1 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 

# Proceedings of Ballast Water Discharge Standards Compliance Subject Matter Expert (SME) Workshop

Distribution Statement: Distribution Statement C: Distribution authorized t o U.S. Government Agencies and their contractors (Adm inistrative/Operational Use) (25 May 2011). Other requests shall be referred to the Technical Director, USCG R&D Center, New London, CT 06320.

Destruction Notice: For unclassified, limited documents, destroy by any method that will prevent disclosure of contents or reconst ruction of the document.


### August 2011Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

ii UNCLAS | CG-926 R&DC | G. E. Roderick| RDC | August 2011 

# N O T I C E

This document is disseminated under the sponsorship of the Department of Homeland Security in the interest of information ex change. The United States Government assumes no liability for its cont ents or use thereof.

The United States Government does not endorse produ cts or manufacturers. Trade or manufacturers’ names appear herein solely because they are considered essential to the object of this report.

This report does not constitute a standard, specifi cation, or regulation.

United States Coast Guard Research & Development Center

1 Chelsea Street New London, CT 06320Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

iii UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 Technical Report Documentation Page

1. Report No. 2. Government Accession Number 3. Recipient’s Catalog No.

4. Title and Subtitle Assessment of Approaches for Compliance and Enforcement of Ballast Water Discharge Regulations: Proceedings of Ballast Water Disc harge Standards Compliance Subject Matter Expert Workshop 5. Report Date 26 August 2011

6. Performing Organization Code Project No. 410131

7. Author(s) Mr. Brandon Lee, Mr. Mario Tamburri and Ms. Sue Kim 8. Performing Report No. R&DC UDI #

9. Performing Organization Name and Address ABS Consulting 1525 Wilson Blvd., Suite 625 Arlington, VA 22209 U.S. Coast Guard Research and Development Center

1 Chelsea Street New London, CT 06320 10. Work Unit No. (TRAIS)

11. Contract or Grant No. HSCG32-11-J-200082

12. Sponsoring Organization Name and Address U.S. Department of Homeland Security Commandant (CG-533) United States Coast Guard

2100 Second St. SW Washington, DC 20593-0001 13. Type of Report & Period Covered Final

14. Sponsoring Agency Code Commandant (CG-533) U.S. Coast Guard Headquarters Washington, DC 20593-0001

15. Supplementary Notes The R&D Center’s technical point of contact is Gail Ro derick, (860) 271-2658, Gail.E.Roderick@uscg.mil

16. Abstract (MAXIMUM 200 WORDS)
This report describes the results of inquiries and analys es in support of HSCG23-08-Q-MSR068, Study of Current Ballasting Operations Conducted in U.S. Waters. In keeping with the C oast Guard’s mission, the report supports a range of Coast G uard decisions to manage the risk of biological invasions, f ocusing primarily on the existing ballasting operations.

The USCG is charged with developing protocols and regulat ions for verifying compliance with both Phase One and Pha se Two ballast water discharge standards. It has proposed a two phase approach for implementing regulation of balla st water,
with the Phase One a set of standards similar to stand ards in the IMO Ballast Water Convention and Phase Two st andards potentially 1,000 times stricter than the Phase One standards . In this work, a compliance framework will be developed a nd refined with the input from research and from SMEs addr ess any technologies or concepts that address complianc e testing and

verification.

17. Key Words Ballast Water, Technologies, Compliance,

Enforcement 18. Distribution Statement Distribution Statement C: Distribution authorized to U.S . Government Agencies and their contractors (Administrative/Operatio nal Use) (25 May 2011). Other requests shall be referred to the Technic al Director, USCG R&D Center, New London, CT 06320.

19. Security Class (This Report) UNCLAS 20. Security Class (This Page) UNCLAS 21. No of Pages 25 (Total) 22. PriceProceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

iv UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 

### EXECUTIVE SUMMARY

This report describes the proceedings of the Ballast Wat er Discharge Standards Compliance Subject Matter Expert (SME) Workshop held 28 – 30 June 2011 at the ABSG Consultin g office in New London, CT in support of HSCG32-11-J-200082, Assessment of Approaches for Compli ance and Enforcement of Ballast Water Discharge Regulations. In keeping with the Coast G uard’s mission, the report provides a preliminary framework and input from SMEs on verifying compliance with proposed regulation and the proper functioning of ballast water treatment systems. It is intended to support a range of U.S. Coast Guard (USCG) decisions regarding potential regulatory steps in support of their marine environmental protection mission. It primarily focuses on the indices and meas ures that can be utilized to determine compliance with ballast water discharge standards.

The USCG is charged with developing protocols and regulations for verifying compliance with ballast water discharge standards. It has proposed a two-phase approach f or implementing regulation of ballast water, with Phase One including a set of discharge standards sim ilar to those found in the International Maritime Organization (IMO) Ballast Water Convention and potent ial future Phase Two standards approximately 1,000 times stricter than the Phase One standards. In this work, a compliance framework will be developed and refined, with input from research and SMEs. The frame work will seek to address any methods, technologies or concepts that might be employed in co mpliance monitoring and verification for vessels’ discharge to meet the current Phase One ballast water standard or the other potential standards proposed.

The ABSG Consulting team has developed a compliance monit oring framework to identify the requisite methods, instruments and approaches for determining complianc e for the various ballast water treatment types. The compliance framework presented includes three levels, which provide increasing levels of

confidence:

1. Measures of treatment performance; 2. Indirect measures of exceedance of the discharge st andard; and 3. Direct measures of compliance with the discharge sta ndard.

Compliance level 1 would utilize indirect or indicative system performance and/or water quality measures that demonstrate appropriate treatment operations, or co nditions have been met, for various ballast water treatment approaches. While level 1 measures may not direct ly establish compliance with numerical discharge standards, they can determine if equipment is o perating as certified or per manufacturer’s specifications. While these measures can be collected in dependently by inspectors, the majority should be integrated into the ballast water treatment systems t hemselves, and allow for inspectors to review instrument outputs. Some level 1 parameters might also be measure d independently by inspectors in appropriately collected samples of water before or during discharge.

