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, DCProtocol for the Verification of Ballast Water Treatment Technologies September 2010

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### 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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### 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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### 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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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

v

### 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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Version 5.15.3.3 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 Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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viiList 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 Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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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.

m 3 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

μ g/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 C enter 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 D escription

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 m 3 per minute

(370

– 4,490 gpm) or a total tank volume within a range of 20

– 14,500 m 3 (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 cond itions 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 10 5 organisms/m 3

Protists Tetraselmis sp. Green microalgae 10 3 organisms/mL

Bacteria Geobacillus sp

. Geobacillus sp. 10 3 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., 10 2 zooplankton/l is acceptable for 10 5 zooplankton/m 3 ).

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 r ate 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 p hysical 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.

17Some 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 expensive 1 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 Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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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

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

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.

19Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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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.

21Table 4. Minimum Criteria for Challenge Water Total Living Populations

1 Organism Size Class Total Concentration Diversity

≥ 50 μm

≥ 10 μm and < 50μm

<10 μm 10 5 3 organisms/m

10 3 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 m 3 per hour (880 gallons

per minute) and an available volume per test cycle of at least 400 m 3 . 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 m 3 shall be processed in each BE test cycle. The recommended minimum

volume for in-tank testing is 200 m 3 (~52,800 gallons). The TF shall provide test and control

ballast tank configurations of at least 200 m 3 . 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 m 3 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 m 3 , 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 m 3 /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 m 3 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 m 3 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:



### Q

iso 

### D = D

iso m 

### Q

m

25Protocol for the Verification of Ballast Water Treatment TechnologiesSeptember 2010Version 5.1Treatment Sample TankBWTSPump≥3 m3≥ 30 m3≥ 30 m3Challenge Water 450 tonsStandard Test OrganismsBallast Tank≥ 200 m3Control HoldingTank≥ 200 m3DischargeDischargeFlowSplitterControl Sample TankChallenge WaterSampleFigure 1. Sampling design example for in-tank treatment.26Protocol for the Verification of Ballast Water Treatment TechnologiesSeptember 2010Version 5.1Pump≥ 30 m3≥ 30 m3≥3 m3Standard Test OrganismsTreatmentHolding Tank≥ 200 m3Control HoldingTank≥ 200 m3FlowSplitterBWTSChallenge WaterSamplePost TreatmentChallenge Water 450 tonsPost TreatmentSample(≥ 30 m3)Control SampleHolding Sample TankDischargeDischargeTankFigure 2. Sampling design example for in-line treatment.27Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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where D iso and D

m are the diameters of the sample port opening and the main flow in the line to

be sampled, respectively; and Q iso and Q

m 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 a ny 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

occu r 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.

30Table 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. Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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31Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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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.

32Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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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 m 3 amongst the BE test

cycles. These O&M test cycles are equivalent to ~50 hours of operation at 200 m 3 per hr (or

~65 hours of operation at 150 m 3 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 m 3

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 m 3 /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.

33Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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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/m 3

≥ 10 μm and < 50μm 100 organisms/mL

< 10 μm 5 x 10 2 /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.

34Protocol 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.

35Protocol 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

36Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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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==ParameterTable 9.Min. SampleVolume (mL)1Sample Volumes, Containers and ProcessingSample3Processing/ PreservationContainers3Maximum HoldingTime

CoreParametersElectronicin situdata(Temperature, pH, Salinity,etc.)2Total suspended solidsDissolved organic carbonParticulate organic carbonDissolved oxygenPhytoplankton Enumeration(Live/Dead Analysis)4Zooplankton Enumeration(Live/Dead Analysis) LowConcentration/DischargeBacteriaNA1002550030033 mconcentrated to1000 mL33 mconcentrated to1000 mL1000NA250 mL HDPE orglass40-mL glass vialWhatman GF/F infoil300 mL glassBOD bottleDark 1000-mLHDPE bottle1 L flask1 L sterile HDPEMaintain 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 asdescribed in the protocolNo preservation; Sub-sample into well plate (20 1-mL wellsobserved). Observe with dissecting microscope and probeorganisms to determine live/dead status. Fix with Lugol’s solutionfor total counts.Plate on appropriate media.NA1 week28 days28 days24 hoursProcess immediatelyProcess immediatelyProcess immediately.

Auxiliary ParametersDissolved inorganic nutrientsChlorophyllaandphaeopigments4040060-mLpolyethylenebottleWhatman GF/F infoilPass through a NucleporeTM (Whatman Inc., Piscataway, NJ)membrane filter; freeze filtrate until analysis.Pass through GF/F; fix with a saturated MgCO3solution; freezefilter until analysis.28 days4 weeksGF/F: pre-ashed glass fiber filter.1Volume processed for analysis; volumes are quantitative.2Conductivity, temperature, pressure, dissolved oxygen, chlorophyll a fluorescence, transmissometry.3Name brand items (e.g., NucleporeTM, WhatmanTM) may be substituted with comparable items from a different manufacturer.4Dinoflagellate methods are under development.Protocol for the Verification of Ballast Water Treatment TechnologiesSeptember 2010Version 5.138Table 10. Recommendation for Water Quality Sample Analysis MethodsParameterUnits InstrumentMethod/ReferenceDissolved ammoniumμMAutoanalyzer APHA Standard Method No. 4500-NH3 / 20theditionEPA Method No. 349.0 http://www.epa.gov/nerlcwww/m349_0.pdfDissolved inorganic nitrate andinorganic nitriteμMAutoanalyzer ESS Method No. 220.3 http://www.epa.gov/glnpo/lmmb/methods/methd220.pdfAPHA Standard Method Nos. 4500-NO2-B and 4500-NO3-F, 19theditionEPA Method No. 353.4 http://www.epa.gov/nerlcwww/m353_4.pdfDissolved inorganic phosphateμMAutoanalyzer ESS Method No. 310.1 http://www.epa.gov/glnpo/lmmb/methods/methd310.pdfEPA Method No. 365.5 http://www.epa.gov/nerlcwww/m365_5.pdfDissolved inorganic silicateμMAutoanalyzer EPA Method 366.0 http://www.epa.gov/nerlcwww/m366_0.pdfDissolved organic carbonμMCarbon AnalyzerAPHA Standard Method No. 5310-C, 20theditionASTM Method Nos. D6317, D2579, D4129, D4839, D513-02 and D5790LMMB Method No. 096 http://www.epa.gov/glnpo/lmmb/methods/docanal2.pdfLMMB Method No. 014 http://www.epa.gov/glnpo/lmmb/methods/pocdoc2.pdfEPA Method No. 440.0 http://www.epa.gov/nerlcwww/m440_0.pdfParticulate organic matterμMCarbon analyzerCHN AnalyzerorLMMB Method No. 097 http://www.epa.gov/glnpo/lmmb/methods/pocanal2.pdfAPHA Standard Method No. 5310-C, 20theditionLMMB Method No. 014 http://www.epa.gov/glnpo/lmmb/methods/pocdoc2.pdfEPA Method No. 440.0 http://www.epa.gov/nerlcwww/m440_0.pdfChlorophylla/phaeopigmentsμg/LFluorometer EPA Method 445.0 http://www.epa.gov/nerlcwww/m445_0.pdfEPA Method No. 446.0 http://www.epa.gov/nerlcwww/m446_0.pdfEPA Method 447.0 http://www.epa.gov/nerlcwww/m447_0.pdfASTM Method No. 3731-87 (1998)Total suspended solidsMg/L5-place balanceESS Method No. 340.2 (LMMB Method No. 065)http://www.epa.gov/glnpo/lmmb/methods/methd340.pdfAPHA Standard Method No. 2540D (1998)EPA Method 160.2 http://www.epa.gov/region09/qa/pdfs/160_2.pdfDissolved oxygenMg/L Radiometer TitraLabEPA Method No. 360.1 (Probe Method)APHA Standard Method No. 4500-0G (Probe Method)Protocol for the Verification of Ballast Water Treatment TechnologiesSeptember 2010Version 5.139Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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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 μ M <5 times MDL 1

Sample replicates Nitrate, nitrite, Sample replicates

Three (3) sample replicates phosphate ≤ 2% PD 2

per treatment cycle 0.01 μ M

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 μ M ≤ 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 μ M ≤ 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 μ g/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.

40Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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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 m 3 concentrated to 1 L) assuming 20 subsamples of 1 mL. Given

these assumptions, 30 m 3 must be sampled to enumerate 10 organisms/m 3 , 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 = 6 0.5 /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.

421Protocol for the Verification of Ballast Water Treatment TechnologiesSeptember 2010Version 5.1Table 12. Density Confidence Intervals for Poisson Distributions Using the Chi-Square Statistic2Scaled Densities and Confidence IntervalsCount Data(Assumes whole water sample volume is concentrated to 1L,with analysis of 20 one-mL subsamples from the concentrate)Whole Water SampleVolume (V) =3V = 1 m333V = 3 mV = 10 mV = 30 m3V = 60 m

Organism Count 95% Upper BoundUpperBound /Count95%MeanUpperDensityBound(m-3) (m-3)95%95%95%MeanMeanMeanUpperUpperUpperDensityDensityDensityBoundBoundBound(m-3) (m-3) (m-3) (m-3) (m-3) (m-3)95%MeanUpperDensityBound-3)(m-3) (m03.0001500500150502.514.744.745023717795242814.026.303.1510031533105103131025.237.752.5815038850129153951336.549.152.2920045867153204671537.6510.512.1025052683175255381848.8611.841.973005921001973059102059.9713.151.8835065711721935661222611.0814.431.8040072213324140721324712.0915.711.7545078515026245791526813.11016.961.7050084816728350851728814.11118.211.6655091018330355911830915.21219.441.62600972200324609720321016.21320.671.5965010332173446510322341117.21421.891.5670010942333657010923361218.21523.101.5475011552503857511525 381319.21624.30 1.5280012152674058012227411320.31725.501.5085012752834258512728421421.21826.691.4890013353004459013330441522.21927.881.4795013943174659513932461623.22029.061.451000145333348410014533481724.243Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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

N = 1 3 5

Concentration (individuals/m 3 )

0.01 60,000 3 ) 1 Sample Volume Required (m

20,000 1 2,000

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

sho uld 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

44Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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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, CellTracker TM 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.

45Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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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

bacteria 6 . Use of multiple types of media enables measurement of the response of different

portions of the ambient bacterial community 7 . 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.

47Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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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 m 3 , and the minimum sample

collection volume associated with each transfer is 3 m 3 . 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 beryllin a, Larval Survival and Growth (EPA Method 1006.0):

http://www.epa.gov/OST/WET/disk1/ctm13.pdf )

49Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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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/m 3 ; ≥ 50μm

(live/dead) × × × ×

Ambient organisms/mL;

≥ 10 an d < 50μm (live/dead) × × × ×

Ambient organisms/mL;

< 10 μm (live/dead) × × × ×

Sea Urc hin, Arbacia punctulat a, 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

50Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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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 t o 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

51Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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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 m 3 , gallons or pounds per m 3 , 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 m 3 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.

54Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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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 fo r 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 m anager will review t he

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.

60Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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## Chapter 9

Environmental, Health, and S afety 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.

61Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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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 BalPure TM

Ballast Water Treatment System, NRL Letter Report 6130/6098, Washington, DC.

62Protocol for the Verification of Ballast Water Treatment Technologies September 2010

Version 5.1

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.

63Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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## Appendix A: Quality Assurance Project Plan Outline

64Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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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 C hecks



### 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

Reco veries for laboratory control samples are calculated as follows:

67100( foundconce ntration ) % Recovery = (A-2)

trueconcen tration

For acceptable analytical accuracy under the verification testing program, the recoveries reported

duri ng 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

X

average = the arithmetic mean of the recovery values Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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Stand ard Deviation is calculated as follows:



### ( X

i 

### − X ) 2

Standard Deviation = (A-4)



### n

− 1

where: X i = the individual recovery values

X = the arithmetic mean of the recovery values

n = the number of determinations

The QAP P 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.

69Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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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 ± 0.5 o 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.

71Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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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/cm 2 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 Br 2 /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/cm 2 (72 seconds); 25

mJ/cm 2 (180 seconds); 50 mJ/cm 2 (360 seconds); and 100 mJ/cm 2 (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/cm 2 (80 seconds); 25 mJ/cm 2 (200 seconds); 50 mJ/cm 2 (400 seconds); and 100

mJ/cm 2 (800 seconds). Additional higher dosages were tested in the zooplankton study since

some of the species were tolerant of 100 mJ/cm 2 ; 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 (O 3 ) 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- p

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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

R 2 = 0.9981

0 1 2 3 4 5 6 7 8 9

0 1 2 3 4 5 6 7

TimeConcentration (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 Cl 2 /L) for synthetic seawater treated with

ozone versus time using the Enaly ozone generator.

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Ozone Concentrations for Freshwater

Treatments vs. Time

y = 0.4923Ln(x) - 0.4348

R 2 = 0.9763

0 0.2 0.4 0.6 0.8

1 1.2

0 5 10 15 20 25

TimeConcentration (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 Br 2 /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 CO 2 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.

752.5 Ozone diffusion in saltwater

y = 0.5033x - 0.2673

R 2 = 0.9914

2 3 4 5 6

Time (min)TRO mg/L as Br2O3mg/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

R 2 = 0.9517

0 1 2 3 4 5 6

Time (min) Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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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 10 4 to 10 5

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 37 o 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 10 6 cells/mL. The bacteria

suspension was then distributed into sample containers to yield a final concentration of 10 4 to 10 5

cells/mL.

In the second method, G. stearothermophilus and B. subtilis spore suspensions were purchased at

the vendor-reported spore concentrations of 10 7 and 10 8 spores/mL. Spores were diluted to an

estimated 10 6 spores/mL in PBS. An aliquot of the PBS spore suspension was then added to the

sample containers to have a final concentration of 10 4 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 37 o C,

and G. stearothermophilus plates were incubated at 55 o 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 10 4 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 10 4 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 10 4 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 10 4 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; CaCl 2 , 0.04 g/100 mL; MgSO 4, 0.04 g/100 mL;

Na 2 HPO 4 , 1.42 g/100 mL; KH 2 PO 4 , 1.36 g/100 mL. Ten (10) mL of each stock solution was

added to 950 mL dH 2 O. PPY was made with proteose peptone (20.0 g), yeast extract (2.5 g),

and dH 2 O (1 L). MY75S comprised sterile filtered natural seawater (750 mL), dH 2 O (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.

