6$# +)# $6+04+%$# () F"2"&"6 @"4)%()2 

@*+'1$&-7 !*$ )4'1$& "/ #$+# @",$,"#- 8)+4,0((N @",$,"#(%$- +)# +#40%-= 3+- @"4)%$#5 %*$ 

-+',0$- /(G$# () [42"0>- ("#()$ -"04%(")5 +)# %*$ )4'1$& "/ 0(6()2 @",$,"#- #$%$&'()$# 1B 

-41%&+@%(")7 

!*$ 3+%$& 3+- *$0# () %*$ 1+00+-% %+)D /"& %3" *"4&- 8+- %*$ )$% %"3 3+- +)+0B.$#= +)# %*$) 

#&+()$# +% /0"3 &+%$ "/ OQOc 0 '()I; 8;<;O 2,'=5 3(%* d: 'O 8;P99d 2+0= #(6$&%$# %" /0"3 %*&"42* 

%*$ /(0%$& -D(# +% + &+%$ "/ c:c 0 '()I; 8;9< 2,'=5 3(%* ;;c 0 '()I; 8O< 2,'= %*&"42* $+@* "/ %*$ Q 

*"4-()2- @")%+()()2 /(0%$& 1+2- 8+00 /0"3- +&$ +,,&"G('+%$ +6$&+2$-=7 !*$ /(&-% %3" /(0%$& 

*"4-()2- () %*$ /(0%$& -D(# 3$&$ $',%B 8$G@$,% /"& %*$ '$%+0 0()$&- 3(%* O '' *"0$-=N %*$ Q 

*"4-()2- #"3)-%&$+' $+@* *$0# + 9:I?' /(0%$& 1+2 3(%* %*$ -$+'- 204$# %3(@$ ") %*$ ()-(#$ 3(%* 

ORS :9<< -$+0+)%7 K/%$& %*$ #&+() ",$&+%(")5 /(0%$& 1+2- 3$&$ &$'"6$# /&"' %*$ *"4-()2-5 &()-$# 

3(%* +'1($)% -$+3+%$&5 +)# %*$(& /(0%&+)#- @")-"0(#+%$# ()%" + 9 0 2&+#4+%$# @B0()#$&7 !*$ 

@B0()#$& 3+- 2$)%0B ()6$&%$# :G5 +)#5 4-()2 ;< '0 -$&"0"2(@+0 ,(,$%%$-5 %*&$$ &$-$+&@*$&- $+@* 
;; 

(''$#(+%$0B +)# -('40%+)$"4-0B %""D + ;< '0 -+',0$ /&"' %*$ @$)%$& "/ %*$ @B0()#$& +)# 

#(-,$)-$# %*$(& -+',0$- ()%" + :< '0 @$)%&(/42$ %41$7 !*$ ()6$&-(") 3+- &$,$+%$#5 +)# %*$ 

&$-$+&@*$&- %""D + :I'0 -+',0$ +)# ,0+@$# (% ()%" %*$(& @$)%&(/42$ %41$5 1&()2()2 $+@* %41$>- 

6"04'$ %" ;: '07 !*(- ,&"@$-- 3+- 4)#$&%+D$) %" $)-4&$ -41-+',0$- 3$&$ &$,&$-$)%+%(6$ "/ %*$ 

-+',0$ () %*$ 9 0 2&+#4+%$# @B0()#$&7 `4$ %" %*$ *(2* @")@$)%&+%(")- "/ ,0+)D%")5 -$#('$)%5 +)# 

#$1&(-5 (% 3+- )$@$--+&B %" #(04%$ %*$ -+',0$- ;<G 4-()2 /(0%$&$# -$+3+%$& 1$/"&$ %*$B 3$&$ 

+)+0B.$# () %*$ -+'$ '+))$& +- %*$ -+',0$- /&"' %*$ ,0+)D%") %"37 

3.4.4 Effect of Crowding on Ambient Zooplankton 

!*$ $//$@% "/ @&"3#()2 #4&()2 -+',0()2 3+- $6+04+%$# 1B @"',+&()2 8;= + -+',0$ -('40+%()2 + 

,+&@$0 "/ 3+%$& d< 'O '$$%()2 %*$ ."",0+)D%") #(-@*+&2$ -%+)#+&# 8;< 0(6()2 "&2+)(-'- 'IO= +)# 

@")@$)%&+%$# #4&()2 -+',0()2 %" + 6"04'$ "/ ; 0 8f d<< ."",0+)D%$&- 0I;= %" 89= + @")%&"0 -+',0$ 

3(%* + '4@* 0"3$& @")@$)%&+%(") "/ ."",0+)D%") 8;< 0I;=7 C(G *4)#&$# "& %$) @",$,"#- 8Acartia 

tonsa5 ,4&@*+-$# /&"' [(6$V",$,"#-7@"'5 C$+%%0$5 \KN -+0()(%B f OO7:=5 3$&$ '+)4+00B @"4)%$# 

+)# +##$# %" + 1$+D$& @")%+()()2 ; 0 "/ +&%(/(@(+0 -$+3+%$& 8-+0()(%B f Oc=7 F$+D$&- 3$&$ ,0+@$# () 

+) ()@41+%"& -$% +% 9:gV +)# (004'()+%$# 1B /04"&$-@$)% 1401- 8P9 ?R X()-%$()- 'I9 -

I;=5 2$)%0B 

+$&+%$#5 +)# ()@41+%$# /"& c *7 Y&$6("4- 3"&D +% UE[e\ *+- -*"3) #($I"// "@@4&- () -+',0$- 

*$0# 0")2$& %*+) -(G * 8E(0$B $% +075 9<<d=5 -" %*$ ."",0+)D%") *+)#0()2 %('$ +)# ()@41+%(") %('$ 

3+- + %"%+0 "/ d *7 K/%$& ()@41+%(")5 %*$ @",$,"#- 3$&$ &$@"6$&$# /&"' %*$ 1$+D$&- 4-()2 + :<I

?' -($6$7 !*$ #$+# A. tonsa 3$&$ @"4)%$#7 U$G%5 %*$ -+',0$- 3$&$ /(G$# 3(%* [42"0>- ("#()$ 

-"04%(")5 %*$ %"%+0 )4'1$& "/ @",$,"#- @"4)%$#5 +)# %*$ )4'1$& "/ 0(6()2 @",$,"#- 3+- 

#$%$&'()$# 1B -41%&+@%(")7 F$@+4-$ %*$ -+',0$ *+)#0()2 %('$ 3+- %"" 0")2 %" ,&$,+&$ +)# 

+)+0B.$ &$,0(@+%$ -+',0$- () + -()20$ #+B5 %*$ $G,$&('$)% 3+- &$,$+%$# %*$ /"00"3()2 #+B7 A) %*+% 

%&(+05 ")0B :P9 Acartia tonsa 3$&$ +6+(0+10$5 -" 9Q 1&()$ -*&(', 8Artemia franciscana= 3$&$ 

+##$# %" 1&()2 %*$ %&$+%'$)% 1$+D$&>- %"%+0 ."",0+)D%") @"4)% %" d<<7 F$@+4-$ )" A. franciscana 

3$&$ +##$# %" %*$ @")%&"0 -+',0$5 %*$ -4&6(6+0 3+- #$%$&'()$# ")0B /"& A. tonsa () 1"%* 

%&$+%'$)%-7 

4 Results 

4.1 Microbeads 

4.1.1 Microbead Disintegration 

!*$ @"4)%- "/ '(@&"1$+#- 8;< :< ?' '(@&"1$+#- +)# ;< ;:< ?' '(@&"1$+#-= +% %*$ -%+&% "/ %*$ 

%&(+0 +)# P #+B- 0+%$& 3$&$ %*$ -+'$ () 1"%* %*$ /(0%$&$# -$+3+%$& +)# %*$ !B,$ AA 3+%$& 8#+%+ )"% 

-*"3)=7 !*$&$/"&$5 '(@&"1$+#- #(# )"% #(-()%$2&+%$ ") %*$ %('$ -@+0$- 4-$# /"& %&(+0-7 

4.1.2 Method of Microbead Counting 

!*$ )()$ %&(+0- @"',+&()2 j#&B> %" j3$%> @"4)%()2 "/ '(@&"1$+#- -*"3$# %*$ 3$% '$%*"# %" 1$ 

-4,$&("& 8a(24&$ 9=7 A) ")0B ")$ "/ /(6$ %&(+0- 4-()2 %*$ #&B '$%*"#5 '(@&"1$+# &$@"6$&B 3+- i 

h<TN *"3$6$&5 () +00 /"4& %&(+0- 4-()2 %*$ 3$% '$%*"#5 '(@&"1$+# &$@"6$&B 3+- i h<T7 U" 1(+- 
;9 

3+- +%%&(14%$# %" +)B &$-$+&@*$&-5 +- +00 %*&$$ &$-$+&@*$&- +@*($6$# 1"%* 2""# +)# ,""& &$@"6$&B7 

M$)@$/"&%*5 '(@&"1$+#- 3$&$ @"4)%$# ")0B () %*$ 3$% '+))$&7 

a(24&$ 97 E$@"6$&B +/%$& '(@&"1$+#- 3$&$ @"4)%$# 4-()2 %*$ j#&B> +)# j3$%> '$%*"#-7 L&+)2$ 

1+&- &$,&$-$)% #&B %&(+0-5 +)# 104$ 1+&- &$,&$-$)% 3$% %&(+0-N )4'1$&- +1"6$ 1+&- ()#(@+%$ %*$ 

,$&@$)% '(@&"1$+# &$@"6$&BN 0$%%$&- ") %*$ GI+G(- 0+1$0- ()#(@+%$ %*$ &$-$+&@*$&>- ()(%(+0-7 

4.1.3 Intercalibration 

\*$) ;Q F"2"&"6 %&+B- 3(%* + -$+3+%$& -+',0$ 3$&$ @"4)%$# 1B %3" "1-$&6$&- %" #$%$&'()$ %*$ 

#(//$&$)@$ /&"' %*$ '$+) "/ %*$ @"4)%-5 %*$ 2&+)# '$+) 3+- ;9T 8C` f ;cTN #+%+ )"% -*"3)=7 

\*$) ")$ "1-$&6$& @"4)%$# %*$ -+'$ %&+B 3(%* + -$+3+%$& -+',0$ /(6$ %('$-5 %*$ )4'1$& "/ 

'(@&"1$+#- @"4)%$# 6+&($# "6$& %*&$$I/"0#J :5 :5 c5 95 P '(@&"1$+#-7 

!*$ ()%$&@+0(1+%(") -*"3$# 2""# +2&$$'$)% 1$%3$$) "1-$&6$&-5 14% 1$@+4-$ #(//$&$)% @"4)%- 