As a relatively quick estimate of whether discharge fr om a vessel is significantly above required limits, compliance level 2 would focus on bulk estimates of bioma ss. Exceedance can be assessed using various biological, chemical, and optical measures, currently a vailable or in development, that are related to live organism abundances. Some of these measures might only pr ovide indications of gross exceedance, while others may provide finer resolution. These measures can a lso be collected autonomously or by inspectors.Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

v UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 However, the various indirect measure thresholds, at w hich the discharge from a vessel is considered out of compliance, will take additional consideration and study to establish.

If it is suspected that a vessel discharge will exceed the discharge standards, compliance level 3 could be used to make direct measurements of the various regulated biological categories. Compliance level 3 would involve detailed biological testing for live organisms, simi lar to evaluations currently conducted under treatment system certification testing, and provide the highest level of scientific certainty and statistical confidence. While several of the methods and approaches for level 3 compliance monitoring currently exist and are employed in full scale shipboard testing for type approval, several logistical challenges remain to be resolved prior to implementation for assessment of ve ssel compliance.

To further refine the compliance monitoring framework, t he ABSG Consulting team has organized, convened, and facilitated a 3-day workshop on 28 – 30 June 2011 with SMEs from the field and representation from the various pertinent governmental b odies like the USCG, Environmental Protection Agency (EPA), Maritime Administration (MARAD), and Na val Research Laboratory (NRL) to provide input on the compliance verification framework and other i nsights on the subject. For each compliance level, the recommendations are based on either the tr eatment type for compliance level 1 or by the organism category being measured for compliance levels 2 and 3.

Through this workshop, the ABSG Consulting team has liste d the discharge category and list the measures to be utilized for each level and has captured the method, i nstrument or analysis necessary. For each instrument, the participants in the workshop indicated th e level of deployment readiness for each technology and the signal relevance of each measurement. In addit ion, the advantages, disadvantages, and requirements for operational utilization were discussed for each lev el and for each instrument.Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

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vii UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 

### TABLE OF CONTENTS

1 Background ..........................................

................................................... ........................................... 1

2 Scope ..............................................

................................................... .................................................. 2

3 Methodology ...........................................

................................................... ......................................... 2

3.1 Literature Review ...................................

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

3.2 Compliance Monitoring Framework ......................

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

3.3 SME Workshop ........................................

................................................... ................................. 4

3.3.1 Technology Readiness Levels .........................

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

3.4 Report .............................................

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

4 Findings ...........................................

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

4.1 Compliance Level 1 – Measures of Treatment Performance

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

4.1.1 Approved Treatment Components .........................

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

4.1.2 Potential Treatment Components .......................

................................................... .................. 16

4.2 Compliance Level 2 – Indirect Measures of Exceedance ..

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

4.3 Compliance Level 3 – Direct Measures of Compliance ...

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

5 Recommendations and Conclusions .........................

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

APPENDIX A. Subject Matter Expert Workshop Agenda ................. ........................................... A-1

APPENDIX B. Subject Matter Expert Workshop Attendees ............ ............................................. B-1

APPENDIX C. Technology Readiness Levels .............................

................................................... .. C-1

APPENDIX D. Measurements .......................................

................................................... ............... D-1

APPENDIX E. Instrument or Analysis of Measurement ................ ................................................ E- 1

APPENDIX F. Subject Matter Expert Workshop Speakers .............. ............................................ F-1

APPENDIX G. Subject Matter Expert Workshop Presentations .......... ......................................... G-1Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

viii UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 

### LIST OF TABLES

Table 1: Ballast Water Discharge Standards .............

................................................... ................................. 1 Table 2: Department of Defense TRL Levels .............

................................................... ............................... 8 Table 3: Compliance Level 1: Measures of Treatment Perfo rmance ............................................ ............... 10 Table 4: Compliance Level 1: Measures of Treatment Perfo rmance (Potential) ................................. ......... 11 Table 5: Compliance Level 1: Recommendations ............

................................................... ........................ 12 Table 6: Compliance Level 2: Indirect Measures of Exceeda nce ............................................... ................. 18 Table 7: Compliance Level 2: Recommendations ............

................................................... ........................ 20 Table 8: Compliance Level 3: Direct Measures of Discharge Standard ........................................... ............ 21 Table 9: Compliance Level 3: Recommendations ............

................................................... ........................ 23Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

ix UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 

### LIST OF ACRONYMS, ABBREVIATIONS, AND SYMBOLS

Acronym Definition ABSG American Bureau of Shipping Group ATP Adenosine Triphosphate BWDS Ballast Water Discharge Standard BWMS Ballast Water Management System CDOM Colored Dissolved Oxygen Matter CFR Code of Federal Regulations CFU Colony Forming Unit CMFDA 5-Chloromethylfluorescein Diacetate COTR Contracting Officer’s Technical Representative CTC 5-Cyano-2,3 Ditolyl Tetrazolium Chloride

CWA Clean Water Act DNA Deoxyribonucleic Acid DO Dissolved Oxygen DoD Department of Defense DPD N,N Diethyl-1,4 Phenylenediamine Sulfate EPA Environmental Protection Agency ETV Environmental Technology Verification

FDA Fluorescein Diacetate

GFI Government Furnished Information

GVP General Vessel Permit HPLC High Performance Liquid Chromatography IMO International Maritime Organization LOPC Laser Optical Plankton Counter

MARAD Maritime Administration

MSD Marine Sanitation Device

m-TEC Membrane Thermotolerant E. coli NASA National Aeronautics and Space Administration NRL Naval Research Laboratory

ORP Oxidation Reaction Potential OWS Oily Water Separator

PA Peracetic Acid PAM Pulse Amplitude Modulated qPCR Quantitative Polymerase Chain Reaction R&DC, RDC Coast Guard Research and Development Center