82 Table 3. Preparation of inorganic medium WC.

1. Prepare 7 Stock Solutions:

a. NaNO 3 : 85.01 g/L

b. CaCl 2 · 2H 2 O: 36.76 g/L

c. K 2 HPO 4 : 8.71 g/L

d. MgSO 4 · 7H 2 O 36.97 g/L

e. Na 2 O 3 Si· 9H 2 O 14.21 g/L

f. NaHCO 3 12.6 g/L

g. Trace Metal Mix

MnCl 2 · 4H 2 O 0.18 g/L

ZnSO 4 · 7H 2 O 0.022 g/L

(NH 4 ) 6 Mo 7 O 24 · 4 H 2 O 0.0046 g/L

CoCl 2 · 6H 2 O 0.012 g/L

CvSO 4 · 5H 2 O 0.01 g/L

H 3 BO 3 0.006 g/L

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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 μ L 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 Br 2 /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 Br 2 /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 Na 2 SeO 3 has

been added to a final concentration of 10 -8 M and the concentration of CuSO 4 . 5H 2 O 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 20 o 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 20 o C 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.

86Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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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

87Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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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.

88Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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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

89Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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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

90Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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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.

91Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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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/cm 2 50 mL Final exposures of 50, 100, 200,

and 400 mJ/cm 2 – 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 Br 2 /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 N 2 (95%) and CO 2 (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).

92Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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Thermal Treatment (Phase I)

For zooplankton, only three temperatures were tested, 35 o , 40 o , and 45 o C. 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 20 μ m 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).

93Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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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/cm 2 (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.

94Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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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.

95Protocol for the Verification of Ballast Water Treatment Technologies September 2010

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## Appendix C: Design and Preliminary Use of a Commercial Filter

Skid to Capture Organisms ≥ 50 μ m in Minimum Dimension

(Nominally Zooplankton) for Evaluating Ships’ Ballast Water

Management Systems at Land-based Test Facilities

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19b. TELEPHONE NUMBER (Include area code) 22-06-2010 NRL Letter Report March 2009-April 2010

Design and Preliminary Use of a Commercial Filter Skid to Capture

Organisms Ż 50 μ

m 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

202-404-2123Science and Engineering



## Design and Preliminary Use of a Commercial Filter Skid to

Capture Organisms ≥ 50 μm in Minimum Dimension

(Nominally Zooplankton) for Evaluating Ship s’ Ballast

Water Management Systems at Land-Based Test Facilities

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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

3'V'T O0-+9 !JJ)9:A 0J 8:$+H;)AA 8:)); B+;:)% N0,A+HLA''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' U

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

V'2'T Q):E0" 0J Q+9%0D)$" /0,H:+HL'''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' 22

V'2'3 >H:)%9$;+D%$:+0H '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' 2T

V'T B+;:)% M$LA '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' 2T

V'T'2 B+;:)% M$L B+);" O%+$;A_@$:)% B;0# 6$:)'''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' 2T

V'T'T B+;:)% M$LA_B+);" $H" ($D0%$:0%< K$;+"$:+0H GA+HL Q+9%0D)$"A ''''''''''''''''''''''''''''' 23

V'T'3 B+;:)% M$LA_8)$*A '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' 2W

V'T'V B+;:)% M$LA_O0-+9+:< 0J 8)$;$H: 0H :E) 8)$*A '''''''''''''''''''''''''''''''''''''''''''''''''''''''''' 2W

V'3 B+;:)% 8F+"''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' 21

V'3'2 B+;:)% 8F+" 7)A+LH ''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' 21

V'3'T B+;:%$:+0H =%)$ 0J B+;:)% 8F+" IA' c;$HF:0H 5): ''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' TT

V'3'3 B;0# K);09+:< :E%0,LE :E) B+;:)% 8F+" IA' c;$HF:0H 5): ''''''''''''''''''''''''''''''''''''''''''''' TT

V'3'V O0-+9 !JJ)9:A 0J 8:$+H;)AA 8:)); B+;:)% N0,A+HLA''''''''''''''''''''''''''''''''''''''''''''''''''''''''' TV

V'3'W /0*^$%+A0H 0J b00^;$HF:0H +H :E) M$;;$A: O$HF IA' :E) B+;:)% 8F+" '''''''''''''''''''''''''' TW

V'3'1 !JJ)9: 0J /%0#"+HL 0H =*D+)H: b00^;$HF:0H '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' TW

W 7+A9,AA+0H ''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' TW

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`

=^^)H"+- Y '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' V4++ +

=^^)H"+- ` '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' V2

=^^) H"+- U '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' V2



### Table of Figures

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' ''''''''''''''''''''''''''''''''''''''''''''''''''''''' 1

B +L,%) T' 6)90I)%< $J:)% *+9%0D)$"A #)%) 90,H:)" ,A+HL :E) e"%<f $H" e#):f *):E0"A' X%$HL)

D$%A %)^%)A)H: "%< :%+$;A. $H" D;,) D$%A %)^%)A)H: #): :%+$;Ad H,*D)%A $D0I) D$%A +H"+9$:) :E)

^)%9)H: *+9%0D)$" %)90I)%<d ;)::)%A 0H :E) -Z$-+A ;$D);A +H"+9$:) :E) %)A)$%9E)%fA +H+:+$;A' ''''''''' 2T

B +L,%) 3' 6)90I)%< )JJ+9+)H9< 0J *+9%0D)$"A +H ;$D0%$:0%< :%+$;A ,A+HL TW g* J+;:)% D$LA [BM\'

M;,). %)". $H" L%))H D$%A %)^%)A)H: W4 g*. 244 g*. $H" 2W4 g* *+9%0D)$"A. %)A^)9:+I);<' '''''' 23

B+L,%) V' 6)90I)%< )JJ+9+)H9< 0J *+9%0D)$"A +H J+);" :%+$;A' GH;)AA H0:)". J+;:)% D$LAf *)AE #$A

TW g*' M;,). %)". $H" L%))H D$%A %)^%)A)H: W4 g*. 244 g*. $H" 2W4 g* *+9%0D)$"A.

%)A^)9:+I);<' h i $ H0:$D;) $*0,H: 0J L%))H $;L$) #$A 90;;)9:)" +H J+;:)% D$LA' '''''''''''''''''''''''''' 2V

B +L,%) W' 6)90I)%< )JJ+9+)H9< 0J W4 g* *+9%0D)$"A +H ;$D0%$:0%< $H" J+);" :%+$;A 90H",9:)" $J:)%

+*^%0I)*)H:A #)%) *$") :0 :E) *+9%0D)$" ^%0:090;' 5,*D)%A +H ^$%)H:E)A)A %)^%)A)H:

%)^;+9$:)Ad BM i J+;:)% D$L' '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' 2W

B+L ,%) 1' B+;:)% AF+" ")A+LH J0% J;0#Z:E%0,LE A$*^;+HL 0J "+A9E$%L)" D$;;$A: #$:)%' '''''''''''''''''' 21

B +L,%) Y' !$:0H O0^;+H)j J+;:)% E0,A+HL' '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' 2Y