&$-40%$# 3*$) + -+',0$ 3(%* -$+3+%$& 8+)# @"&&$-,")#()2 #$1&(-= 3+- @"4)%$# &$,$+%$#0B5 (% 3+- 

)$@$--+&B %" @")#4@% 6+0(#+%(") %&(+0-k() 1"%* %*$ 0+1"&+%"&B +)# %*$ /($0#k4-()2 /&$-*3+%$&5 -" 

'(@&"1$+#- 3"40# 1$ 6(-(10$7 

4.2 Filter Bags 

4.2.1 Filter Bag Field Trials—Water Flow Rate 

!*$ /(0%$& 1+2- @0"22$# (''$#(+%$0B () %*$ /(&-% %&(+0-5 3*(@* 3$&$ @")#4@%$# +% &$0+%(6$0B *(2* 

/0"3 &+%$- 8$7275 ;:< 2,'N K,,$)#(G 9=7 C41-$H4$)% %&(+0- -*"3$# + /0"3 &+%$ "/ 9: 2,' +00"3$# 

-4-%+()$# /0"3 3(%* 0"3 ,&$--4&$ #(//$&$)%(+0- +@&"-- /(0%$& 1+2- *+6()2 &$0+%(6$0B -'+00 '$-* 

-(.$- 8:< ?' "& 9: ?'=7 A) -41-$H4$)% %&(+0-5 %*$ /0"3 &+%$ 3+- -$% +% 9: 2,'7 \(%* %*$ 

77 76

88

95

80

91 90

95 96

70

75

80

85

90

95

100

Microbead Recovery (%)

Trials and Observers
;O 

M+B3+&# 4)(%-5 + : @' 89”= *"-$ -4,,0($# 3+%$& %" %*$ -B-%$'N +% 9: 2,'5 3+%$& /0"3()2 %*&"42* 

%*$ ,(,$ 3"40# %&+6$0 +% <7PQ ' -I; 8;7P ',*=7 

4.2.2 Filter Bags—Field and Laboratory Validation Using Microbeads 

E$@"6$&B $//(@($)@($- "/ :< ?' '(@&"1$+#- () %*$ 0+1"&+%"&B &+)2$# /&"' c;T I QcT 3*$) %*$ 

'(@&"1$+#- 3$&$ @"4)%$# () %*$ 3$% /+-*(") +)# %*$ /(0%$& 1+2-> -$+'- 3$&$ 4)204$# "& 204$# 

()-4//(@($)%0B 3(%* R+&()$ W"",S 8a(24&$ O=7 b-4+00B5 )" '(@&"1$+#- 3$&$ /"4)# () %*$ /(0%&+%$N 

") ")$ "@@+-(")5 P '(@&"1$+#- 3$&$ /"4)# 8;<< 3$&$ +##$# %" %*$ /(0%$& 1+2N &$@"6$&B 

$//(@($)@($- $G@04#$ '(@&"1$+#- /"4)# () %*$ /(0%&+%$=7 E$@"6$&B "/ %*$ :<I?' '(@&"1$+#- 

2$)$&+00B (',&"6$# +/%$& %*$ '(@&"1$+#- 3$&$ @"4)%$# () %*$ 3$% /+-*(") +)# %*$ -$+'- 3$&$ 

204$# '"&$ %*"&"42*0B7 \*$) OR :9<<S 3+- 4-$#5 &$@"6$&B $//(@($)@B "/ %*$ -'+00$-% 

'(@&"1$+#- &+)2$# /&"' Q9T I ;;<T 8'$+) f hcT=7 !*$ 0+&2$& %3" -(.$ @0+--$- -*"3$# 

$G@$00$)% &$@"6$&B $//(@($)@($-5 +% 0$+-% h:T5 3(%* ")$ $G@$,%(") 8QPT5 !&(+0 9;=7 K 0+1"&+%"&B 

%&(+0 4-()2 :< ?' '(@&"1$+#- () + :< ?' /(0%$& 1+2 3(%* -$+'- 204$# %3(@$ ") %*$ ()-(#$ 3(%* OR 

:9<<S -$+0+)% B($0#$# + 9<T &$@"6$&B $//(@($)@B 8#+%+ $G@04#$# /&"' a(24&$ O=7 

a(24&$ O7 E$@"6$&B $//(@($)@B "/ '(@&"1$+#- () 0+1"&+%"&B %&(+0- 4-()2 9: ?' /(0%$& 1+2- 8aF=7 F04$5 

&$#5 +)# 2&$$) 1+&- &$,&$-$)% :< ?'5 ;<< ?'5 +)# ;:< ?' '(@&"1$+#-5 &$-,$@%(6$0B7 
;c 

K0%*"42* %*$ %+)D %*+% +@%$# +- + /&$-*3+%$& &$-$&6"(& /"& /($0# %&(+0- 3+- @0$+)$# 1$/"&$*+)#5 + 

-'+00 +'"4)% "/ 2&$$) +02+$ /&"' %*$ %+)D +)# %*$ ,(,()2 () %*$ -B-%$' 3+- @"00$@%$# +)# 

@")@$)%&+%$# () %*$ /(0%$& 1+2- 8a(24&$ c5 !&(+0 ;N +00 %&(+0- $G@$,% %*$ /(&-% ")$ *+# + 9:I?' /(0%$& 

1+2 ,0+@$# 4,-%&$+' %" /(0%$& '+%$&(+0 /&"' %*$ 3+%$&=7 `4&()2 -+',0$ +)+0B-(-5 &$'"6()2 +02+$ 

/&"' ")$ F"2"&"6 @"4)%()2 @*+'1$& 3(%* '+)B '(@&"1$+#- +)# &$I@"4)%()2 (% 0$# %" + 9<T 

()@&$+-$ () %*$ )4'1$& "/ '(@&"1$+#- &$@"6$&$# 8!&(+0 P=7 !*$ 0(%%0$ 1(% "/ +02+$ () %*$ -+',0$-5 (% 

-$$'-5 "1-@4&$# %*$ :< ?' '(@&"1$+#-5 '+D()2 &$@"6$&($- +,,$+& %" 1$ 0"3 $6$) %*"42* 

'(@&"1$+#- 3$&$ 0(D$0B @+,%4&$# () %*$ /(0%$& 1+2-7 !*$ %+)D 3+- &(2"&"4-0B -@&411$# %" &$'"6$ 

+02+$5 +)# %*$ $G,$&('$)% 3+- &$,$+%$# 3(%* 9<< :< ?' +)# 9<< ;:< ?' '(@&"1$+#-7 !*$ 

&$-40%- 3$&$ 1$%%$&J hOT +)# ;<<T &$@"6$&B $//(@($)@B 8:< ?' +)# ;:< ?' '(@&"1$+#-5 

&$-,$@%(6$0B=N %*$&$ 3+- )"%(@$+10B 0$-- +02+$ () %*$ -+',0$ %*+) () ,&$6("4- $G,$&('$)%-7 

C41-$H4$)% %&(+0- 3(%* 6$&B @0$+) %+)D- -*"3$# &$@"6$&B $//(@($)@($- l QPT "/ :< ?' 

'(@&"1$+#-7 E$@"6$&B $//(@($)@B "/ 0+&2$& 8;<< ?' +)# ;:< ?'= '(@&"1$+#- ,&("& %" %&(+0 P 

&+)2$# /&"' PcT %" ;<;T 8%*$ 0+%%$& &$,&$-$)%- + @"4)%()2 $&&"& "& + '(@&"1$+# 0$/%"6$& /&"' + 

,&$6("4- %&(+05 +0%*"42* /(0%$& 1+2- 3$&$ $G+'()$# /"& &$-(#4+0 '(@&"1$+#- ") + #(--$@%()2 

'(@&"-@",$ ,&("& %" $+@* %&(+0=7 a"00"3()2 !&(+0 P5 $//(@($)@($- 3$&$ l hhT7 A) +00 %&(+0-5 )$+&0B 

+00 "/ %*$ '(@&"1$+#- 3$&$ &$@"6$&$# /&"' %*$ /(0%$& 1+2 %" 3*(@* %*$B *+# 1$$) +##$#N () ")$ 

()-%+)@$5 ;O '(@&"1$+#- 3$&$ /"4)# () %*$ /(0%$& 1+2 #"3)-%&$+' "/ (%7 

a(24&$ c7 E$@"6$&B $//(@($)@B "/ '(@&"1$+#- () /($0# %&(+0-7 b)0$-- )"%$#5 /(0%$& 1+2-> '$-* 3+- 

9: ?'7 F04$5 &$#5 +)# 2&$$) 1+&- &$,&$-$)% :< ?'5 ;<< ?'5 +)# ;:< ?' '(@&"1$+#-5 &$-,$@%(6$0B7 

m f + )"%+10$ +'"4)% "/ 2&$$) +02+$ 3+- @"00$@%$# () /(0%$& 1+2-7 
;: 

F$@+4-$ '(@&"1$+# &$@"6$&B $//(@($)@($- ()@&$+-$# +/%$& (',&"6$'$)%- 3$&$ '+#$ %" %*$ 

,&"%"@"0 /"& %&(+0- () %*$ 0+1"&+%"&B 8(7$75 @"4)%()2 4-()2 %*$ 3$% '$%*"# +)# -$+0()2 %*$ /(0%$& 

1+2-> -$+'-= +)# %*$ /($0# 8(7$75 &$'"6()2 +02+$ /&"' %*$ &$-$&6"(&- 4-$# %" *"0# /&$-*3+%$&=5 %*$ 

#+%+ @"00$@%$# +/%$& (',&"6$'$)%- 3$&$ @"',(0$# %" 2(6$ + %&4$ ,(@%4&$ "/ '(@&"1$+# &$@"6$&B 

8a(24&$ :=7 \(%* %*$ $G@$,%(") "/ %*$ 0+1"&+%"&B %&(+0 4-()2 :< ?' '(@&"1$+#- () + :<I?' /(0%$& 

1+25 %*$ +6$&+2$ &$@"6$&B $//(@($)@B 3+- i QhT7 

4.2.3 Filter Bags—Seams 

L/ %*$ -$6$) -$+0+)%- $G+'()$#5 R+&()$ W"",S +,,$+&$# 1$-% /"& -$+0()2 %*$ /(0%$& 1+2-> -$+'- 

1$@+4-$ (% 3+- %*$ '"-% ,0(+10$ 14% )"% -%(@DB7 K/%$& 1$()2 4-$# () /($0# %$-%-5 *"3$6$&5 %*$ @0$+& 

-$+0+)% 1$2+) %" /0+D$ +,+&%5 +00"3()2 '(@&"1$+#- %" 1$@"'$ %&+,,$# () %*$ -$+0+)% +)# 

,"%$)%(+00B 4)@"6$&()2 %*$ &$0+%(6$0B 0+&2$ *"0$- +0")2 %*$ 1+2-> -$+'-7 ORZ :9<< 3+- &$I

$6+04+%$#5 +)# +/%$& 4-()2 (% () + /($0# %&(+0 +)# )"%(@()2 )" 1&$+D#"3)5 (% 3+- 4-$# () +00 

-41-$H4$)% $G,$&('$)%-7 [(D$ R+&()$ W"",5 (% 3+- +0-" ,0(+10$ 14% )"% -%(@DB5 +)# (% 3+- 3*(%$5 

-" &$# '(@&"1$+#- 3$&$ 6(-(10$ +0")2 %*$ -$+'-7 

4.2.4 Filter Bags—Toxicity of Sealant on the Seams 

!*$&$ 3+- )" +,,+&$)%5 )$2+%(6$ $//$@% "/ %*$ ORZ :9<< -$+0+)% ") Artemia franciscana "& 