RNA Ribonucleic Acid SME Subject Matter Expert

SOW Statement of Work TRL Technology Readiness Level

TRO Total Residual OxidantProceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

x UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 Acronym Definition

U.S. United States

USCG United States Coast Guard

UV Ultraviolet

UV Ultraviolet VHF Very High Frequency VOC Volatile Organic CompoundProceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

1 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 

### 1 BACKGROUND

Ballasting is a necessary function of vessels. Vesse ls take on ballast water for a variety of reasons, including, but not limited to, adjusting trim and reversing the e ffects of discharged cargo on stability. Vessels also discharge ballast water for similar rea sons. Longitudinal and transverse loadings must also be kept balanced to ensure stability. Additionally, there are structural requirements for balanced loading to ensure that bending moments and stresses within do not com promise the strength limitations of the ship. Many ships have been lost as a result of uneven loading. As variations occur in weight differentials on a ship, variable ballast—and ballast water in particular—is necessary to ensure and environmentally sound transport. This ballast water, however, has inherent da ngers of its own.

Because ballast water systems vary significantly by ship type and even by ship, only a general description is provided here. In general, ballast water systems are compr ised of one or more sea chests, from which one or more pumps draw water, typically via filters. These pum ps are connected to tanks used for ballast via one or more manifolds, which allow the direction of flow to an d from the designated tanks, or overboard. The same systems are used to both fill and drain tanks as needed, with an overboard discharge separate from the sea chest used for draining the tank. As water from one part of the world is transported to another and discharged, the issue of Non-indigenous species (NIS), so metimes known as aquatic nuisance species (ANS) are brought to United States waters. While not all these species survive relocation, many do—and some thrive. Of these, a number have caused significant ecological and economic damage. It has been estimated that NIS may cost the United States over one hundred billion dollars per year.

These environmental consequences have resulted in a range o f regulations relating to ballast water discharge. The discharge standards utilized for this study are based on the protocols and regulations for verifying compliance proposed by the USCG. The USCG has pr oposed a two phase approach for implementing regulation of ballast water, with the Ph ase One a set of standards similar to standards in the IMO Ballast Water Convention and Phase Two standards pot entially 1,000 times stricter than the Phase One standards (Table 1). To comply with the standards, vessel s have begun installing ballast water treatment systems to treat ballast water to comply with the acc epted organism levels at discharge.

Table 1: Ballast Water Discharge Standards

Organisms >50 μm

in minimum

dimension Organisms 10-50 μm

in minimum

dimension Bacteria Viruses

per m 3 per ml per ml per ml

IMO D-2 10 10 no limit no limit

USCG Phase 1 10 10 no limit no limit

USCG Phase 2 0.01 0.01 10 100Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

2 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 Diverse ballast water treatment systems are being deve loped to meet the IMO and USCG discharge standard (see Efficacy of Ballast Water Treatment Systems: a Report by th e EPA Science Advisory Board, 2011) . Prior to use on vessels, each treatment system must go thro ugh extensive laboratory, land-based and shipboard testing. Only after the treatment systems has be demo nstrated to safely, effectively and reliably meet the discharge standards is it provided with a Type Approval cert ificate for use in routine vessel operations.

This report addresses the monitoring of vessel discharges to estimate or determine compliance using a three level framework. This compliance framework has been deve loped and refined, with input from research and SMEs. The framework addressed methods, technologies or con cepts that might be employed in compliance monitoring and verification for vessels’ discharge to meet the current Phase One ballast water standard or the other potential standards proposed. Input from SMEs was gathered during the SME workshop held 28 – 30 June, 2011 at the ABSG Consulting office in New London, CT

.



### 2 SCOPE

The goal of this workshop was to gather expert insight an d advise on methods, instruments and approaches that can be used in a three level compliance framework. To achieve these goals, the workshop included:

1. A review of the USCG proposed rule making and the EPA Vess el General Permit (VGP);

2. The current state of ballast water treatment systems ; 3. Current ballast water treatment certification testin g methods and approaches;

4. Presentation of a proposed three-level compliance monit oring framework;

5. Aggregation of measurements, methods, instruments and anal yses per treatment type or regulated discharge category; and

6. Assessment of each method, instrument or analysis.



### 3 METHODOLOGY

Our technical approach incorporated literature reviews, SME ev aluations, and a report. Highlights included the following:

• Coordinating with the Contracting Officer’s Technical Repr esentative (COTR) to obtain Government Furnished Information (GFI);

• Conducting a literature review to develop a list of potential ballast water discharge treatment systems and a list of performance factors and measures, instrument s and analyses;

• Developing a compliance verification framework to evaluate technologies and to determine what measures to be utilized for ensuring compliance; and

• SME workshop held 28 – 30 June, 2011 in New London, CT at the ABS G Consulting office to elicit feedback on the framework and input into factors and meth ods. The workshop agenda is included as Appendix A.Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

3 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 

### 3.1 Literature Review

The initial step in the methodology was to obtain and re view all literature available relating to ballast water treatment technologies. We reviewed information provided by the Government and from other sources like the EPA Science Advisory Board, American Bureau of Shippi ng, the Alliance for Coastal Technologies, and from Lloyd’s Register. The literature review produced four outputs that were then used for the

evaluation:

• List of current and potential ballast water treatment te chnologies.

• List of standards or regulations that may apply to vessel s concerning the discharge of ballast water.

• Performance evaluation criteria, preliminary measures, instruments, and analyses were identified.

• Preliminary criteria for each of the performance meas ures selected and inputted into the compliance

verification framework to evaluate the identified techno logies and compliance.



### 3.2 Compliance Monitoring Framework

The ABSG Consulting team has developed a compliance monit oring framework to identify the requisite methods, instruments and approaches for determining complianc e for the various ballast water treatment types. The compliance framework presented includes three levels, which provide increasing levels of

confidence:

1. Measures of treatment performance; 2. Indirect measures of exceedance of the discharge st andard; and 3. Direct measures of compliance with the discharge sta ndard.