B +L,%) `' 5<;0H *0H0J+;$*)H: *)AE J+;:)% D$L A)9,%)" +H $H !$:0H O0^;+H)j J+;:)% E0,A+HL' '' 2`

B +L,%) U' c;$H I+)# 0J :E) ^%0:0:<^) J+;:)% AF+" $: 56(?@' M;,) $%%0# AE0#A #$:)% )H:)%+HL

:E) J+;:)% AF+"d <);;0# $%%0# AE0#A #$:)% )-+:+HL :E) AF+"' '''''''''''''''''''''''''''''''''''''''''''''''''''''''''' 2`

B +L,%) 24' k$*$"$ 57cZ`4 =+% "+$^E%$L* ^,*^ $A +HA:$;;)"' '''''''''''''''''''''''''''''''''''''''''''''''''' T4

B +L,%) 22' c%)AA,%) "%0^ 0J J+;:)% AF+" IA' J;0# %$:) [J+L,%) J%0* !$:0H B+;:%$:+0H. ((/\' '''''''''' T2

B +L,%) 2T' B;0# I);09+:< *0"); J0% #$:)% J;0# :E%0,LE $H !$:0H O0^;+H)j B+;:)% N0,A+HL $: W4

L^*. )H:)%+HL :E0,LE :E) :0^ 0J :E) E0,A+HL $H" )-+:+HL $: :E) D0::0*' '''''''''''''''''''''''''''''''''''''' T3

B+L,%) 23' B;0# I);09+:< *0"); +;;,A:%$:+0H J0% #$:)% J;0# :E%0,LE $H !$:0H O0^;+H)j B+;:)%

N0,A+HL $: TW L^*. )H:)%+HL :E0,LE :E) :0^ 0J :E) E0,A+HL $H" )-+:+HL $: :E) D0::0*' ''''''''''''''' TV2



## 1 Introduction

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/0H:%0; $H" Q$H$L)*)H: 0J 8E+^Af M$;;$A: @$:)% $H" 8)"+*)H:A. #E+9E )A:$D;+AE)A A:$H"$%"A J0%

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=*0HL :E) 9%+:)%+$ J0% D$;;$A: #$:)% "+A9E$%L) +A :E) ")HA+:< 0J 0%L$H+A*A l W4 g* [H0*+H$;;<

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e^%)I)H:. *+H+*+C) $H" ,;:+*$:);< );+*+H$:) :E) %+AFA :0 :E) )HI+%0H*)H:. E,*$H E)$;:E. ^%0^)%:<

$H" %)A0,%9)A $%+A+HL J%0* :E) :%$HAJ)% 0J E$%*J,; $a,$:+9 0%L$H+A*A $H" ^$:E0L)HAf. +A $;A0

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M)9$,A) :E) ^%0D$D+;+:< 0J J+H"+HL $ ;+I+HL C00^;$HF:)% +H $ #$:)% A$*^;) *)):+HL :E) ^%0^0A)"

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A$*^;) 0% $ A,DZA$*^;)\. :E) ;)I); 0J ")A+%)" ^%)9+A+0H. $H" :E) %);)I$H: "+A9E$%L) A:$H"$%" [)'L'.

Z3 Z3 m 24 I+$D;) 0%L$H+A*A * 0% m 4'2 * \' c%)I+0,A #0%F $: :E) 5$I$; 6)A)$%9E ($D0%$:0%< +H ?)<

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+H +H"+I+",$; E0,A+HLA_$ 90HJ+L,%$:+0H 90**0H;< ,A)" +H #$:)%Z:%)$:*)H: J$9+;+:+)A_9$H D)

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## 2 Objectives

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H,*D)% 0J viable [;+I+HL\ 0%L$H+A*A [>QX. T44W\'



## 3 Experimental Approach

=;; ;$D0%$:0%< $H" J+);" )-^)%+*)H:A #)%) 90H",9:)" $: :E) M$;;$A: @$:)% O%)$:*)H: O)A: B$9+;+:<

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#)$:E)%. $H" "+JJ+9,;: :0 90HJ+L,%) +H $ J;0#Z:E%0,LE A$*^;+HL $^^$%$:,A A,+:$D;) J0% ;$%L)

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J+;:)% D$LA. )$9E )H9;0A)" +H $ A:$+H;)AA A:)); E0,A+HL' N)%). #) ")A9%+D) ^%);+*+H$%< :%+$;A :0

)I$;,$:) :E) %):)H:+0H )JJ+9+)H9< 0J J+;:)% D$LA ,A+HL *+9%0D)$"A. :E) ")A+LH $H" 90HA:%,9:+0H 0J

:E) J+;:)% AF+". $H" +H+:+$; I$;+"$:+0H :%+$;A'



### 3.1 Ballast Water Treatment Test Facility (BWTTF) Description

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)A:$D;+AE)" $: 56(?@. #E)%) %)A)$%9E +A 90H",9:)" :0 ^%0I+") :)9EH+9$; L,+"$H9) J0% :E) !OKfA

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+H %)I+)#\' OE) M@OOB +A ;09$:)" 0H B;)*+HL ?)< $: O%,*D0 c0+H: =HH)-. 5$I$; =+% 8:$:+0H

?)< @)A:. B(' =A ^$%: 0J :E) 5$I$; 6)A)$%9E ($D0%$:0%<fA /)H:)% J0% /0%%0A+0H 89+)H9) $H"

!HL+H))%+HL. +: J,H9:+0HA $A $ A9+)H:+J+9 :)A: ^;$:J0%* J0% :E) A:$H"$%"+C)" $AA)AA*)H: 0J

:)9EH0;0L+)A ")A+LH)" :0 %)",9) 0% );+*+H$:) $a,$:+9 H,+A$H9) A^)9+)A +H AE+^D0$%" D$;;$A: #$:)%'

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)-^)%+*)H:A $H" ")I);0^ *):E0"A :0 +HJ0%* :E) !OK I)%+J+9$:+0H ^%0:090; $H" :E)%)D< E$A $

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+")

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3 3 D$;;$A: :$HF. $ 2W2 * [V4.444 L$;\ D$;;$A: :)A: :$HF. $H" $ 3UV * [24V.444 L$;\ "+A9E$%L) :$HF'

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:)A: 90H"+:+0HA. 90H",9: A$*^;+HL. $H" $H$;<C) A$*^;)A +H :E) ;$D0%$:0%<' =;; #$:)% J%0*

)-^)%+*)H:A +A :%)$:)" ^%+0% :0 J+H$; "+A9E$%L) :0 %)*0I) $;; $"")" 9E$;;)HL) 90*^0H)H:A $H" $H<

D<Z^%0",9:A 0J $H< :%)$:*)H: A<A:)*A :)A:)"' O0 $990**0"$:) <): ,H")J+H)" $H" ,H+")H:+J+)"

:)9EH0;0L+)A. :E) A<A:)* 9$H D) %)90HJ+L,%)" :0 $990**0"$:) :%)$:*)H: A<A:)*A $: ,^;+J:. +HZ

:$HF. 0% "+A9E$%L) ;09$:+0HA +H :E) J;0# ^$:E'

/0H:%0; 0J $;; A<A:)*A $H" +HA:%,*)H:$:+0H +A ^%0I+")" D< :E) N0H)<#);; c;$H:A9$^) >H",A:%+$;