+'1($)% @",$,"#-7 A) 1"%* %&(+0-5 () +00 &$,0(@+%$-5 %*$ ,$&@$)%+2$ "/ 0(6()2 "&2+)(-'- 3+- +% 0$+-% 

a(24&$ :7 E$@"6$&B $//(@($)@B "/ :< ?' '(@&"1$+#- () 0+1"&+%"&B +)# /($0# %&(+0- @")#4@%$# 

+/%$& (',&"6$'$)%- 3$&$ '+#$ %" %*$ '(@&"1$+# ,&"%"@"07 U4'1$&- () ,+&$)%*$-$- 

&$,&$-$)% &$,0(@+%$-N aF f /(0%$& 1+27 
;d 

h<T 8K,,$)#(G O=7 A) %*$ Artemia franciscana $G,$&('$)%5 1$@+4-$ %*$ #+%+ 3$&$ )"% #(-%&(14%$# 

)"&'+00B5 + R+))I\*(%)$B E+)D C4' !$-%- 3+- 4-$#5 +)# )" -(2)(/(@+)% #(//$&$)@$ 3+- /"4)# 

8, f <7ch=7 A) %*$ @",$,"# $G,$&('$)%5 + %I%$-% -*"3$# )" -(2)(/(@+)% #(//$&$)@$ 1$%3$$) 

%&$+%'$)% +)# @")%&"0 2&"4,- 8, f <7:c=7 \*$) +00 "/ %*$ A. franciscana &$-40%- 8/&"' 1"%* 

@")%&"0 +)# %&$+%'$)% 2&"4,-= 3$&$ +6$&+2$# ()%" + 2&+)# '$+) +)# @"',+&$# %" %*$ 2&+)# '$+) 

"/ %*$ @",$,"# &$-40%-5 %*$ )4'1$&- 3$&$ )$+&0B (#$)%(@+0 8hQ7OT +)# hQ7;T5 &$-,$@%(6$0B=7 

4.3 Filter Skid 

4.3.1 Filter Skid Design 

K/%$& ()6$-%(2+%()2 @"''$&@(+00B +6+(0+10$ /(0%&+%(") #$6(@$-5 %*$ X+%") !",0()$Z !B,$ AA 

*"4-()2 8X+%") a(0%&+%(")5 [[V5 A-$0()5 U_= 3+- @*"-$)7 !" @+,%4&$ ."",0+)D%") $//$@%(6$0B () 

0+&2$ 6"04'$- "/ 3+%$&5 %*$ /(0%$& -D(# 3+- #$-(2)$# 3(%* ;< @B0()#&(@+0 /(0%$& *"4-()2-5 $+@* 

@")%+()()2 + '$%+0 /(0%$& 1+-D$% 3(%* *"0$- O 'm (0.12”= () #(+'$%$& 8a(24&$ dN -@*$'+%(@ () 

K,,$)#(G c +)# ",$&+%(")- '+)4+0 () K,,$)#(G :=7 K- 3+%$& $)%$&$# %*$ -D(#5 (% 3+- -,0(% %" 

/0"3 ()%" ")$ "/ %3" ,&$/(0%$&-5 3*(@* @")%+()$# )" /(0%$& 1+2-5 14% %*$ '$%+0 /(0%$& 1+-D$%- -$&6$# 

+- ,&$/(0%$&- %" @"00$@% 0+&2$ "&2+)(-'-5 -$#('$)%5 +)# &4-% /0+D$- /&"' %*$ 1+00+-% %+)D7 

a"00"3()2 $+@* ,&$/(0%$&5 3+%$& /0"3 3+- -,0(% +2+()5 ()%" %3" /(0%$& *"4-()2-5 %*4- $6$)0B 

#(-%&(14%()2 %*$ 3+%$& /0"3 1$%3$$) %*$ *"4-()2- +)# &$#4@()2 %*$ /(0%&+%(") 0"+# /"& $+@* /(0%$& 

1B + /+@%"& "/ /"4&7 X+@* "/ %*$ /"4& *"4-()2- 3+- /"00"3$# 1B +)"%*$& /(0%$& *"4-()25 3*(@* 3+- 

()%$)#$# %" @+,%4&$ +)B%*()2 "&2+)(-'- %*+% '+B *+6$ 2")$ %*&"42* %*$ /(&-% -$% "/ *"4-()2-7 K00 

/(0%$& *"4-()2-5 $G@04#()2 %*$ ,&$/(0%$&-5 @")%+()$# + /(0%$& 1+2 #$-(2)$# %" @+,%4&$ +00 "&2+)(-'- 

2&$+%$& %*+) :<I?' '()('4' #('$)-(") () $+@* $G,$&('$)%7 K D$B /$+%4&$ "/ %*$ X+%") #$-(2) 

(- %*$ %(2*% -$+0 +'")2 %*$ /(0%$& 1+25 *"4-()25 +)# *"4-()2 @"6$&7 

a(24&$ d7 a(0%$& -D(# #$-(2) /"& /0"3I%*&"42* -+',0()2 "/ #(-@*+&2$# 1+00+-% 3+%$&7 
;P 

4.3.1.1 Filter Housings 

!*$ @B0()#&(@+0 X+%") !",0()$Z 1+2 /(0%$& *"4-()2-5 @")-%&4@%$# "/ @"&&"-(")I&$-(-%+)% O;d 

-%+()0$-- -%$$05 *+# #('$)-(")- "/ ;Q @m (7”= #(+'$%$& G h; @m (36”= 0")2 8a(24&$ PN #&+3()2 

@&$+%$# 4-()2 C"0(#\"&D- 9<;< O` `$-(2) C"/%3+&$5 V")@" RK=7 !*$ *$(2*% +)# "&($)%+%(") 

"/ %*$ *"4-()2- @"40# 1$ 6+&($# 4-()2 %*$(& -4,,"&% 0$2-7 

 

a(24&$ P7 X+%") !",0()$Z /(0%$& *"4-()27 

\+%$& $)%$&$# %*$ -(#$ "/ %*$ *"4-()2 %*&"42* + : @m (2”= /0+)2$# @"))$@%(") +)# /0"3$# "6$& 

%*$ %", "/ %*$ /(0%$& 1+25 -$+0()2 (% () ,0+@$7 !*(- #$-(2) &$-40%$# () + '()('+0 6"04'$ "/ 

4)/(0%$&$# 0(H4(# 8(7$75 1$%3$$) %*$ *"4-()2>- 0(# +)# %*$ %", "/ %*$ /(0%$& 1+2= +)# ,&"6(#$# 

",%('4' -$+0()2 "/ %*$ /(0%$& 1+2 -" + '()('+0 +'"4)% "/ 3+%$& 1B,+--$# %*$ /(0%$& 1+27 

K0%*"42* $+@* "/ %*$ X+%") !",0()$Z /(0%$& *"4-()2- +&$ '+)4/+@%4&$# /"& + '+G('4' /0"3 &+%$ 

"/ dQ; 0 'I; 8;Q< 2,'= +)# ;<5:<< 2 @'I9 8;:< ,"4)#- ,$& -H4+&$ ()@*5 ,-(= '+G('4' ,&$--4&$5 

%*$ /(0%$& -D(# 3+- #$-(2)$# -" $+@* "/ %*$ *"4-()2- 3"40# &$+0(.$ +,,&"G('+%$0B h: 0 '()I; 89: 

2,'= #4&()2 )"&'+0 ",$&+%(") 8-$$ -$@%(") O7O7; /"& &+%(")+0$=7 K##(%(")+00B5 %*$ '+)4/+@%4&$&>- 

",$&+%(") ()-%&4@%(")- ()#(@+%$# 0"3I/0"3 @")#(%(")- 0$+# %" 2""# /(0%&+%(") &$-40%-7 FB -,0(%%()2 

%*$ /0"3 /"4& 3+B-5 (% 3+- ,"--(10$ %" -0"3 6$0"@(%B %*&"42* %*$ -D(# 1B + /+@%"& "/ /"4&7 

K '+() @"',")$)% "/ %*$ /(0%$& -D(# (- %*$ /(0%$& 1+2- %*+% 3$&$ -)+,,$# ()%" ,0+@$ () %*$ $(2*% 

/(0%$& *"4-()2- +/%$& %*$ %3" ,&$/(0%$&-7 !*$ '+%$&(+0 "/ %*$ /(0%$& 1+2- 3+- @*"-$) %" 8;= '('(@ 

%*$ /(0%&+%(") $//(@($)@B "/ ,0+)D%") )$%-5 3*(@* +&$ @"''")0B 4-$# 1B %$-% /+@(0(%($-5 +)# 89= 

@+,%4&$ "&2+)(-'- +)# +00"3 %*$' %" 1$ 2$)%0B &$'"6$# /&"' %*$ /(0%$& 1+2 -4&/+@$ 3(%*"4% 

()@&$+-()2 '"&%+0(%B7 a$0%5 '(@&"/(1$&5 +)# '")"/(0+'$)% '$-*$- 3$&$ @")-(#$&$#5 +)# )B0") 

'")"/(0+'$)% /(0%$& 1+2- 1$-% '$% %*$ @&(%$&(+ 8X+%") ,+&% )4'1$& FURLO:Y9Y5 a(24&$ QN %*$ 

/(0%$& 1+2>- ,"0B,&",B0$)$ &()2 (- -)+,,$# ()%" ,0+@$ +% %*$ %", "/ %*$ /(0%$& *"4-()27 !*$ O '' 

*"0$- () %*$ '$%+0 0()$& +&$ 6(-(10$ %*&"42* %*$ /(0%$& 1+2>- '$-*=7 
;Q 

a(24&$ Q7 UB0") '")"/(0+'$)% '$-* /(0%$& 1+2 -$@4&$# () +) X+%") !",0()$Z /(0%$& *"4-()27 

4.3.1.2 Piping, Valves, and Diaphragm Pump 

K00 ,(,()2 1$%3$$) %*$ /(0%$& *"4-()2- 3+- : @' 82”= #(+'$%$&5 C@*$#40$ c<5 Y^V 8C,$+&-S 

R+)4/+@%4&()2 V"',+)B5 CB0'+&5 VK=5 3(%* 204$# /(%%()2- 8a(24&$ h=7 Y(,()2 3+- @"))$@%$# %" 