Compliance level 1 would utilize indirect or indicative system performance and/or water quality measures that demonstrate appropriate treatment operations, or co nditions have been met, for various ballast water treatment approaches. While level 1 measures may not direct ly establish compliance with numerical discharge standards, they can determine if equipment is o perating as certified or per manufacturer’s specifications. While these measures can be collected in dependently by inspectors, the majority should be integrated into the ballast water treatment systems t hemselves, and allow for inspectors to review instrument outputs. Some level 1 parameters might also be measure d independently by inspectors in appropriately collected samples of water before or during discharge.

As a relatively quick estimate of whether discharge fr om a vessel is significantly above required limits, compliance level 2 would focus on bulk estimates of bioma ss. Exceedance can be assessed using various biological, chemical, and optical measures, currently a vailable or in development, that are related to live organism abundances. Some of these measures might only pr ovide indications of gross exceedance, while others may provide finer resolution. These measures can a lso be collected autonomously or by inspectors. However, the various indirect measure thresholds, at w hich the discharge from a vessel is considered out of compliance, will take additional consideration and study to establish.

If it is suspected that a vessel discharge will exceed the discharge standards, compliance level 3 could be used to make direct measurements of the various regulated biological categories. Compliance level 3 would involve detailed biological testing for live organisms, simi lar to evaluations currently conducted underProceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

4 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 treatment system certification testing, and provide the highest level of scientific certainty and statistical confidence. While several of the methods and approaches for level 3 compliance monitoring currently exist and are employed in full scale shipboard testing for type approval, several logistical challenges remain to be resolved prior to implementation for assessment of ve ssel compliance.



### 3.3 SME Workshop

The compliance verification framework was presented to SMEs at the workshop held 28 – 30 June, 2011 in the ABSG Consulting office in New London, CT. SMEs inc luded representation from the various pertinent governmental bodies like the USCG, EPA, MARAD, and NRL. Other SMEs included leaders in the fields of ballast water treatment and invasive species and rela ted disciplines such as wastewater and drinking water treatment; aquatic toxicology; water quality instru mentation; genetics and molecular biology; and environmental policy and enforcement.

The discussion on the first day of the workshop, 28 June 2011, began with an overview of current regulatory practices. A representative from the USCG Office of Ope rating and Environmental Standards discussed the current status of the ongoing process towards developing a f ederal ballast water management program and discharge standards. The current USCG proposed rules for the discharge of ballast water involve two phases. Phase One discharge standards would be the same as those the IMO D-2 and Phase Two would, in essence, be 1,000 times more stringent (Table 1).

The ABSG Consulting team has listed the discharge categ ory and list the measures to be utilized for each level and has captured the instrument or analysis necessar y. For each instrument, the participants in the workshop indicated the level of readiness for each tech nology and the signal relevance of each measurement based on the 9-point Technology Readiness Levels (TRL) established by the Department of Defense (DoD). 1 See Appendix C for the complete list and the description of each level. In addition, the advantages, disadvantages, and requirements for operational utilizat ion were discussed for each level and for each

instrument.

A goal of the workshop was to discuss what realistic approa ches, methods and instruments are available, or in development, that could measure vessel compliance f or the Phase One standard. Additional discussion focused on whether Phase One methodology and tools for m easuring compliance would be translatable to the standards established for Phase Two. By discussing th e confidence intervals of potential methodology and available measurement tools; USCG will be in a better position to determine how the compliance regime should be structured.

1 Technology Readiness Levels in the Department of Defense , Source: DOD (2006), Defense Acquisition Guidebook.Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

5 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 Additional material on the first day of the workshop incl uded a presentation by an EPA Water Permits Division representative on the current and upcoming modifi cations to the VGP. The presentation provided background on the Clean Water Act, the EPA’s VGP program and the relative ballast water obligations. The Clean Water Act permit effluence limits are technolog y based (driven by best management and best professional judgment practices) and water quality based. Th e precedence established by EPA was discussed relative to the methodology for establishing c ompliance and appropriate measurement technology and methodologies.

Representatives from the USCG gave a presentation outl ining vessel onboard inspection procedure. Two regulatory case studies were highlighted: oily water separ ators and marine sanitation devices. The practice of port state control inspection was brought up as a mean s to remove substandard shipping practices in domestic waters. Applicability of port state control ins pections to ensure ballasting compliance with

international standards was discussed. Also of interes t was the USCG hierarchical approach to compliance characterized by oily water separator shipboard inspection.

A representative from the Maritime Environmental Resourc e Center, University of Maryland, presented an overview of current ballast water management systems. The fundamental criteria and challenges of treating ballast water, as well as the different categories of ballast water management systems and the current ty pe approved systems, were specified. The main questions that a rose during this presentation were: how to monitor compliance for ballast water management syste ms; the issue of detecting gross exceedance with discharge regulations; and, how to directly quantify the d ischarge regulations.

The EPA Environmental Technology Verification (ETV) Pro gram generic protocols for verifying ballast water treatment technology was presented by a represen tative from the Naval Research Laboratory, Key West. The main discussion was centered on possibility o f adapting methods and approaches in the ETV protocols, to verify performance of ballast water treatm ent systems are land-based test facilities, for use i n compliance monitoring. The ETV protocols were developed with lengthy stakeholder input and have been validated for their intended purpose.

A review of international testing efforts was also gi ven by a representative from the Royal Netherlands Institute for Sea Research. In this review, the follow ing topics were introduced with an international perspective: common standards and uniform implementation; inv olvement of public and private stakeholders; encouragement of technological innovation; an d practical constraints to compliance

enforcements.