/0H:%0; $H" =,:0*$:+0H A<A:)* [N0H)<#);;. Q0%%+A O0#HAE+^. 5&\. #E+9E E$A D))H 9,A:0*+C)"

D< )HL+H))%A $: 56(?@ :0 ^%0I+") M@OOBZA^)9+J+9 "$:$ $9a,+A+:+0H. 0^)%$:+0H$; 90H:%0;. $H"

A<A:)* *0H+:0%+HL #+:E $;$%* $H" +H:)%;09F J,H9:+0H$;+:<' OE) +H",A:%+$; 90H:%0; E$%"#$%)

90HA+A:A 0J A:$H"$;0H) 90H:%0; ^$H);A #+:E ^%0L%$**$D;) ;0L+9 90H:%0;;)%A. %);$<A. $H" $H$;0LZ:0Z

"+L+:$; 90HI)%:)%A 90HH)9:)" :0 $ %)",H"$H: 90*^,:)% A)%I)% A<A:)* :0 ;0L $;; $9:+0HA $H" "$:$' =

90*^,:)% +H $ 90H:%0; %00* $"]$9)H: :0 :E) D$;;$A: :$HFA #+:E J+I) "+A^;$<A ^%0I+")A :E) ^%+*$%<

0^)%$:0% +H:)%J$9) :0 90H:%0; 0J :E) M@OOB' OE+A 90H:%0; A<A:)* ^%0I+")A $ A)%+)A 0J L%$^E+9$;

90H:%0; A9%))HA J%0* #E+9E :E) 0^)%$:0% 9$H A);)9: $H" I+)# :E) 0I)%$;; A<A:)* #+:E F)<

^$%$*):)%A "+A^;$<)"'



### 3.2 Microbeads

OE) ^%0-+)A ,A)" J0% C00^;$HF:0H #)%) %)". /E%0*08^E)%)ZO 5>8O /)%:+J+)" *+9%0A^E)%)A

[Q+9%0L)H+9A /0%^0%$:+0H. B%)*0H:. /=\' 7)^)H"+HL 0H :E) :%+$;. 0H) 0% :#0 A+C)A 0J

*+9%0D)$"A #)%) ,A)"n #+:E $ H0*+H$; "+$*):)% 0J VU g* [/K i Y'`o\. U1 g* [/K i Y'`o\ 0%

2W4Zg* q 3'1 g* [/K i 1o\' =;; *+9%0D)$"A #)%) 90*^0A)" 0J $ 9%0AAZ;+HF)" ^0;<A:<%)H)

"+I+H<;D)HC)H) 90^0;<*)%' Q+9%0D)$"A #)%) 90,H:)" +H 2W *; M0L0%0I 90,H:+HL 9E$*D)%A 0%

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^)'

GA,$;;<. *+9%0D)$"A #)%) 90,H:)" :E) "$< D)J0%) $H )-^)%+*)H: $H" ^;$9)" +H $ D)$F)% #+:E

O<^) >> #$:)% [WZg* J+;:)%)" $H" :%)$:)" D< %)I)%A) 0A*0A+A $H" ")+0H+C$:+0H\' OE) 90H:)H:A 0J

:E) D)$F)% #)%) ^0,%)" +H:0 :E) ^;$HF:0H H):. J+;:)% D$L. 0% A+)I) #+:E 9$%) :0 *+H+*+C) A^;$AE+HL

$H" ;0AA 0J *+9%0D)$"A' >H+:+$; :%+$;A ,A)" 144 *+9%0D)$"A :0 $^^%0-+*$:) $ "+A9E$%L) I0;,*) 0J

3 Z3 3 14 * *)):+HL :E) >QX C00^;$HF:0H "+A9E$%L) A:$H"$%" [24 C00^;$HF:0H * - 14 * i 144V

*+9%0D)$"A\d A,DA)a,)H: :%+$;A :<^+9$;;< ,A)" 244 0% T44 *+9%0D)$"A [#E+9E $;;0#)" *0%) :%+$;A

:0 D) %,H. $A ;)AA :+*) #$A A^)H: 90,H:+HL *+9%0D)$"A\' >H $;; 9$A)A. :E) D)$F)% 90H:$+H+HL :E)

*+9%0D)$"A #$A %+HA)" 3ZW :+*)A #+:E O<^) >> #$:)%. $H" :E) D)$F)% $H" #$AE #$:)% #)%)

)-$*+H)" J0% *+9%0D)$"A :E$: *$< E$I) $"E)%)" :0 :E) D)$F)% #$;;'

=: :E) )H" 0J ;$D0%$:0%< :%+$;A 0% J+);" %,HA. :E) A+)I) 0% J+;:)% D$LA #)%) %+HA)" #+:E O<^) >> #$:)%

0% $%:+J+9+$; A)$#$:)% [>HA:$H: X9)$Hrd =a,$%+,* 8<A:)*A. >H9'. Q)H:0% XN\ +H:0 D)$F)%A'

8,DA$*^;)A 0J $^^%0-+*$:);< 2W *; #)%) ^+^)::)" +H:0 M0L0%0I 90,H:+HL 9E$*D)%A' O#0

%)A)$%9E)%A 90,H:)" A,DA$*^;)A ,H:+; :E) )H:+%) I0;,*) 0J )$9E D)$F)% #$A 90,H:)"'

=""+:+0H$;;<. A,%J$9)A :E$: :0,9E)" :E) A$*^;) #)%) )-$*+H)" J0% *+9%0D)$"An D)$F)%A. ^+^):

:+^A. $H" J,HH);A'

B0% )$9E M0L0%0I 9E$*D)% $H$;<C)". 90,H:A #)%) *$") D< $ %)A)$%9E)% ,H:+; :#0 A,DA)a,)H:

90,H:A $L%))"' >H *0A: )-^)%+*)H:A. :#0 %)A)$%9E)%A )-$*+H)" $: ;)$A: 0H) 0J :E) M0L0%0I

/E$*D)%A :0 )HA,%) 90,H:A #)%) :E) A$*)' >J :E) J+H$; 90,H: "+JJ)%)" D):#))H :E) %)A)$%9E)%A.

#E+9E #$A %$%) [$^^%0-+*$:);< T4o 0J :E) :+*)\. +: "+JJ)%)" D< 0H) 0% :#0 *+9%0D)$"A' M)9$,A)

+: #$A $AA,*)" *+9%0D)$"A #)%) *,9E *0%) ;+F);< :0 D) ,H")%90,H:)" :E$H 0I)% 90,H:)". :E)

E+LE)A: H,*D)% #$A ,A)" +H :E) J+H$; :$;;<'



### 3.2.1 Microbead Disintegration

@E)H :E) +H+:+$; %)90I)%< %$:)A #)%) ;0# +H J+);" $H" ;$D0%$:0%< :%+$;A. +: A))*)" ,H;+F);<. D,:

^0AA+D;). :E) *+9%0D)$"A "+AA0;I)" +H A)$#$:)% 0% O<^) >> #$:)%' =H $AA$< #$A 90H",9:)"n +H

A)^$%$:) #);;A 0J $ 2TZ#);; ^;$:). 24 W4Zg* *+9%0D)$"A $H" 24 2W4 g* *+9%0D)$"A

#)%) "+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 $

;$D0%$:0%< D)H9E J0% Y "$<A. $H" :E)H *+9%0D)$"A +H )$9E #);; #)%) 90,H:)"'