%*$ /(0%$& *"4-()2- 4-()2 : @' 82”= #(+'$%$& dQ D2 8;:< 01= ^+) C%")$ /0+)2$- 3(%* $%*B0$)$ 

,&",B0$)$ #($)$ R")"'$& 8RI@0+--= &411$& 8XY`R= 2+-D$%-7 

a(24&$ h7 Y0+) 6($3 "/ %*$ ,&"%"%B,$ /(0%$& -D(# +% UE[e\7 F04$ +&&"3 -*"3- 3+%$& $)%$&()2 

%*$ /(0%$& -D(#N B$00"3 +&&"3 -*"3- 3+%$& $G(%()2 %*$ -D(#7 
;h 

!" +00"3 /(0%$& *"4-()2- %" 1$ ()#(6(#4+00B (-"0+%$#5 : @' (2”= #(+'$%$& 6+06$- 3$&$ /(%%$# %" %*$ 

()0$% +)# "4%0$% "/ $+@* /(0%$& *"4-()2 8C,$+&-S R+)4/+@%4&()2 V"',+)B5 '"#$0 V"',+@% 9<<< 

1+00 6+06$-=7 F$@+4-$ +00 (-"0+%(") 6+06$- 3$&$ 0$/% () %*$ ;<<T ",$) ,"-(%(") #4&()2 ",$&+%(")5 

%*$B #(# )"% +## -*$+& /"&@$- %" %*$ "&2+)(-'- #4&()2 -+',0()27 

K% %*$ 1+-$ "/ $6$&B /(0%$& *"4-()25 + %*&$+#$# ;7O @m (0.5”= #(+'$%$& Y^V 1+00 6+06$ 8C,$+&-S 

R+)4/+@%4&()2 V"',+)B= 3+- ()-%+00$# %" -$&6$ +- + #&+() 6+06$7 !*$ 6+06$- &$'+()$# @0"-$# 

#4&()2 /(0%&+%(") ",$&+%(")- 14% 3$&$ ",$)$# +% %*$ $)# "/ + &4) %" #&+() %*$ /(0%$& *"4-()2- /&"' 

%*$ 1"%%"'7 A) %*(- '+))$&5 /(0%$& 1+2- 3$&$ &$'"6$# /&"' %*$ *"4-()2- 3(%*"4% %*$ 1"%%"'- "/ 

%*$ /(0%$& 1+2- /0"+%()2 4,3+&#- +)# ,"%$)%(+00B 0"-()2 ,+&% "/ %*$ -+',0$7 

K %*&$+#$#5 <7dc @' 8<795”= #(+'$%$& Y^V 1+00 6+06$ 3+- ()-%+00$# +%", "/ $+@* /(0%$& *"4-()25 

+)# (% 3+- 4-$# %" '+)4+00B 10$$# +(& %&+,,$# 3(%*() %*$ *"4-()2- ,&("& %" 4-$7 !*(- (',"&%+)% 

-+/$%B /$+%4&$ ,&$6$)%$# +(& /&"' 1$()2 %&+,,$# 3(%*() /(0%$& *"4-()2- +)# ,"%$)%(+00B 14(0#()2 %" 

-$6$&+0 %('$- 2&$+%$& %*+) +%'"-,*$&(@ ,&$--4&$7 !*(- /$+%4&$ +00"3$# %*$ -B-%$' ",$&+%"& %" 

D)"3 3*$) %*$ /(0%$& *"4-()2- 3$&$ /400 "/ 3+%$& 8(7$75 3+%$& $G(%- %*$ 6+06$-=5 %*$&$1B $)-4&()2 

%*$ /(0%$& 1+2- 3$&$ 3$% ,&("& %" -+',0()27 

K n+'+#+ U`YIQ< Q @' 83”= #(+,*&+2' ,4', 8n+'+#+S K'$&(@+5 A)@75 K&0()2%") M$(2*%-5 A[= 

3+- ()-%+00$# %" #&+3 + -+',0$ /&"' %*$ #(-@*+&2$ 0()$ $G(%()2 %*$ 1+00+-% %+)D ()%" %*$ /(0%$& -D(# 

8a(24&$ ;<=7 !*$ ,4', 3+- )$$#$# 1$@+4-$ %*$ #(-@*+&2$ 0()$ 3+- 4)#$& -4@%(") 8+- 3+%$& 3+- 

,4',$# %" + #(-@*+&2$ %+)D /"& /(0%&+%(") ,&("& %" #(-@*+&2$ ()%" +'1($)% 3+%$&-=N %*$&$/"&$5 %" 

@"00$@% + &$,&$-$)%+%(6$ -+',0$ "/ %*$ 0()$5 + #(+,*&+2' ,4', 3+- 1$$) ()-%+00$#7 A% 3+- 

@")-%&4@%$# "/ ,"0B,&",B0$)$ 3(%* +) XY`R &411$& #(+,*&+2' () @")%+@% 3(%* %*$ 3+%$& ,+--()2 

%*&"42* %*$ ,4',7 Y&$6("4- %$-%()2 1B $)2()$$&- +% UE[e\ ") 6+&("4- ,4', %B,$- ") 

"&2+)(-' '"&%+0(%B *+- -*"3) #(+,*&+2' ,4',- %" 1$ %*$ -4,$&("& @*"(@$ 8E(0$B $% +075 9<<h=7 

K0%*"42* (% 3+- @+,+10$ "/ /0"3()2 +% + &+%$ "/ 99< 2,'5 %*$ ,4', 3+- -$% +% ;<< 2,' +)# 

",$&+%$# 1B @"',&$--$# +(& -4,,0($# /&"' + ,"&%+10$ C400+(&S ;Q:CVaR +(& @"',&$--"& 

8R(@*(2+) V(%B5 AU=7 K %('$I+6$&+2$# -+',0$ 3+- &$'"6$# /&"' %*$ #(-@*+&2$ 0()$ /&"' %*$ 

1+00+-% %+)D %" %*$ 0()$ ()%" %*$ /(0%$& -D(# 4-()2 + %$$N +) (-"D()$%(@ -+',0$ ,"&% 3+- )"% 

)$@$--+&B5 +- %*$ -+',0$ 6"04'$ 3+- 2&$+%$& %*+) ;<T "/ %*$ %+)D 6"04'$ 8(7$75 d< 'O "/ 9<< 'ON 

E(@*+&# $% +075 9<<Q=7 
9< 

a(24&$ ;<7 n+'+#+ U`YIQ< K(& #(+,*&+2' ,4', +- ()-%+00$#7 

 

4.3.1.3 Pressure Drop Calculations vs. Flow Rate 

R+()%+()()2 + 0"3 ,&$--4&$ #&", +@&"-- + /(0%$& 1+2 (- @&(%(@+0 1$@+4-$ (% &$#4@$- %*$ @*+)@$- 

"&2+)(-'- +&$ D(00$# #4&()2 -+',0()25 +)# (% $)*+)@$- /(0%&+%(") $//(@($)@B +)# ()@&$+-$- %*$ 

-$&6(@$ 0(/$ "/ %*$ /(0%$& 1+25 %*4- &$#4@()2 %*$ ",$&+%()2 @"-% "/ %*$ -B-%$'7 `4&()2 %*$ #$-(2) 

,*+-$5 %*$ /(0%$& -D(# 3+- +)+0B.$# %" #$%$&'()$ 3*$&$ %*$ 140D "/ %*$ /&(@%(") 0"--$- "@@4&&$#7 

`+%+ /"& %*$ /(0%$& *"4-()2- +)# /(0%$& 1+2- 3$&$ +6+(0+10$ /&"' %*$ '+)4/+@%4&$& 8a(24&$ ;;=7 

!*$ +)+0B-(- 3+- @"',0$%$# 4-()2 #+%+ /&"' 9: ?' /(0%$& 1+2-5 -()@$ #+%+ /"& O: ?' )B0") 

'")"/(0+'$)% /(0%$& 1+2- 3$&$ 4)+6+(0+10$7 K- $G,$@%$#5 %*$ ,&$--4&$ #&", ()@&$+-$# +- %*$ /0"3 

&+%$ ()@&$+-$#7 K% + /0"3 &+%$ "/ h: 0 '()I; 89: 2,'=5 %*$ ,&$--4&$ #&", +@&"-- +) ()#(6(#4+0 

*"4-()2 3(%* + 9: ?' )B0") '")"/(0+'$)% 1+2 3+- -0(2*%5 +,,&"G('+%$0B O: 2 @'I9 8<7: ,-(=7 A% 

(- +)%(@(,+%$# %*$ ,&$--4&$ #&", 3"40# 1$ + 1(% -'+00$& 4-()2 + O:I?' /(0%$& 1+27 
9; 

a(24&$ ;;7 Y&$--4&$ #&", "/ /(0%$& -D(# 6-7 /0"3 &+%$ 8/(24&$ /&"' X+%") a(0%&+%(")5 [[V=7 

F$@+4-$ %*$ -D(# 3+- #$-(2)$# 3(%* + -'+00 /""%,&()%5 %*$&$ 3$&$ -$6$&+0 $01"3- +)# /(%%()2-5 +- 

3$00 +- /0"3 #(&$@%(") @*+)2$-5 3*(@* @")%&(14%$# %" %*$ ,&$--4&$ #&", +@&"-- %*$ -D(#7 \*$) +00 

,&$--4&$ @*+)2$- 3$&$ @+0@40+%$#5 %*$ ,&$--4&$ #&", +@&"-- %*$ $)%(&$ -D(# 3+- dO; 2 @'I9 8h ,-(= 

+% OPh 0 '()I; 8;<< 2,'N @+0@40+%(")- )"% -*"3)=7 

4.3.1.4 Flow Rate, Flow Control, and Volume Measurement through the Filter 

Skid 

!*$ /0"3 &+%$ 3+- '")(%"&$# 4-()2 + Wa C(2)$% 9::; R+2'$%$&5 3*(@* 3+- -(%4+%$# ") %*$ ()0$% 

0()$ %" %*$ -D(# 4-()2 + -+##0$ /(%%()27 !*$ %"%+0 6"04'$ -+',0$# 3+- @+0@40+%$# /&"' + %"%+0(.$& 

()-%+00$# #"3)-%&$+' "/ %*$ -D(# %*+% 4-$# %*$ $0$@%&(@+0 -(2)+0 2$)$&+%$# 1B %*$ R+2'$%$& %" 