The first day of workshop presentations concluded with th e EPA Office of Wastewater Management discussion of developing, validating, and approving Clean Water Act and other EPA methods. Analytical methods are used by the EPA and the States to establish re gulatory priorities and to develop limitations or standards. The USCG may elect to use an analytical met hod at the same robust level as the EPA to establish

ballast water standards and the framework to monitor and de termine compliance. USCG may be able to use EPA methods that are currently in place for wastewate r or drinking water with some modification for ballast water. It was suggested that work done in the water security field may also be applicable to ballast water.Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

6 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 The closing discussion for the first day introduced three proposed levels of compliance monitoring: measures of treatment performance, indirect measure of gross exceedance, and direct measures of discharge

standard. The indirect and direct measures increase in level of confidence in detecting gross exceedance and in logistic constraint levels of effort required and cost. Discussions then began to address in detail the availability, requirements, confidence, limitations and logistic constraints of instrumentation for indirect measures of treatment performance. There was consensus that the first level of compliance monitoring should be the highest priority for the USCG and that mo st, if not all, measurements of treatment system performance should be made directly by the treatment sys tems themselves (as part of normal operational diagnostics) with the data made available to inspectors . In many cases the instruments needed for these measures of treatment performance are off-the-shelf te chnology, but may need some modification and validation for shipboard use and ballast system applicatio n.

On the second day of the workshop, June 29th 2011, discussions be gan by focusing on indirect measures of gross exceedance. Specifically, biological, chemical, o ptical estimates of live organism abundances, collected autonomously or by inspectors, for detecting vessel s in gross-exceedance of the discharge standards. The discussion also covered the availability, requirements, confidence, limitations, logistic

constraints of instrumentation for indirect measures o f gross exceedance, and discussions of emerging or promising novel approaches/technologies.

The first presentation of that day was given by a repres entative of Moss Landing Marine Laboratories as examples of a potential new approach to detect gross exceeda nce using proxies such as FDA stains plus fluorometry, ATP kits and in situ / in vivo chlorophyll fl uorometers. The presentation emphasized that analytical techniques are needed that: (a) shows a reliabl e linear response to living biomass; (b) has adequate sensitivity; (c) relies on simple biochemical protocol; (d) has rapid analysis time; (e) has predictable temperature response; (f) requires only simple portable instrumentation; and, (g) requires minimal technical training. Discussion that followed on i ndirect measures of gross exceedance concluded that analytical kits and instruments (e.g., PAM Fluoro meters) have the potential to detect gross exceedance, however, they need additional development for this specif ic application and rigorous validation before it can be universally employed.

The afternoon of day two focused on direct measures of d ischarge standards. Specifically, on direct measures of live organism abundances in the various regula ted categories, collected autonomously or by inspectors. The discussion also covered the availabilit y, requirements, confidence, limitations, logistic constraints of instrumentation for direct measures, and discussions of emerging or promising novel approaches/technologies. While this level of compliance monitoring would be extremely difficult, timeconsuming and costly, the SMEs concluded that most of the approaches and methods currently used in landbased testing of ballast water management systems (and described in the ETV protocols) could be adaptable for shipboard compliance monitoring if this level of confi dence is required.

On the third day of the workshop, discussions regarding the matrices resumed. The group strived to gain a general consensus on the most promising approaches and requir ed specifications. SMEs generally believed that technology readiness levels (TRL) approach should be employed to assess the maturation of the various analyses, methods or instruments towards full operationa l use in ballast water compliance. It was agreed that the DoD TRL definitions and scale of 1 through 9 is benef icial for this workshop. In general, there was

consensus that future work and research is needed to establi sh the ballast water compliance measurementProceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

7 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 framework. Though, there was general agreement that exist ing and in-development instruments, methods and analyses can be adapted and validated to meet the needs of USCG enforcement of ballast water regulations.

3.3.1 Technology Readiness Levels

Technology Readiness Levels (TRLs) together make up a sys tematic metric, or measurement, system that supports assessments of the maturity of new technologies (materials, components, devices, etc.). TRLs are also used for the consistent comparison of maturity bet ween different types of emerging technology. In general when new technology is invented or conceptualized, it would not fit basic safety regulations to apply it immediately to its planned application. Usually ne w technology is subjected to experimentation, refinement, and increasingly realistic testing. Once t he technology has past enough tests and is proven safe, reliable, and functions by itself in its planned applicati on, it can be incorporated into a system or subsystem.

Each TRL represents a specific timeframe within the ov erall development process of new technology depicted in Table 2. For the purpose of this workshop, we hav e taken these TRLs and measured the readiness of the technologies that may be utilized to m easure and verify compliance with the discharge standard implemented. The workshop attendees agreed to the understanding that TRL 1 is the initial stage of idea conception while TRL 9 is the stage where technologi es were market ready and available. Many of the technologies were market ready, but have not been utili zed in a ballast water system context, thus were deemed to be at a TRL 5 or 6. Technologies proven to operate in a ballast water system context were given a TRL of 7 or higher.Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

8 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 Table 2: Department of Defense TRL Levels 2

TRL Description

1. Basic principles observed and reported Lowest level of technology readiness. Scientific rese arch begins to be translated into applied research and development. Example might include paper studies of a technology's basic properties.

2. Technology concept and/or application formulated Invention begins. Once basic principles are observed, pra ctical applications can be invented. The application is speculative and there is no proof o r detailed analysis to support the assumption. Examples are still limited to paper studies. 3. Analytical and experimental

critical function and/or characteristic proof of concept Active research and development is initiated. This include s analytical studies and laboratory studies to physically validate analytical predictions o f separate elements of the technology. Examples include components that are not yet integrated or representative. 4. Component and/or

breadboard validation in laboratory environment Basic technological components are integrated to establ ish that the pieces will work together. This is relatively "low fidelity" compared to the event ual system. Examples include integration of 'ad hoc' hardware in a laboratory.

5. Component and/or

breadboard validation in

relevant environment Fidelity of breadboard technology increases significantly . The basic technological components are integrated with reasonably realistic suppo rting elements so that the technology can be tested in a simulated environment. Ex amples include 'high fidelity' laboratory integration of components.