### 3.2.2 Method of Microbead Counting

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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<

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"+^^)" +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

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[)$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'

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### 3.3 Filter Bags

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9* t 16” u ;0HL\ #+:E I$%+0,A *)AE A+C)A [244 g*. W4 g*. TW g*. 0% 24 g*\ #)%) ,A)" A+HL;<

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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+;:)%

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/;)**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 9 m (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 9 m (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&$# /"& 9c * 3+- +##$#7h

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. franciscana 5 +)# +'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%$&

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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 %*$

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-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 &()-$#

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(''$#(+%$0B +)# -('40%+)$"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 $+@* %41$>-

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-+',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 +

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3(%* + '4@* 0"3$& @")@$)%&+%(") "/ ."",0+)D%") 8;< 0 I; =7 C(G *4)#&$# "& %$) @",$,"#- 8 Acartia

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#$%$&'()$# 1B -41%&+@%(")7 F$@+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$5 -" 9Q 1&()$ -*&(', 8 Artemia 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$

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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 100Microbead 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

— F ield 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

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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

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+02+$5 +)# %*$ $G,$&('$)% 3+- &$,$+%$# 3(%* 9<< :< ?' +)# 9<< ;:< ?' '(@&"1$+#-7 !*$

&$-40%- 3$&$ 1$%%$&J hOT +)# ;<<T &$@"6$&B $//(@($)@B 8:< ?' +)# ;:< ?' '(@&"1$+#-5

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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

— To xicity 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%$& *"4-()2- 3+- : @' 8 2” = #(+'$%$&5 C@*$#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%$& *"4-()2- 4-()2 : @' 8 2” = #(+'$%$& dQ D2 8;:< 01= ^+) C%")$ /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<79 5” = #(+'$%$& 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 @' 8 3” = #(+,*&+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<< ' O N

E(@*+&# $% +075 9<<Q=79<

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+279;

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 5 0 1 00 150 200 250Pressure 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 250Pressure Drop (psi)

Flow Rate (GPM)

Eaton Topline Filter Housing Pressure Drop ( ps i) vs. Flow (g pm )

Housing pressure drop (psi)

clean filter pressure drop for 25um NMO (psi)

Tota l pr essure drop across housi ng ( psi)

Linear (Total pressure d rop 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< ' 9 7 !*$ -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&)$5 a[=7 A%- -4&/+@$

+&$+ 3+- ;7P9 ' 9 5 )$+&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

+&$+ ()@&$+-$- /&"' %*$ : @' 8 2” = ,(,$ %" %*$ ;Q @' 8 7” = *"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 ',*=79O

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;

79c

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. franciscana 5 , 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('+%$0B d<< @",$,"#- 8 Acartia 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.tonsa N #+%+

)"% -*"3)=7



## 5 Discussion

K /(0%$& -D(# 3+- -4@@$--/400B #$-(2)$# %" -+',0$ 0+&2$ 6"04'$- "/ 3+%$& %" #$%$&'()$ (/ (%-

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7 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 μm

diameter

microbeads

recovered a

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

1 K ,&$6("4- %&(+0 @")#4@%$# 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$=

"& /&$-*3+%$&79h



### 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$$)

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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<

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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+2-7O<



### 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 Art emia franciscana 5 @40%4&$# 1&()$ -*&(',7

Replicate Live

c opepods

(≥ 50 μm) Total

c opepods

(≥ 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%$& CD(# #&+3()2 3(%* #('$)-(")-7O9



### 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,-%&$+' +)# #"3)-%&$+' "/ %*$ XaC7OOV-313V-404V-405V-303EFS HousingsHousings 2" Manual Inlet/Outlet ValvesPrimary Pumps 8 & 9Diaphragm Pump 5Ballast TankDischarge TankV-510TITLEAquatic Nusiance Species Ballast Water Treatment Test FacilityANS BWTTF - NRLKW, Trumbo Point Annex, Key West, FLEaton Filter Skid Schematic OverviewScale: N/ADATE15 June 2010DRAWN BYNRL Code 6136Cameron MoserREVISION00V-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 valveSP-11 Flanged T from 6" main line into EFS sample portSP-114(*%,- 1"*#+"*-++%$4&-1 "-3 *2"-#"1 2"0 /+&%".+,- !* '( )%&$"#!56

5( 7 lose 1/2” 1% *"- 2,%+ 8*.8&3 ,- &*9: ,; +:& "-1"8"1$*. )*+,- <,2."-&= ;".+&% :,$3"-#3 *+

+:&"% 2,3+&%",% &-13 >!"#$%& ? *-1 !"#$%& 5@ A*..,,- BC(

!"#$%& 5( )*+,- <,2."-&= !".+&% D,$3"-# $3&1 "- +:& )*+,- !".+&% /0"1(

6( E2&- &*9: :,$3"-# AF $-39%&G"-# +:& ;,$% ."1 9.*423 *+ +:& *-+&%",% &-1 *-1 .";+"-# +:&

:"-#&1 ."1 AF "+3 :*-1.& >!"#$%& ? ,% !"#$%& 5@ A*..,,- 'C(

B( H-3+*.. * ;".+&% A*# "- &*9: ,; +:& ;".+&% :,$3"-#3 >!"#$%& 6C( <:& ;".+&% A*# G".. 3."1& "-+,

+:& :,$3"-#@ *-1 +:& +,2 2.*3+"9 %"-# ,; +:& ;".+&% A*# 3:,$.1 3&*+ "-+, +:& +,2 ,; +:&

:,$3"-#( <*0& 9*%& G:&- "-3+*.."-# +:& ;".+&% A*#3 +, 2%&8&-+ +:&"% %"22"-# ,- 3:*%2 &1#&3

1$%"-# "-3+*..*+",-(

I( !".. &*9: ,; +:& :,$3"-#3 ;%,4 +:& +,2 $3"-# * :,3& G"+: %$--"-# 3&*G*+&%( !".+&% +:&

3&*G*+&% AF 2.*9"-# +:& &-1 ,; +:& :,3& "- * 'BJ μ 4 4&3: ;".+&% A*# *-1 1"%&9+"-# +:&

G*+&% +:*+ 2*33&3 +:%,$#: +:& ;".+&% A*# "-+, *.. ;".+&% :,$3"-#3(

K( 7.,3& &*9: ,; +:& "-1"8"1$*. ;".+&% :,$3"-#3 AF &-3$%"-# EJ%"-#3 *%& "- +:& 2%,2&%

2,3"+",-@ G:"9: "3 1&+&%4"-&1 AF &-3$%"-# &*9: EJ%"-# ."&3 ;.*+ "- "+3 1&3"#-*+&1 9"%9$.*%

3.,+ >!"#$%& 6C( /:$+ +:& ."1 *-1 %&J+"#:+&- +:& ;,$% ."1 9.*423(5B

!"#$%& 6( !".+&% :,$3"-# G"+: 9,8&% ,2&- *-1 ;".+&% A*# "-3+*..&1L EJ%"-#3 *%& 2%,2&%.F 3&*+&1(

M( E2& n 1⁄4” A.&&1 2,%+ 8*.8&3 *+ +:& *-+&%",% &-1 ,; &*9: :,$3"-# >!"#$%& 5@ A*..,,- 5C(

N( E2&- *.. 4*-$*. *-1 9,-+%,. 8*.8&3 +, 9%&*+& * ;.,G 2*+: ;%,4 +:& A*..*3+ +*-0 +, +:&

1"39:*%#& +*-0 G"+: *- &O9&2+",- ,; +:& )!/ *-1 1"39:*%#& +*-0 "-.&+3@ G:"9: *%& +,

%&4*"- closed

( ?P( <:& )!/ 1%*G3 ;.,G ;%,4 3*42.& 2,%+ ??>!"#$%& '@ /QJ??C G:"9: 9,--&9+3 +, +:& main 6”