@+0@40+%$ %*$ %"%+0 6"04'$ 1B '40%(,0B()2 %*$ /0"3 &+%$ 1B %('$ +)# -4''()2 (% "6$& %*$ $)%(&$ 

%$-% &4) F$@+4-$ %*$ -+',0()2 3+- + @")%()4"4- ",$&+%(")5 &+%*$& %*+) + 1+%@* ,&"@$--5 %*$ 

%"%+0(.$& 3+- 4-$# %" -4' %*$ /0"3 &+%$7 a0"3 3+- @")%&"00$# %*&"42* %*$ /(0%$& -D(# 4-()2 + 

0()$+&5 ,)$4'+%(@I+@%4+%$#5 P @m (3”= #(+'$%$& #(+,*&+2' 6+06$7 A) %4&)5 6+06$ ,"-(%(") 3+- 

@")%&"00$# 4-()2 Y&","&%(")+05 A)%$2&+05 `$&(6+%(6$ 8YA`= /$$#1+@D @")%&"0 6(+ M")$B3$00 

@")%&"0- 0"2(@ 1+-$# ") %*$ R+2'$%$& &$+#()2 %" '+()%+() + /0"3 "/ ;<< 2,' %*&"42* %*$ -D(#7 

 

y = 0.0194x

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 50 100 150 200 250

Pressure Drop (psi)

Flow Rate (GPM)

Eaton Topline Filter Housing Pressure Drop (psi) vs. Flow (gpm)

Housing pressure drop (psi)

clean filter pressure drop for 25um NMO (psi)

Total pressure drop across housing (psi)

Linear (Total pressure drop across housing (psi))

y = 0.0194x

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 50 100 150 200 250

Pressure Drop (psi)

Flow Rate (GPM)

Eaton Topline Filter Housing Pressure Drop (psi) vs. Flow (gpm)

Housing pressure drop (psi)

clean filter pressure drop for 25um NMO (psi)

Total pressure drop across housing (psi)

Linear (Total pressure drop across housing (psi))
99 

4.3.1.5 Cost and Weight of the Filter Skid 

\*$) %*$ @"-% "/ %*$ '+%$&(+0- +)# 0+1"& %" 14(0# %*$ /(0%$& -D(# 3+- %+140+%$#5 %*$ %"%+0 @"-% "/ %*$ 

,&"%"%B,$ 3+- o;h5Q:;5 +)# (% %""D +,,&"G('+%$0B d< *"4&- %" @")-%&4@% (% 8K,,$)#(G d=7 

!*$ -D(# 3+- +)+0B.$# %" #$%$&'()$ (%- 3$(2*% 3(%* +)# 3(%*"4% 3+%$&7 b-()2 %*$ &$-,$@%(6$ 

3$(2*% "/ $+@* "/ %*$ -D(#>- @"',")$)%- /&"' %*$ '+)4/+@%4&$&>- 0(%$&+%4&$5 %*$ 3$(2*% "/ %*$ 

-D(# 3(%*"4% 3+%$& 3+- $-%('+%$# %" 1$ 99Q D2 8:<O 01=N 3*$) -$+3+%$& (- @")%+()$# () +00 "/ %*$ 

*"4-()2- +)# %*$ ,(,()2 -B-%$'5 %*$ %"%+0 3$(2*% "/ %*$ -D(# 3+- +) $-%('+%$# ::d D2 8;99: 01=7 

4.3.2 Filtration Area of Filter Skid vs. Plankton Net 

!*$ $//$@%(6$ -4&/+@$ +&$+ "/ %*$ /(0%$& -D(# 3+- @"',+&$# %" %*+% "/ %B,(@+0 ,0+)D%") )$%7 !*$ 

$//$@%(6$ -4&/+@$ +&$+ "/ + -()20$ /(0%$& 1+2 3(%*() %*$ /(0%$& -D(# 3+- <7cO '9 8@+0@40+%(")- )"% 

-*"3)=7 F$@+4-$ %*$ -+',0$ /0"3 3+- -,0(% ()%" /"4& /(0%$& *"4-()2- () ,+&+00$05 %*$ $//$@%(6$ 

-4&/+@$ +&$+ (- /"4& %('$- %*$ -4&/+@$ +&$+ "/ ")$ *"4-()25 "& ;7P< '97 !*$ -4&/+@$ +&$+ @"40# 

$+-(0B 1$ ()@&$+-$# 1B ()@&$+-()2 %*$ )4'1$& "/ /(0%$& *"4-()2- +&&+)2$# () ,+&+00$07 

A) %*$ ,+-%5 + C$+IW$+& R"#$0 h<<< Y0+)D%") U$% 3(%* + '"4%* ",$)()2 "/ d< @' +)# + 0$)2%* %" 

'"4%* &+%(" "/ OJ; 3+- 4-$# +% UE[e\ 8C$+IW$+& V"&,"&+%(")5 R$01"4&)$5 a[=7 A%- -4&/+@$ 

+&$+ 3+- ;7P9 '95 )$+&0B (#$)%(@+0 %" %*$ /(0%$& -D(# 8@+0@40+%(")- )"% -*"3)=7 

4.3.3 Flow Velocity through the Filter Skid vs. Plankton Net 

!*$ /(0%$& -D(# 3+- #$-(2)$# %" "1%+() #(-@*+&2$ 3+%$& -+',0$- +% + 6"04'$%&(@ 3+%$& /0"3 &+%$ "/ 

;<< 2,'5 +)# %*$ /0"3 %*&"42* %*$ -D(# 3+- ,04'1$# %" -,0(% ()%" + -$% "/ ,&$I/(0%$&- +% + 

6"04'$%&(@ /0"3 &+%$ "/ :< 2,'7 K/%$& %*$ %3" ,&$I/(0%$&-5 %*$ /0"3 -,0(% +2+() %" /0"3 ()%" + -$% 

"/ /"4& /(0%$& *"4-()2- %*+% @")%+()$# O:?' /(0%$& 1+2- %" @+,%4&$ "&2+)(-'- i :< ?'7 !*$ 

6"04'$%&(@ /0"3 &+%$ %" %*$-$ /"4& *"4-()2- 3+- 9: 2,'7 !*$ /0"3 6$0"@(%B "/ %*$ 3+%$& +- (% 

/0"3- %*&"42* %*$ ,&$I/(0%$&- +)# /(0%$&- 3+- @+0@40+%$# +)# @"',+&$# %" 6$0"@(%($- $)@"4)%$&$# () 

%*$"&$%(@+05 *"&(.")%+0 ,0+)D%") %"3- 4-()2 ,0+)D%") )$%-7 

4.3.3.1 Flow Velocity through Two Prefilters (No Filter Bags) 

\+%$& 3+- -4,,0($# %" %*$ /(0%$& *"4-()2- %*&"42* + : @' 89”= ())$& #(+'$%$& Y^V ,(,$7 !*$ 

/0"3 6$0"@(%B "/ %*$ 3+%$& $)%$&()2 %*$ ,&$I/(0%$& *"4-()2- (- ;7:: ' -I; 8O7: ',*= 8K,,$)#(G P5 

XH4+%(") ;=7 

K- %*$ 3+%$& $)%$&- %*$ /(0%$& *"4-()25 %*$ /0"3 6$0"@(%B (- &$#4@$# 1$@+4-$ %*$ @&"-- -$@%(")+0 

+&$+ ()@&$+-$- /&"' %*$ : @' 82”= ,(,$ %" %*$ ;Q @' 87”= *"4-()2 8a(24&$ ;9N '"#$0 @&$+%$# 4-()2 

C"0(#\"&D-=7 V"&&$-,")#()20B5 %*$ /0"3 6$0"@(%B #$@&$+-$- /&"' ;7:: ' -I; 8O7cP ',*= %" <7:; 

' -I; 8;7;O ',*N K,,$)#(G P XH4+%(") 9=7 K- %*$ 3+%$& 0$+6$- %*$ *"4-()25 %*$ /0"3 6$0"@(%B 

&$%4&)- %" ;7:: ' -I; 8O7cP ',*=7 
9O 

a(24&$ ;97 a0"3 6$0"@(%B '"#$0 /"& 3+%$& /0"3 %*&"42* +) X+%") !",0()$Z a(0%$& M"4-()2 +% :< 

2,'5 $)%$&()2 %*"42* %*$ %", "/ %*$ *"4-()2 +)# $G(%()2 +% %*$ 1"%%"'7 

4.3.3.2 Flow Velocity through Housings with Filter Bags 

`"3)-%&$+' "/ %*$ ,&$I/(0%$&-5 %*$ 3+%$& /0"3 -,0(% %" $)%$& %*$ )$G% /"4& *"4-()2-5 3*(@* 

@")%+()$# /(0%$& 1+2-7 !*$ 6"04'$%&(@ /0"3 &+%$ %" $+@* "/ %*$-$ /(0%$& *"4-()2- (- 9: 2,'7 !*$ 

/0"3 6$0"@(%B $)%$&()2 %*$-$ /(0%$&- (- <7PP ' -I; 8;7P ',*N K,,$)#(G P5 XH4+%(") =7 

K- -$$) () %*$ /(&-% -$% "/ ,&$I/(0%$&-5 +- %*$ 3+%$& $)%$&- ()%" %*$ *"4-()25 %*$ /0"3 6$0"@(%B 

#$@&$+-$- 1$@+4-$ %*$ @&"-- -$@%(")+0 +&$+ ()@&$+-$- /&"' : @' %" ;Q @' 89” to 7”, a(24&$ ;O=7 

M$&$5 %*$ /0"3 6$0"@(%B -0"3- /&"' <7PP ' -I; 8;7P9 ',*= %" <79: ' -I; 8<7:d ',*N %*$ 6$0"@(%B "/ 

%*$ 3+%$& 3*$) @")%+@%()2 %*$ /(0%$& 1+2-N K,,$)#(G P5 XH4+%(") c=7 K- %*$ 3+%$& 0$+6$- %*$ 

*"4-()25 (% &$%4&)- %" <7PP ' -I;7 
9c 

a(24&$ ;O7 a0"3 6$0"@(%B '"#$0 (004-%&+%(") /"& 3+%$& /0"3 %*&"42* +) X+%") !",0()$Z a(0%$& 

M"4-()2 +% 9: 2,'5 $)%$&()2 %*"42* %*$ %", "/ %*$ *"4-()2 +)# $G(%()2 +% %*$ 1"%%"'7 

4.3.3.3 Flow Velocity through a Plankton Net 

!*$ /0"3 6$0"@(%B "/ 3+%$& $)%$&()2 + ,0+)D%") )$% +- (% (- %"3$# 1$*()# + 1"+% 3+- @+0@40+%$#7 

a"& %*(- $G+',0$5 (% 3+- +--4'$# %*$ 6$0"@(%B "/ %*$ 1"+% #4&()2 -+',0()2 3+- <7:; I O7; ' -I; 8; 

I d D%-N $7275 K&")5 ;hd:=5 %*$ '"4%* #(+'$%$& "/ %*$ )$% 3+- ; '5 +)# %*$ $G(% #(+'$%$& )$+& %*$ 