6. System/subsystem model or prototype demonstration in a

relevant environment Representative model or prototype system, which is well beyond the breadboard tested for TRL 5, is tested in a relevant environment. Represents a major step up in a technology's demonstrated readiness. Examples include testing a prot otype in a high fidelity laboratory environment or in simulated operational environment.

7. System prototype

demonstration in an operational environment Prototype near or at planned operational system. Represent s a major step up from TRL 6, requiring the demonstration of an actual system prototype in an operational environment, such as in an aircraft, vehicle or space. Examples include te sting the prototype in a test bed aircraft.

8. Actual system completed and 'flight qualified' through

test and demonstration Technology has been proven to work in its final form a nd under expected conditions. In almost all cases, this TRL represents the end of true system development. Examples include developmental test and evaluation of the system in its i ntended weapon system to determine if it meets design specifications.

9. Actual system 'flight proven' through successful mission operations Actual applicati on of the technology in its final form and under missi on conditions, such as those encountered in operational test and evaluation. In almost all cases, this is the end of the last "bug fixing" aspects of true system development. Exampl es include using the system under operational mission conditions.

2 DOD (2006), Defense Acquisition GuidebookProceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

9 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 

### 3.4 Report

The focus of this section is the presentation of a high level summary proceedings and analysis of the information gathered at the SME Workshop. This report shal l include a thorough compilation of the technologies and the measurements, instruments or analy ses and their associated TRL score to ensure compliance with the ballast water discharge standard (B WDS).



### 4 FINDINGS

The SME agreed that the three level compliance monitorin g framework was appropriate. The following

sections are summaries of SME discussions and conclus ions of instruments and methods to address each of the compliance level.



### 4.1 Compliance Level 1 – Measures of Treatment Performance

Compliance level 1 would utilize indirect or indicative system performance and/or water quality measures that demonstrate appropriate treatment operations, or co nditions have been met, for various ballast water treatment approaches. While level 1 measures may not direct ly establish compliance with numerical discharge standards, they can determine if equipment is o perating as certified or per manufacturer’s specifications. While these measures can be collected in dependently by inspectors, the majority should be integrated into the ballast water treatment systems t hemselves, and allow for inspectors to review instrument outputs. Some level 1 parameters might also be measure d independently by inspectors in appropriately collected samples of water before or during discharge.

Most ballast water treatment systems (BWTS) utilize different and multiple combination of methods to treat ballast water to achieve compliance with the BWDS. Ins tead of addressing each BWTS and their resulting combinations, this report breaks down the treatment syste ms into the various treatment components. Most

BWTS utilizes one or a combination of the treatment c omponents found in Table 3 below. For Compliance level 1, the measurements of each component are measure d, instead of the different combinations of each

BWTS.

In Table 3 below, ballast water treatment components a re listed, followed by the indicative measurement to show the proper functioning/operations of the equipment, an d then what parameter(s) would be directly measured. The treatment components in Table 3 are methods currently utilized in BWTS that has received Type Approval Certification from an Administration (in accordance with the IMO ballast water convention).

• Column A – Treatment Component: individual process to remov e, kill or inactivate organisms in

ballast water

• Column B – Indicative Parameter: the quantification/dete rmination of mechanical, physical or chemical parameters.

• Column C – Instrument or Method: Observation, instrument , sensor, sample, analytical kit, etc. used

to make a measurement.

• Column D – Estimated Technology Readiness Level (TRL) fo r this specific applicationProceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

10 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 Table 3: Compliance Level 1: Measures of Treatment Perf ormance Treatment Component Indicative Parameter Instrument or Method TRL

Cavitation Acoustic Passive acoustic sensor or acoustic interferom etry 7

Pressure Pressure sensors (before/after) 9

Coagulation (flocculent) Dose of coagulant used System diagnostics 9

Turbidity Turbidity sensor 8

Deoxygenation pH (if CO 2 is used) pH sensor 7

Oxygen sensor DO sensor 8

Electrochlorination (NaOCl) TRO Amperometric sensor 8

ORP ORP sensors 8

Chlorine Inline DPD kits, automated 8

Power supply, voltage, current System power diagnostics 9

Conductivity/Salinity Conductivity and temperature sensor 9

CDOM (before /after) Fluorometer 6

Filters Water clarity Sight glass, water sample, turbidity se nsor 8

Flow rate/Pressure Pressure sensors (before/after) 9

Back flush frequency System diagnostics 9

Hydrocyclone Water clarity of treated water Sight glass, water sampl e, turbidity sensor 8

Filter flush rate System diagnostics 9

Power consumption System diagnostics of voltage and current 9

Water clarity of waste stream Sight glass, water sampl e, turbidity sensor 8

Ozone TRO Amperometric sensor 8

ORP ORP sensors 8

Power consumption System diagnostics of voltage and current 9

Conductivity/Salinity Conductivity and temperature sensor 9

CDOM (before /after) Fluorometer 6

Peracetic acid Hydrogen peroxide Chemical-specific sensor 7

Peracetic acid Chemical-specific sensor 7

pH pH sensor 7

CDOM (before /after) Fluorometer 6

UV, UV+TiO2 Power System diagnostics of voltage and current 9

Lamp life System maintenance 9

UV Output sensor UV sensor 8

Rate of flow Flow meter 9

CDOM (before /after) Fluorometer 6Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

11 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 Table 4 lists other proposed treatment components, which a re in development.

Table 4: Compliance Level 1: Measures of Treatment Perf ormance (Potential)
Treatment Component Indicative Parameter Instrument or Method TRL

Alkylamines pH pH sensor 7

Alkylamines Chemical

- specific kit 7

Chlorine dioxide Chlorine dioxide In

- line chlorine dioxide kit or polarographic sensor 8 Electric pulse Power consumption System diagnostics of voltage and current 9

Heat Temperature Thermistor 9

Menadione Menadione Sample analysis with HPLC 7 Plasma pulse Power consumption System diagnostics of voltage and current 9

Heat Thermistor 9

Shear Acoustic P assive acoustic sensor or acoustic interferometry 7

Pressure Pressure sensors (before/after) 9

Ultrasound Power consumption System diagnostics of voltage and current 9

Acoustic sensing P assive acoustic sensor or acoustic interferometry 7

For each of these technologies, the indicators for eac h of the treatment types can be incorporated into the BWTS and/or be an inline process for validation and verif ication of functionality as per manufacturer’s specifications. If a BWTS has multiple components, eac h component would optimally utilize a measure to ensure functionality.