A*..*3+ +*-0 1"39:*%#& ."-& AF * < ;"++"-#( /.,G.F ,2&- +:& )!/ "-.&+ 8*.8& +,

*22%,O"4*+&.F BPR $-+". G*+&% $-1&% :&*1 2%&33$%& ;%,4 +:& A*..*3+ +*-0 #&-&%*+&3 *

3+&*1F 3+%&*4 ;.,G"-# ;%,4 +:& ,2& n 1⁄4” ;".+&% :,$3"-# A.&&1 2,%+3@ "-1"9*+"-# * ;$.. 2%"4&

"3 *9:"&8&1(

*( S+ +:"3 2,"-+@ "; * :,$3"-# ."1 "3 -,+ 2%,2&%.F 3&*.&1@ * .&*0 >1$& +, * .,,3& ."1

9.*42 ,% $-*."#-&1 EJ%"-#C G".. A& *22*%&-+( <:"3 3"+$*+",- 4$3+ A& %&4&1"&1 AF

9.,3"-# +:& )!/ "-.&+ 8*.8&@ "3,.*+"-# +:& .&*0"-# :,$3"-#3 *+ +:&"% "-1"8"1$*. "-.&+3

*-1 ,$+.&+3 AF 9.,3"-# 2” 8*.8&3 >!"#$%&3 ? *-1 'C@ *-1 +:&- 9,%%&9+"-# +:& "33$&(

A( T&J,2&- :,$3"-# "-.&+ *-1 ,$+.&+ 8*.8&3 +:&- %&2&*+ 3+&2 M(

??( <:%,++.& A*90 +:& 4*-$*. )!/ "-.&+ 8*.8& $-+". +:& ;.,G "- 3+&2 ?P "3 %&1$9&1 +, *

4"-"4$4@ +:&- 9.,3& *.. A.&&1 2,%+ 8*.8&3(

?'( E2&- +:& )!/ "-.&+ 8*.8& 9,42.&+&.F( Q%&33$%& ;%,4 +:& A*..*3+ +*-0 G".. &U$*."V&

+:%,$#:,$+ +:& 3F3+&4 $23+%&*4 ,; +:& 1"39:*%#& +*-0 "-.&+ 8*.8& 3$9: +:*+ ,2&-"-# "+

G,$.1 "-8,0& ;.,G@ "(&(@ +:"3 8*.8& 3:,$.1 A& +:& .*3+ ,A3+*9.& 2%&8&-+"-# :&*1 2%&33$%&

;%,4 +:& A*..*3+ +*-0 ;%,4 9*$3"-# * #%*8"+FJ"-1$9&1 ;.,G "-+, +:& 1"39:*%#& +*-0(

?5( 7:&90 +:& ;$&. .&8&. ;,% 2,%+*A.& *"% 9,42%&33,% +, &-3$%& +:&%& "3 *- *42.& *4,$-+ ;,%

+:& 1&3"%&1 %$- +"4&(5I

?6( D,,0 $2 +:& 3⁄4” *"% :,3& ;%,4 +:& 1"*2:%*#4 2$42 +, +:& 2,%+*A.& *"% 9,42%&33,%( <:&

3⁄4” *"% :,3& :*3 7:"9*#,J+F2& +G"3+J.,90 ;"++"-#3( <:&3& ;"++"-#3 3:,$.1 *."#- *-1 4*+&

9,%%&9+.F(

?B( <:& 1"*2:%*#4 2$42@ 2,G&%&1 ;%,4 :"#:J2%&33$%& *"% #&-&%*+&1 AF +:& 9,42%&33,%@

;&&13 "-+, *-1 "3 9,-+%,..&1 A"-*%".F $3"-# *- &.&9+%"9*..F *9+$*+&1 3,.&-,"1 8*.8& .,9*+&1

+,G*%1 +:& +,2 &-1 ,; +:& 2$42( )-3$%& +:& 2,G&% 2.$# ;%,4 +:& 3,.&-,"1 8*.8& "3

2.$##&1 "-+, * 2,G&% %&9&2+*9.& -&*% +:& 1"*2:%*#4 2$42(

?I( /+*%+ +:& 9,42%&33,% *-1 +:&- ,2&- +:& 9,%%&32,-1"-# 4*-$*. 8*.8&3 +:*+ ;&&1 *"% +, +:&

1"*2:%*#4 2$42 >!"#$%& '@ W"*2:%*#4 Q$42C(

?K( S+ +:& XY<<! "- Z&F Y&3+@ * D,-&FG&.. Q.*-+39*2& 7,-+%,. *-1 S$+,4*+",- 3F3+&4 "3

"-+&#%*+&1 3$9: +:*+ *.. 3G"+9:&3@ 3&-3,%3@ *-1 4*9:"-&%F 3&-1 "-2$+3 *-1 %&9&"8& ,$+2$+3

;%,4 * 2%,#%*44*A.& .,#"9 9,-+%,..&% +:*+ 9*- A& 4,-"+,%&1@ ,2&%*+&1@ *-1 9,-+%,..&1

;%,4 * :$4*-J4*9:"-& "-+&%;*9& >D[HC "- * 9&-+%*."V&1 9,-+%,. %,,4( \3"-# *- "1&-+"9*.