@"# $)# 3+- ;< @'7 F$@+4-$ "/ (%- @")(@+0 -*+,$5 %*$ )$% %$)#- %" @")@$)%&+%$ "&2+)(-'- -%&+()$# 

/&"' %*$ 3+%$& -+',0$ () %*$ @"# $)# +)# 4-$- -4&/+@$ /(0%&+%(") %" -$,+&+%$ "&2+)(-'- +)# 

,+&%(@0$- i O: ?'7 !*$ $)%&+)@$ 6$0"@(%($- "/ %*$ 3+%$& $)%$&()2 %*$ ,0+)D%") )$% /"& -,$$#- ; D%5 

9 D%-5 +)# d D%- +&$ <7:; ' -I;5 ;7<O ' -I;5 +)# O7; ' -I;5 &$-,$@%(6$0B7 !*$ 6$0"@(%B "/ 3+%$& 

$)%$&()2 %*$ /(0%&+%(") *"4-()2- () %*$ /(0%$& -D(# 8<7PP ' -I;5 K,,$)#(G P5 XH4+%(") = (- -('(0+& %" 

+ *"&(.")%+0 ,0+)D%") %"3 1$%3$$) <7:; ' -I; I ;7<O ' -I; 8; I 9 D%-=7 

4.3.4 Toxic Effects of Stainless Steel Filter Housings 

C%+()0$-- -%$$0 #(# )"% ()#4@$ '"&%+0(%B ") ;9I*"4& "0# Artemia franciscana )+4,0(( "& +'1($)% 

."",0+)D%") 8i :<?'= "6$& + 97: * $G,"-4&$ %('$5 +- %*$&$ 3+- )" -(2)(/(@+)% #(//$&$)@$ 1$%3$$) 

%*$ ,$&@$)%+2$ "/ 0(6()2 "&2+)(-' () %*$ @")%&"0 +)# %&$+%'$)% 2&"4,- 8K,,$)#(G QN %I%$-% , f 

<7:h /"& A. franciscana5 , f <7OO /"& ."",0+)D%")=7 K0%*"42* %*$ 2&+)# '$+) /&"' +00 A.

franciscana '$+-4&$'$)%- 81"%* @")%&"0 +)# %&$+%'$)% 2&"4,-= 3+- 2&$+%$& %*+) %*$ 2&+)# '$+) 
9: 

/&"' +00 +'1($)% ."",0+)D%") 8hh7;T 6-7 QP7QT=5 %*$&$ 3+- )" -%+%(-%(@+0 #(//$&$)@$ 1$%3$$) 

%*$' 8R+))I\*(%)$B E+)D C4' !$-%5 , f <7;:=7 

4.3.5 Comparison of Zooplankton in the Ballast Tank vs. the Filter Skid 

K -+',0$ @"00$@%$# 4-()2 + ,0+)D%") )$% %"3 /&"' + 1+00+-% %+)D *"0#()2 9<< 'O

 "/ +'1($)% 

-$+3+%$& 3+- @"',+&$# %" + %('$I+6$&+2$#5 d<I'O -+',0$ @"00$@%$# +- %*$ %+)D 3+- #&+()$#7 !*$ 

,$&@$)%+2$ "/ 0(6()2 ."",0+)D%") 3+- )$+&0B (#$)%(@+0 8K,,$)#(G h=7 !*$ '$+) ,$&@$)%+2$ "/ 

0(6()2 @",$,"# )+4,0(( 3+- hQ7OT +)# hP7QT 8,0+)D%") %"3 +)# /(0%$& -D(#5 &$-,$@%(6$0B=5 +)# /"& 

@",$,"# +#40%- +)# @",$,"#(%$-5 (% 3+- Qc79T +)# QO7PT 8,0+)D%") %"3 +)# /(0%$& -D(#5 

&$-,$@%(6$0B=7 

4.3.6 Effect of Crowding on Ambient Zooplankton 

\*$) +,,&"G('+%$0B d<< @",$,"#- 8Acartia tonsa= "& ;< @",$,"#- 3$&$ +##$# %" ; 0 "/ +&%(/(@(+0 

-$+3+%$& +)# ()@41+%$# /"& c*5 %*$ ,$&@$)%+2$ "/ 0(6()2 @",$,"#- 3+- l h<T () $+@* %&$+%'$)% 

/"& 1"%* %&(+0-J hQ7<T +)# ;<<T 8d<<5 ;< A. tonsa= +)# hP7:T +)# h<T 8d<<5 ;< A.tonsaN #+%+ 

)"% -*"3)=7 

5 Discussion 

K /(0%$& -D(# 3+- -4@@$--/400B #$-(2)$# %" -+',0$ 0+&2$ 6"04'$- "/ 3+%$& %" #$%$&'()$ (/ (%- 

."",0+)D%") @")@$)%&+%(") '$$%- + 1+00+-% 3+%$& #(-@*+&2$ -%+)#+&# "/ p ;< "&2+)(-'- 'IO7 

b-()2 "//I%*$I-*$0/ @"',")$)%-5 (% 3+- 14(0% %" '$$% %*$ &$H4(&$'$)%- "/ *+6()2 + 0"3 3+%$&I/0"3 

&+%$5 -$0/I@")%+()$# )$%-5 +)# + -'+00 /""%,&()%7 V+0@40+%(")- "/ %*$ /(0%&+%(") -4&/+@$ +&$+ +)# 

/0"3 6$0"@(%B -*"3 %*$ -D(# (- @"',+&+10$ %" + -%+)#+&# ,0+)D%") )$%5 +)# %*$ )4'1$& "/ /(0%$& 

*"4-()2- () %*$ -D(# @"40# 1$ ()@&$+-$# %" +00"3 '"&$ -4&/+@$ +&$+7 !*$ %&+#$ "//5 "/ @"4&-$5 (- + 

2&$+%$& *+)#0()2 %('$ "/ %*$ -+',0$5 +- '"&$ /(0%$& 1+2- 3"40# &$H4(&$ &()-()27 

A)(%(+0 %&(+0- 4-()2 ."",0+)D%") ,&"G($- 8'(@&"1$+#- :< ?' () #(+'$%$&= %" 6+0(#+%$ /(0%$& 1+2-> 

$//(@($)@($- &$6$+0$# (--4$- %*+% 3$&$ +##&$--$#J %*$ '$%*"# /"& @"4)%()2 '(@&"1$+#- 3+- 

(',&"6$#5 %*$ /(0%$& 1+2-> -$+'- 3$&$ -$+0$# %" ,&$6$)% '(@&"1$+#- /&"' -0(,,()2 %*&"42* %*$ 

*"0$-5 +)# %*$ -'+00 +'"4)% "/ +02+$ /"4)# () *"0#()2 %+)D- 4-$# () /&$-*3+%$& /($0# %&(+0- 3+- 

&$'"6$# %" $)-4&$ +00 '(@&"1$+#- 3$&$ 6(-(10$ 3(%*() -+',0$-7 !*$ 0+%%$& ,"()% (004-%&+%$- %*$ 

(',"&%+)@$ "/ $',0"B()2 /04"&$-@$)% -%+()- "& '"6$'$)% "& 1"%* %" H4+)%(/B ."",0+)D%")7 

L%*$&3(-$5 ."",0+)D%") '+B 1$ 4)#$&@"4)%$# 3*$) -'+00 "&2+)(-'- +&$ "1-@4&$# 1B #$+# 

"&2+)(-'-5 -$#('$)%5 +)# #$1&(- () -+',0$-7 K0%*"42* )"% +##&$--$# () %*(- -%4#B5 %*$ -+'$ (- 

%&4$ "/ %*$ ,&"%(-% 8i ;< ?' +)# p :< ?'= -(.$ @0+--7 A) %*$ $)#5 '(@&"1$+#- +,,&"G('+%()2 %*$ 

0"3$& $)# "/ %*$ ."",0+)D%") -(.$ @0+-- @"40# 1$ &$@"6$&$# /&"' /(0%$& 1+2- () /($0# +)# 

0+1"&+%"&B %&(+0- 3(%* 2""# $//(@($)@B5 i QPT7 

!" +##&$-- @")@$&)- %*+% %*$ -$+0+)% 4-$# %" /4-$ %*$ /(0%$& 1+2-> -$+'- +)# %*$ -%+()0$-- -%$$0 "/ 

%*$ /(0%$& *"4-()2- '+B 1$ %"G(@ %" ,0+)D%") +- %*$B +&$ -$H4$-%$&$# 8+01$(% /"& + -*"&% %('$= () %*$ 
9d 

/(0%$& 1+2- 3(%*() %*$ *"4-()2-5 -*"&%I%$&' %"G(@(%B %$-%- 3$&$ @")#4@%$#7 F"%* %"G(@(%B %$-%- 

$G,"-$# %*$ %$-% "&2+)(-' Artemia franciscana +)# +'1($)% ."",0+)D%$&- %" %*$ ,"%$)%(+0 

%"G(@+)%7 U" (''$#(+%$ "& +,,+&$)% )$2+%(6$ $//$@% ") %*$ @&4-%+@$+)- 3+- $6(#$)%7 A) $+@* 

()-%+)@$5 )" -(2)(/(@+)% #(//$&$)@$ 1$%3$$) %&$+%'$)% "& @")%&"0 2&"4,- 3+- #$%$@%$#7 

^+0(#+%()2 )$%-> "& /(0%$&-> &$%$)%(") $//(@($)@($- (-5 +- /+& +- 3$ D)"35 +) 4)@"''") ,&+@%(@$5 

1"%* () "@$+)"2&+,*(@ &$-$+&@* () 2$)$&+05 +)# () 1+00+-% 3+%$& %&$+%'$)% %$-%()2 -,$@(/(@+00B7 

E$2+&#()2 %*$ 0+%%$& @+-$5 (% -$$'- $-,$@(+00B &$0$6+)% 2(6$) %*$ ,"%$)%(+0 /"& /()$- %" 1$ 0$6($# 

3*$) 1+00+-% 3+%$& '+)+2$'$)% -B-%$'- $G@$$# + #(-@*+&2$ -%+)#+ R(@&"1$+#- +&$ )"% + 

,$&/$@% ,&"GB /"& 0(6()2 "&2+)(-'-5 3*(@* '+B 1$ -H4$$.$# %*&"42* + )$% '"&$ &$+#(0B %*+) 

,"0B-%B&$)$ -,*$&$-7 !*$ '(@&"1$+#- #"5 *"3$6$&5 &$,&$-$)% + 2""# '$%&(@5 +- %*$B @+) 1$ 

,4&@*+-$# +% %*$ 0"3$& -(.$ @0+-- 8$7275 3(%* + #(+'$%$& "/ :< ?'= +)# *+6$ )" -,()$- "& -$%+$ %" 