Based on the lists developed in Tables 3 and 4, the SME gene rated Table 5 that considers the usefulness of the various measurements in compliance monitoring of bal last water discharge and the time required until it has reached true operational readiness for this specific a pplication (i.e., TRL 9). The results of Table 5 can be used to prioritize approaches to compliance level 1.

Column A – Treatment Type, individual process to remove, k ill or inactivate organisms in ballast water.

Column B – Measurement, the quantification/determination o f mechanical, physical or chemical parameters.

Column C – BWMS internal (should be replaced with “Instrum ent or Analysis”), observation, instrument, sensor, sample, analytical kit, etc. used to make a me asurement.

Column D – Signal Relevance, describes how pertinent, conn ected, or applicable the measurement is in determining ballast water management systems (BWMS) eff ective operations or quantifying fundamental changes to water quality.

Column E - Compliance Operational Readiness, estimates of time required for the Instrument or Analysis to be operational (including specific shipboard ballast water Standard Operating Procedures), validated, and implemented.Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

12 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 Table 5: Compliance Level 1: Recommendations
Treatment Type Measurement BWMS internal Signal

Relevance Compliance Operational

Readiness Immediate (< 1 yr), medium (2-3 yr), long (> 4 yr)

Cavitation and Acoustic Acoustic sensors medium medium

Shear Pressure Pressure sensors (before/after) high Immediate Coagulation (flocculent) Sample of water for dose analysis low medium Dose of inert gas treatment monitoring high immediate Deoxygenation pH pH sensor high immediate/medium (inert gas) Oxygen sensor DO sensor high immediate/medium

Deoxygenation Dose of nitrogen gas Treatment monitoring high immediate/medium (nitrogen) DO DO sensor high immediate/medium

TRO Amperometric sensors medium immediate/medium

ORP ORP sensors low immediate/medium

DPD Inline DPD kits, automated high immediate/medium Voltage and

current Power monitoring diagnostics high immediate Conductivity/ salinity Conductivity, temperature sensors high immediate

Electrochlorination (NaOCl) CDOM (before /after) CDOM fluorometers low long

Visual inspection pipe sight glass or water sample low immediate

Flow rate Flow meters medium immediate

Pressure Pressure high immediate

Back flush frequency treatment monitoring high immediate

Filters Optical clarity of

water turbidity

sensors/transmissometers low medium Optical clarity of

water turbidity

sensors/transmissometers low medium

Back flush frequency treatment monitoring high immediate

Voltage and current Power monitoring diagnostics high immediate

Hydrocyclone Turbidity of waste stream turbidity sensor low medium

TRO amperometric sensors medium immediate/medium

ORP ORP sensors low immediate/medium

DPD Inline DPD kits, automated high immediate/medium Voltage and

current Power monitoring diagnostics high immediate Conductivity/ salinity conductivity+temperature sensors high immediate

Ozone CDOM (before /after) CDOM fluorometers low longProceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

13 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 Treatment Type Measurement BWMS internal Signal

Relevance Compliance Operational

Readiness Immediate (< 1 yr), medium (2-3 yr), long (> 4 yr)

TRO amperometric sensors medium medium

ORP ORP sensors medium medium

hydrogen peroxide, peracetic acid peracetic acid sensors high medium

Dose of peracetic

acid treatment monitoring high immediate

pH pH medium immediate/medium

Peracetic acid CDOM (before /after) CDOM fluorometers low long

Voltage and current Power monitoring diagnostics high immediate

Lamp life treatment monitoring high immediate

UV dose UV sensor high immediate

Rate of flow Flow meters high immediate

UV and

UV+TiO2 CDOM (before /after) CDOM fluorometers low long

pH pH sensor medium medium

Alkylamines Alkylamines Alkylamines specific kits high ?

Chlorine dioxide Chlorine dioxide Chlorine dioxide kits high medium Deoxygenation (vacuum) DO DO sensor high immediate/medium

Electric/Plasma pulse Voltage and current Power monitoring diagnostics high immediate

Heat Temperature Thermistors high immediate

Menadione Menadione Lab HPLC high medium

Voltage and current Power monitoring diagnostics high immediate

Ultrasound Acoustic signals Acoustic sensors high immediate

4.1.1 Approved Treatment Components

The following are short summaries of the treatment compo nents outlined in Table 3. These technologies currently are found in ballast water management systems that have received a Type Approval Certification from an Administration (in accordance with the IMO b allast water convention). Complete descriptions of these treatment approaches can be found in other review documents including (e.g., EPA SAB 2011, Albert et al. 2010). These indicators or measurements are: direct , indirect, or indicative systems operations or water quality measures; collected autonomously and integrat ed into fundamental BWMS diagnostics; and demonstrates effective and reliable system operations an d/or appropriate treatment conditions have been

met.Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

14 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 4.1.1.1 Cavitation

Cavitation is the creation of cavities, or microbubbles, near lower pressure regions in a liquid caused by rapid changes in pressure, typically by subjecting the wate r to high velocities, followed by the subsequent implosion following the sudden decrease in pressure. Equipme nt such as high velocity propellers, venture pipes, or slit plates can generate cavitation bubbles. The creation and rupturing of these cavitation bubbles create hydrodynamic forces and ultrasonic oscillations th at can collapse and implode organisms in the

water.

Two approaches can be used to quantify a cavitation component of treatment systems, Acoustic sensors may be calibrated to estimate cavitation intensity and press ure sensor before and after the cavitation device could provide indication of the functionality of the system. The instrumentation for these pressure measurements can be incorporated into the overall system design. Th ey are generally currently available, cost effective, in-use on ships, but may require some minimal additional development and validation.