,% 3"4".*% 3F3+&4 "3 %&U$"%&1 +, &-+&% 2%,2&% 2*%*4&+&%3 ;,% 1&3"%&1 2%&33$%& *-1 A*..*3+

+*-0 1"39:*%#& *-1 )!/ 3*42."-# ;.,G 3&+ 2,"-+3 "-+, 9,-+%,. ,2&%*+",- "-2$+ ;"&.13 "- +:&

D[H( W&2&-1"-# ,- +:& .,9*+",- ,; +:& 9,-+%,.."-# ;.,G 4&+&%@ +:& 3&+ 2,"-+ 3:,$.1 A&

*1]$3+&1 +, *99,$-+ ;,% 3*42."-# ;.,G +, *9:"&8& +,+*. 1&3"%&1 ;.,G@ &(#(@ "; +:& 2%"4*%F

2$423 *%& 9,-+%,..&1 $3"-# * ;.,G 4&+&% $23+%&*4 ;%,4 G:&%& 3*42."-# ;.,G %&],"-3

4*"- ;.,GL +:& 3&+ 2,"-+ 3:,$.1 A& +:& +,+*. 1&3"#-&1 ;.,G 4"-$3 +:& 3*42."-# ;.,G(

E2&%*+",-

?( Q*%+"*..F ,2&- +:& "-.&+ 9,-+%,. 8*.8& +, +:& 1"39:*%#& +*-0 >!"#$%& '@ ^*.8& 6PBC $3"-# +:&

D[HL 8&%";F +:*+ ;.,G "3 "-"+"*."V&1 ;%,4 +:& A*..*3+ +*-0 +, +:& 1"39:*%#& +*-0(

Note: H; ."++.& ,% -, ;.,G ,A3&%8&1@ *- "33$& -&&13 ;$%+:&% "-8&3+"#*+",- AF +:&

3F3+&4 ,2&%*+,%( 7*$3&3 4*F "-9.$1& 8*.8&3 3&+ "- *- "42%,2&% 2,3"+",-@ *-

"-,2&%*A.& ;.,G 4&+&%@ ,% * :"#:&% .&8&. ,; G*+&% "- +:& 1"39:*%#& +*-0 +:*- +:&

A*..*3+ +*-0(

'( X&#"- +:& 1"*2:%*#4 2$42 *-1 3*42."-# *$+,4*+",- ;%,4 +:& D[H AF 9."90"-# _/+*%+`

$-1&% +:& *22%,2%"*+& 9,-+%,. ,2&%*+",-3( Q$423 *-1 8*.8&3 G".. *9+$*+& +, *9:"&8&

1&3"%&1 3&+ 2,"-+3(

5( 7,-+"-$*..F 4,-"+,% )!/ 2%&33$%& *-1 ;.,G +, &-3$%& ;".+&% A*#3 1, -,+ 9.,#L 2%&33$%&

3:,$.1 -,+ &O9&&1 5P 2,$-13 2&% 3U$*%& "-9:(

/:$+1,G-

?( S3 :&*1 2%&33$%& ;%,4 +:& A*..*3+ +*-0 1&9%&*3&3@ +:& 2%"4*%F 2$423 >!"#$%& '@ Q$423 M

*-1 NC G".. 3+*%+ +, ,8&%2,G&% +:& 3$9+",- ;%,4 +:& 1"*2:%*#4 2$42 1%*G"-# G*+&% ;%,4

+:& 3*42.& 2,%+ >!"#$%& '@ /QJ??C( <:"3 3"+$*+",- 4$3+ A& 2%&8&-+&1 AF 9.,3&.F

4,-"+,%"-# )!/ ;.,G *3 +:& A*..*3+ +*-0 .&8&. A&9,4&3 .,G >%&.*+"8& +, +:& +,+*. ;.,G *-1

+*-0 1"4&-3",-3@ "(&(@ *22%,O"4*+&.F ?P 4"-$+&3 %&4*"-"-# *+ +:& 9$%%&-+ ;.,G %*+& *-1

%&4*"-"-# 8,.$4&C $-+". "+ 3$11&-.F A&#"-3 +, 1&9%&*3& 1%*4*+"9*..F( S+ +:"3 2,"-+a

*( /:$+ 1,G- +:& 2%"4*%F 2$423 >!"#$%& 'C(

A( 7.,3& +:& 2%"4*%F 2$423` "-.&+ 9,-+%,. 8*.8& >!"#$%& '@ ^*.8& 5P5C +, "3,.*+& *..

;.,G +:%,$#: )!/(

9( E2&- +:& 9,-+%,. 8*.8& +, +:& 1"39:*%#& +*-0 "-.&+ +, ?PPR >!"#$%& '@ ^*.8& 6PBC(

1( b,%4*. ;.,G +:%,$#: )!/ 3:,$.1 A& %&3+,%&1 *-1 3+*A.&(5K

'( W&2&-1"-# ,- +*-0 1"4&-3",-3@ ;.,G G".. 9,-+"-$& ;,% * 2&%",1 $-+". "+ *#*"- A&#"-3 +,

1&9%&*3& 1%*3+"9*..F( S+ +:"3 2,"-+a

*( 7.,3& +:& *$+,4*+&1 1"*2:%*#4 8*.8& >!"#$%& '@ ^*.8& 5?5C *+ +:& ,$+.&+ ,; +:&

1"*2:%*#4 2$42(

A( H44&1"*+&.F 3:$+ 1,G- +:& 1"*2:%*#4 2$42(

9( H44&1"*+&.F "3,.*+& )!/ ;%,4 +:& 3F3+&4 AF 9.,3"-# +:& 3"O 2” A*.. 8*.8&3 *+ +:&

;*%+:&3+ $23+%&*4 *-1 1,G-3+%&*4 ;".+&% "-.&+3 *-1 ,$+.&+3 >!"#$%& BC(

!"#$%& B( X*.. 8*.8&3 >"- %&1 9"%9.&3C 9.,3&1 "44&1"*+&.F *;+&% +:& 1"*2:%*#4 2$42 "3 3:$+ 1,G-(

5( H3,.*+& +:& %&4*"-"-# :,$3"-#3 AF 9.,3"-# *.. 2” A*.. 8*.8&3(

6( T&.&*3& 2%&33$%& *+ +:& 2,3+&%",% & nd 1/2” dr *"- 8*.8&3 3$9: +:*+ ;.,G 9:*--&.3 +:%,$#:

+:& ;".+&%3 *-1 ,$+ +:%,$#: +:& 1%*"- 8*.8&3(

B( E-9& 2%&33$%& %&.&*3&3@ ,2& n the 1⁄4” A.&&1 8*.8&3 *+ +:& +,2 ,; +:& :,$3"-#3( <:&

%&4*"-"-# G*+&% G".. 1%*"- ,-+, +:& #%,$-1 $-+". -, 4,%& G*+&% &O"+3 +:& :,$3"-#3(

I( E2&- +:& ."1 ,; &*9: :,$3"-# AF $-39%&G"-# ."1 9.*423 *-1 .";+"-# *+ +:& :*-1.&(

<:& ;".+&% A*#3 *%& %&*1F ;,% &O+%*9+",-(5M



### Appendix 6

)*+,- !".+&% /0"1 A".. ,; 4*+&%"*.3 *-1 9*.9$.*+",- ,; G&"#:+(



### Equipment Quantity Cost Total CostFilter Housing

Eaton 316SS Topline TM Housing 10 $1,707.00 $17,070.00Filter Bags

Filter Specialist Incorporated 35μm size 2 NMO

35 with plastic snap rings 10 $20.43 $204.30PVC 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 $24.205N 

### Equipment Quantity Cost Total CostWood 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 $4.00Stainless hardware 5/8-11x3" 316 stainless steel hex cap screw

p artially threaded 100 $4.32 $432.00

5/8-11 316 Stainless steel hex cap nut 2 2 $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 $46.90Cam-LockFittings

Aluminum Cam-and-Groove Hose Coupling

Plug, PFA Adapter, 3 Coupling Size, 3" Pipe Size 2 $62.30 $124.60

Cost of Equipment Components $19,851.57Labor

H ours to build skid per design drawing 80 $150.00 $12,000.00

Total Discharge Skid Cost $19,851.576P



### 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 9 m (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&(

)U$*+",- 6( !.,G 8&.,9"+F 9*.9$.*+",- ;,% 'BJ#24 ;.,G "- *- ?M 94 > 7” C 1"*4&+&% ;".+&% :,$3"-#(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

z oopl

(≥ 50 μm) Total

z oopl

(≥ 50 μm) % living

z oopl

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 franciscana L 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",-

.