@0()2 %" + )$% "& /(0%$&7 K 0"2(@+0k+)# )$@$--+&Bk)$G% -%$, (- %" @"',+&$ &$%$)%(") $//(@($)@($- 

"/ )+%4&+0 +H4+%(@ @"''4)(%($- 1$%3$$) %*$ /(0%$& -D(# +)# ,0+)D%") )$%-7 !*"-$ %&(+0- +&$ 

4)#$&3+B +% UE[e\7 

6 Acknowledgements 

\(00(+' jF_> e()$$ +)# F+&&") C%&()2*+' *$0,$# @")-%&4@% %*$ /(0%$& -D(# +)# &4) /($0# %&(+0-5 +)# 

3$ +&$ 2&+%$/40 /"& %*$(& +--(-%+)@$7 \$ +,,&$@(+%$ +)# +@D)"30$#2$ %*$ -4,,"&% "/ %*$ bC V"+-% 

W4+&# X)6(&")'$)%+0 C%+)#+&#- `(6(-(") 8VWI:99c= /"& /4)#()2 %*(- &$-$+&@*7 

7 References 

K&") \5 K*0-%&"' XM5 F+&B FR@e5 Fq K\M5 V0+&D$ \` 8;hd:= !"3()2 @*+&+@%$&(-%(@- "/ 

,0+)D%") -+',0()2 2$+&7 [(')"0 L@$+)"2& OJOOIOc< 

a$#$&+0 E$2(-%$& 89<<h= C%+)#+&#- /"& [(6()2 L&2+)(-'- () C*(,-r F+00+-% \+%$& `(-@*+&2$# () 

 b7C7 \+%$&-N `&+/% Y&"2&+''+%(@ X)6(&")'$)%+0 A',+@% C%+%$'$)%5 Y&","-$# E40$ +)# 

U"%(@$5 Pc aE ccdO;IccdP9 89Q K424-% 9<<h=7 U+%(")+0 K&@*(6$- +)# E$@"&#- 

K#'()(-%&+%(")5 \+-*()2%")5 `V 

A)%$&)+%(")+0 R+&(%('$ L&2+)(.+%(") 89<<c= V")6$)%(") F\RsVLUasOd jA)%$&)+%(")+0 

V")6$)%(") /"& %*$ V")%&"0 +)# R+)+2$'$)% "/ C*(,-> F+00+-% \+%$& +)# C$#('$)%-5 9<<c> 

A)%$&)+%(")+0 R+&(%('$ L&2+)(.+%(") 89<<:= E$-"04%(") RXYV7;9:8:O= jW4(#$0()$- /"& K,,&"6+0 

"/ F+00+-% \+%$& R+)+2$'$)% CB-%$'- 8WQ=> 

[$'($4G X_5 W&+)% _5 \($& !5 `&+D$ [5 E"11()- C5 F4&)- e7 A) &$6($37 W$)$&(@ Y&"%"@"0 /"& %*$ 

^$&(/(@+%(") "/ F+00+-% \+%$& !&$+%'$)% !$@*)"0"2($-5 ^$&-(") c797 C41'(%%$# %" %*$ XYK 

X)6(&")'$)%+0 !$@*)"0"2B ^$&(/(@+%(") Y&"2&+' 

[$'($4G5 X_5 W&+)% _5 \($& !5 E"11()- C5 E(0$B C5 MB0+)# \5 `+6(- [5 `")"3(@D !5 C%&()2*+' 

F5 e()$$ \_5 F&"3) F5 X6$&$%% E 89<<Q+= Y(0"% X)6(&")'$)%+0 !$@*)"0"2B ^$&(/(@+%(") 
9P 

8X!^= !$-% E$,"&% "/ %*$ C$6$&) !&$)% `$U"&+ F+0Y4&$!R F+00+-% \+%$& !&$+%'$)% CB-%$'5 

UE[ [$%%$& E$,"&% d;O<sd<hQ5 \+-*()2%")5 `V 

[$'($4G X_5 E"11()- C5 F4&)- e5 E+%@0(// C5 M$&&()2 Y 89<<Q1= X6+04+%(") "/ E$,&$-$)%+%(6$ 

C+',0()2 /"& E+&$ Y",40+%(")- 4-()2 R(@&"1$+#-7 E$,"&% U"7 VWI`I<OI<h5 b)(%$# C%+%$- 

V"+-% W4+&# E$-$+&@* +)# `$6$0",'$)% V$)%$&5 W&"%")5 V! 

E(@*+&# E^5 W&+)% _a5 [$'($4G X_ 89<<Q= K)+0B-(- "/ F+00+-% \+%$& C+',0()2 Y"&% `$-(2)- 

4-()2 V"',4%+%(")+0 a04(# `B)+'(@-7 E$,"&% U"7 VWI`I<;I<Q (--4$# 1B %*$ bC V"+-% 

W4+&# E$-$+&@* +)# `$6$0",'$)% V$)%$&5 W&"%")5 V! 

E(0$B CV5 W&+)% _a5 [$'($4G X_5 E"11()- CM5 MB0+)# \F 89<<d= F("0"2(@+0 ()I%+)D -+',0()2 

+)# -+',0$ #$2&+#+%(") /"& -%+)#+&#(.$# 1+00+-% 3+%$& %&$+%'$)% %$@*)"0"2B -+',0()27 

K1-%&+@% () %*$ Y&"@ ;c%* A)%$&)+%(")+0 V")/$&$)@$ ") KH4+%(@ A)6+-(6$ C,$@($-5 e$B 

F(-@+B)$5 a0 

E(0$B CV5 [$'($4G X_5 W&+)% _a5 E"11()- CM5 MB0+)# \F5 `+6(- [X 89<<h= ^+0(#+%(") "/ 

1("0"2(@+0 '$%*"#- /"& /400I-@+0$ %&$+%'$)% %$-%()27 K1-%&+@% () Y&"@ ;d%* A)%$&)+%(")+0 

V")/$&$)@$ ") KH4+%(@ A)6+-(6$ C,$@($-5 R")%&$+05 t4$1$@5 V+)+#+ 
9Q 

Appendix 1 

`+%+ /&"' ()(%(+0 /($0# %&(+0- @")#4@%$# +% UE[e\ @"',+&()2 '(@&"1$+# &$@"6$&B $//(@($)@B 

1$%3$$) + ,0+)D%") )$% +)# /(0%$& 1+2- +&&+)2$# () -$&($-7 

Type of

Filtration

Parameter

Mesh size Dimensions Flow rate 

(gpm)

Number of 

50 μmdiameter 

microbeads 

recovereda

Y0+)D%") 

)$%

O: ?' )$% 3(%* %3" /(0%$& 

1+2- () -$&($- +- ,&$/(0%$&-J 

:< ?' 8/(&-% () -$&($-= +)# 

9: ?' 8-$@")#=1

hh @m (39”= 

#(+'$%$& +% %*$ %", 

+)# ;d9 @m (64”= 

0")2 

;<< ;:<sd<< 

89:T=

a(0%$& 1+2- 

89 () -$&($-= 

;<< ?' 8/(&-%= +)# 9: ?' 

'$-* 8-$@")#=

;Q @m (7”= #(+'$%$& 

+% %*$ %", +)# c; 

(16”= @' 0")2 

9: ;dQsd<< 

89QT=

+

!*$ d<< '(@&"1$+#-5 () !B,$ AA 3+%$& () + 1$+D$&5 3$&$ 2$)%0B ,"4&$# ()%" %*$ @"# @4, "/ %*$ 

,0+)D%") )$% "& %*$ /(&-% /(0%$& 1+2 () -$&($-7 K/%$&3+&#-5 %*$ 1$+D$& 3+- $G+'()$# /"& &$-(#4+0 

'(@&"1$+#-5 +)# () 1"%* @+-$-5 < 3$&$ /"4)#7 

1K ,&$6("4- %&(+0 @")#4@%$# 3(%* +'1($)% -$+3+%$& ,4',$# %*&"42* %*$ ,0+)D%") )$% 3(%* )" 

,&$/(0%$&- () ,0+@$ B($0#$# + -+',0$ -" 0"+#$# 3(%* #$1&(- +)# -$#('$)% %*+% (% 3"40# *+6$ %+D$) 

#+B- %" +)+0B.$ +00 "/ (%7 A) %*$ QT "/ -+',0$ +)+0B.$#5 ; '(@&"1$+# 8"/ d<< +##$#= 3+- /"4)#7 

K00 -41-$H4$)% %&(+0- 4-$# ,&$/(0%$&- 3(%* +'1($)% -$+3+%$& 8+- 3+- %*$ @+-$ /"& #+%+ () %*(- %+10$= 

"& /&$-*3+%$&7 
9h 

Appendix 2 

E$-40%- "/ ,&$0('()+&B %&(+0- %" #$%$&'()$ %*$ +,,&",&(+%$ /0"3 &+%$ %*&"42* %*$ /(0%$& 1+2- %" 

+00"3 '+G('4' /0"3 +)# '()('4' @0"22()27 

Mesh in 

first bag 

in series 

(μm)*

Mesh in 

second 

bag in 

series 

(μm)

Flow 

rate 

(l min-1)

(gpm)

Duration 

of the 

run 

(min)

Outcome

9: 9: :dQ 

8;:<= 

h !*$ 1+2- @0"22$# +)# 3+%$& /0"3 )$+&0B -%",,$# 

+0'"-% (''$#(+%$0BN + %"%+0 "/ QP 2+0 8OO< 0= 

/0"3$# 

:< :< cPc 

8;9:=

cd !*$ 1+2- @0"22$#5 ,&$--4&$ #(//$&$)%(+0 1$%3$$) 

/(0%$& *"4-()2- ()@&$+-$# 8Y; f :7O ,-(5 Y9 f 

;h7;5 YO f :h7c +% %*$ $)# "/ %*$ &4)=5 +)# %*$ 

/0"3 &+%$ -0"3$# %" d97; 2,'N OO;9 2+0 8;95::< 

0= /0"3$# 

;<< 9: h: 

89:= 

dc !*$ ,&$--4&$ #(//$&$)%(+0 &$'+()$# -0(2*% 8Y; f 

dc7Q ,-(5 Y9 f dO7h5 YO f dc7O +% :O '()=N ;cQ: 

2+0 8:d9P 0= /0"3$# 3(%* 0(%%0$ @0"22()2 "/ 1+2- 

:< 9: h: 

89:=

Q< !*$ ,&$--4&$ #(//$&$)%(+0 &$'+()$# -0(2*% 8Y; f 

d:79 ,-(5 Y9 f dO7h5 YO f dc7O +% P< '()=N ;cd< 

2+0 8::OO 0= /0"3$# 3(%* 0(%%0$ @0"22()2 "/ 1+2- 

9: 9: h: 

89:= 

P9 !*$ ,&$--4&$ #(//$&$)%(+0 &$'+()$# -0(2*% 8Y; f 

dc7:5 Y9 f dO795 YO f dO7O=N ;QOO 2+0 8dhcd 0= 

/0"3$# 3(%* 0(%%0$ @0"22()2 "/ 1+2- 

mK00 $G,$&('$)%- 3$&$ @")#4@%$# 4-()2 M+B3+&# a(0%&+%(") 4)(%- 3(%* c;I@' 0")2 /(0%$& 1+2-7 
O< 

Appendix 3 

`+%+ /&"' %"G(@(%B %$-%- $6+04+%()2 %*$ $//$@% "/ ORZ :9<< -$+0+)% ") 1&()$ -*&(', Artemia 

franciscana 8%", %+10$= +)# +'1($)% ."",0+)D%") 81"%%"' %+10$= +/%$& %3" *"4&- "/ $G,"-4&$ %('$7 

Replicate Live 

Af

Total

Af

% living 

Af

V;m PQ Ph hQ7P 

V9 d< d< ;<<7< 

VO cO cO ;<<7< 

Vc cQ cQ ;<<7< 

!; ch :c h<7P 

!9 :d :d ;<<7< 

!O :< :< ;<<7< 

!c dd dQ hP7; 

mV f @")%&"0 2&"4,5 ! f %&$+%'$)% 2&"4,5 K/ f Artemia franciscana5 @40%4&$# 1&()$ -*&(',7 

Replicate

Live 

copepods 

(≥ 50 μm)

Total 

copepods

(≥ 50 μm)

% living 

copepods

V;m 9< 9< ;<<7< 

V9 ;h 9< h:7< 

VO ;h 9< h:7< 

Vc 9< 9< ;<<7< 

!; 9< 9< ;<<7< 

!9 ;h 9< h:7< 

!O ;h ;h ;<<7< 

!c 9< 9< ;<<7< 

mV f @")%&"0 2&"4,5 ! f %&$+%'$)% 2&"4, 
O; 

Appendix 4 

X+%") a(0%$& CD(# #&+3()2 3(%* #('$)-(")-7 
O9 

Appendix 5 

X+%") a(0%$& CD(# ",$&+%(")- '+)4+07 

!*$-$ ()-%&4@%(")- +&$ -,$@(/(@ %" ",$&+%(")- 4-()2 %*$ X+%") a(0%$& CD(# 8XaC5 -*"3) () a(24&$ ;= 

+)# ,$&/"&'$# +% %*$ U+6+0 E$-$+&@* [+1"&+%"&B 1+00+-% 3+%$& %&$+%'$)% %$-% /+@(0(%B 8F\!!a= () 

e$B \$-%5 a0"&(#+7 !*$ ,&"@$#4&$- %B,(@+00B 4-$# () %&(+0- 3(%* %*$ XaC +&$ 1&"+#0B @+%$2"&(.$# 

()%" ,&$,+&+%(")5 ",$&+%(")5 +)# -*4%#"3)7 

a(24&$ ;7 X+%") a(0%$& CD(# -*"3()2 +(& 10$$# +)# #&+() ,"&%- +)# *"4-()2 '+)4+0 ()0$% +)# 

"4%0$% 1+00 6+06$-7 

L)0B + %&+()$# -B-%$' ",$&+%"& -*+00 ,$&/"&' ",$&+%(")- -,$@(/($# () %*(- #"@4'$)%N (',&",$& 

-$%4, "& ",$&+%(") @+) u$",+&#(.$ ,$&-"))$0 -+/$%B "& #+'+2$ $H4(,'$)%7 

Y&$,+&+%(") 

;7 ^$&(/B %*$&$ (- -4//(@($)% @+,+@(%B () %*$ #(-@*+&2$ %+)D %" &$@$(6$ %*$ $)%(&$ 6"04'$ "/ 

3+%$& @")%+()$# () %*$ 1+00+-% %+)D7 

97 A-"0+%$ %*$ XaC 1B @0"-()2 %*$ '+)4+l 3” butter/0B ()0$% +)# "4%0$% 6+06$- 8a(24&$ 95 

^+06$- :;< +)# c<c5 &$-,$@%(6$0B=5 3*(@* +&$ 4,-%&$+' +)# #"3)-%&$+' "/ %*$ XaC7
OO 

V-313

V-404

V-405

V-303

EFS Housings

Housings 2" Manual Inlet/

Outlet Valves

Primary Pumps 8 & 9

Diaphragm Pump 5

Ballast Tank Discharge Tank

V-510

TITLE

Aquatic Nusiance Species Ballast Water Treatment Test Facility 

ANS BWTTF - NRLKW, Trumbo Point Annex, Key West, FL

Eaton Filter Skid Schematic Overview

Scale: N/A

DATE

15 June 2010

DRAWN BY

NRL Code 6136

Cameron Moser

REVISION

00

V-303 Primary pumps inlet control valveV-313 Automated diaphragm valveV-404 EFS outlet - manual 3" butterfly valveV-405 Discharge tank inlet control valveV-510 EFS inlet - manual 3" butterfly valve

SP-11 Flanged T from 6" main line into EFS sample port

SP-11

4(*%,- 1"*#+"*-++%$4&-1 "-3 *2"-#"1 2"0 /+&%".+,- !* '( )%&$"#!
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5M 

Appendix 6 

)*+,- !".+&% /0"1 A".. ,; 4*+&%"*.3 *-1 9*.9$.*+",- ,; G&"#:+( 

 

Equipment Quantity Cost Total Cost 

Filter Housing 

Eaton 316SS ToplineTM Housing 10 $1,707.00 $17,070.00 

Filter Bags 

Filter Specialist Incorporated 35μm size 2 NMO 

35 with plastic snap rings 10 $20.43 $204.30 

PVC Piping and Fittings 

2" Spears van stone flange socket style 28 $7.59 $212.52 

PVC/EPDM COMPACT B VLV 2" S 14 $50.51 $707.14 

2" schedule 40 socket Tees 10 $1.50 $15.00 

2" x 2" x 3/4" reducing TEE Soc x Soc x FIPT 

schedule 40 20 $2.50 $49.95 

2" Soc x Soc schedule 40 ELL 30 $1.22 $36.45 

3/4" plug 20 $0.52 $10.35 

PVC/EPDM COMPACT B VLV 1/2" S 12 $14.18 $170.10 

3" Spears van stone flange socket style 10 $12.95 $129.54 

3" Soc x Soc schedule 40 ELL 10 $4.42 $44.18 

Low-Pressure PVC Ball Valve 1/2" NPT Female, 

White (Same as 4876K11) 10 $5.96 $59.60 

Thk-Wall Dk Gray PVC Thrd-One-End Pipe 

Nipple 1/2" Pipe Size, 2" Length, Schedule 80 20 $1.21 $24.20 
5N 

 

Equipment Quantity Cost Total Cost 

Wood Skid

4x8 treated plywood 3/4" 2 $32.00 $64.00 

2x6 treated 8' board 3 $5.50 $16.50 

4x4 treated 8' board 2 $9.50 $19.00 

Wood Screws 40 $0.10 $4.00 

Stainless hardware 

5/8-11x3" 316 stainless steel hex cap screw 

partially threaded 100 $4.32 $432.00 

5/8-11 316 Stainless steel hex cap nut 22 $8.00 $176.00 

Type 316 SS Type A SAE Flat Washer 5/8" Screw 

Size, 1-5/16" OD, .07"-.13" Thick, Packs of 10 20 $8.54 $170.80 

Type 316 Stainless Steel Split Lock Washer 5/8" 

Screw Size, 1.08" OD, .15" min Thick, Packs of 

10 

12 $5.37 $64.44 

Type 316 Stainless Steel Hex Head Cap Screw 

5/8"-11 Thread, 3-1/4" Length, Packs of 1 10 $4.69 $46.90 

Cam-Lock 

Fittings 

Aluminum Cam-and-Groove Hose Coupling 

Plug, PFA Adapter, 3 Coupling Size, 3" Pipe Size 2 $62.30 $124.60 

Cost of Equipment Components $19,851.57 

Labor 

Hours to build skid per design drawing 80 $150.00 $12,000.00 

Total Discharge Skid Cost $19,851.57 
6P 

Appendix 7 

7*.9$.*+",-3 ,; ;.,G 8&.,9"+F "- +:& ;".+&% 30"1 *-1 1$%"-# * :F2,+:&+"9*. 2.*-0+,- +,G( 

)U$*+",- ?( !.,G 8&.,9"+F 9*.9$.*+",- ;,% BP #24 "- * B 9m (2”C 1"*4&+&% 2"2&( 

)U$*+",- '( !.,G 8&.,9"+F 9*.9$.*+",- ;,% BP #24 "- *- ?M 94 >7”C 1"*4&+&% ;".+&% :,$3"-#( 

)U$*+",- 5( !.,G 8&.,9"+F 9*.9$.*+",- ;,% 'BJ#24 ;.,G "- * B 94 >2”C 1"*4&+&% 2"2&( 

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6? 

Appendix 8 

T&3$.+3 ,; +,O"9"+F +&3+3 &8*.$*+"-# +:& &;;&9+ ,; 3+*"-.&33 3+&&. ,- A%"-& 3:%"42 Artemia 

franciscana *-1 *4A"&-+ V,,2.*-0+,- ;,% '(B : ,; &O2,3$%&( 

Replicate Live 

Af

Total 

Af

% living 

Af

Live 

zoopl 

(≥ 50 μm)

Total 

zoopl

(≥ 50 μm)

% living 

zoopl

7?c 'I6 'IB NN(I 56 5M MN(B 

7' 'I' 'IK NM(? ?B 'P KB(P 

<? 5B5 5B6 NN(K 56 5M MN(B 

<' ''N '5' NM(K 'M 55 M6(N 

<5 56M 5BP NN(6 5P 5P ?PP 

c 7 d 9,-+%,. #%,$2@ < d +%&*+4&-+ #%,$2@ S; d Artemia franciscanaL V,,2. d *4A"&-+ 

V,,2.*-0+,- e BP f4.

Appendix 9 

T&3$.+3 ,; +:& 9,42*%"3,- A&+G&&- ."8"-# 9,2&2,13 "- +:& A*..*3+ +*-0 *-1 +:& ;".+&% 30"1( 

( 

Replicate Live 

nauplii

Total 

nauplii

% living

nauplii

Live 

A + C

Total

A + C

% living 

A + C

Q<?c M5 MI NI(B 5N 65 NP(K 

Q<' ?P6 ?P6 ?PP 'M 5I KK(M 

!/? 'N5 'NN NM KN NK M?(6 

!/' 'KN 'MI NK(I NM ??6 MI 

c Q< d 2.*-0+,- +,GL !/ d ;".+&% 30"1L S g 7 d *1$.+ *-1 9,2&2,1"+& 3+*#&3( S.. 9,2&2,13 G&%& e BP f4 

"- 4"-"4$4 1"4&-3",-.