4.1.1.1 Coagulation/flocculation
Coagulation is the utilization of a flocculent to combine with colloids present in the water to come out of suspension. Flocculants aggregate suspended particles to form flocs, increasing the sedimentation or the filterability of small particles. Many flocculants ar e multivalent cations such as aluminum, iron, calciu m, or magnesium. Flocculants are also manufactured by creati ng long-chain polymer flocculants like modified polyacrylamides.

Two indicators can be used to indicate treatment perfor mance: dose of coagulant used and water clarity. A dosage meter and a turbidity sensor can be incorporated in to the overall treatment system design.

4.1.1.2 Deoxygenation
Deoxygenation via inert gas is the method by which dissolved oxygen is replaced with inactive gases like nitrogen and CO 2 . Removing oxygen creates lethal hypoxic conditions; howeve r, typically requires holding times of three of four days for the reduction of organis ms to reach discharge standard levels.

To quantify changes produced by deoxygenation systems would simpl y require the use of dissolved oxygen (DO) sensors. DO sensors are currently available, wi dely used in water quality and oceanography measurements, and may require only minimal additional de velopment and validation. If the deoxygenation systems utilize CO 2 as one of the inert gases, pH sensors can also estima te treatment effectiveness. Similarly pH sensors are currently available, widely u sed in water quality and oceanography measurements, but will likely require significant additional developmen t and validation.

4.1.1.3 Electrochlorination (NaOCl)
Electrochlorination, or electrolysis, utilizes an ele ctrical current through ballast water in an electrolyt ic chamber in a manner that creates free chlorine, sodium hypochlorite, hydroxyl radicals, and potentially other unwanted residual compounds. This process is limited to b rackish and seawater with salinity above approximately 10 psu but the sodium hypochlorite level produced can effectively kill most aquatic organisms. Depending on initial concentrations of sodium hy pochlorite produced, organic content of treated water and holding time, neutralization of treated water prior to discharge may also be required.Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

15 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 For systems that utilize electrochlorination, the fo llowing indicators can be used to measure system functionality: Total Residual Oxidant (TRO), Oxidation Re action Potential (ORP), chlorine levels, power use (voltage and current), water conductivity/salinity, an d Colored Dissolved Oxygen Matter (CDOM). TRO, measured by an amperometric sensor, ORP, measured by an ORP sensor, and chlorine, measured by an inline DPD kit. These approaches are in wide use but l ikely require some additional development and validation. CDOM can indicate the level of remaining o rganic matter, which may be utilized in a feedback

mechanism.

4.1.1.4 Filters
Filters capture and physically remove organisms and particle s. The size of the particle captured is determined by the size of the interstitial space of the filter. Filters often utilize a back-flushing cycle to remove and discard captured particles. Filters are often ut ilized as the first stage of ballast water treatment, typically upon ballast water intake. Filters often cre ate pressure gradients and reduce the flow rate accordingly.

To quantify operations during ballasting, pressure sensors be fore and after the filter systems can quantify pressure differentials. Back flushing rate can also be logged by the treatment systems and provide indications that filters are operating appropriately. Fi nally, a variety of water clarity estimates or meas ures (from a simply sight glass to a turbidity sensor) could pr ovide qualitative estimate of filter effectiveness.

4.1.1.5 Hydrocyclone (Cyclonic Separation)
Cyclonic separation utilizes centrifugal forces to separa te particles from the water. Particles with a specif ic gravity greater than water will be separated and removed f rom the mater. Particles and organisms with similar density to water are not easily removed.

Like filters, the operations hydrocyclones can be exami ned using back flushing rate logged by the treatment systems water clarity estimates or measures.

4.1.1.6 Ozone
Ozone is an oxidizing biocide that oxidizes on contact wi th an organism. Ozone will also interact with chemicals present in sea water to create bromine compo unds, creating additional biocides. Ozone generators utilize ambient air through an electric discharge field t o create ozone that is injected into ballast water.

Because ozone is also an oxidizing chemical treatment, several of the approaches used for electrochlorination can be used to quantify ozone treatme nt performance, including TRO sensors (also called ozone sensors), ORP sensor, power use (voltage a nd current), water conductivity/salinity, and Colored Dissolved Oxygen Matter (CDOM). CDOM can indicate the level of remaining organic matter, which may be utilized in a feedback mechanism.

4.1.1.7 Peracetic Acid
Peracetic acid is an organic compound (CH 3 CO 3 H) that acts as an oxidizing agent. Peracetic acid can b e created in multiple methods, but sold in solution to main tain stability of the chemical. The cell membrane of organisms is disrupted by the hydroxyl radical (HO-).Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

16 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 Sensors and inline monitoring systems (membrane and polarogr aphic sensor) are available to quantify concentrations of peracetic acid and while in wide use wo uld likely require some additional development and validation for this specific application.

4.1.1.8 Ultraviolet (UV) Light and UV+TiO 2 Ultraviolet radiation changes and breaks down cellular unit s like the cell membrane or genetic material and causes cell function failure, which may result in cell death or the disruption of cell reproduction. The

utilization of TiO 2 as a catalyst increases the oxidation rate by creatin g hydroxyl radicals and oxide ions. Efficacy is dependent upon variables like the distance fro m the light source, turbidity of the water, time requirement of treatment and power utilization of the UV light source.

UV treatment performance can be monitored by UV light s ensors placed in the treatment chamber and by quantifying system power use. Additionally, routine bulb re placements can be utilized to ensure performance as per manufacturer specifications.

4.1.2 Potential Treatment Components

In this section, the treatment components listed are th ose that are in utilized in a BWTS but have not yet received Type Approval Certification from an Administr ation (in accordance with the IMO ballast water convention).

4.1.2.1 Alkylamines
An Alkylamine is a compound that has an alkyl group bonded to the nitrogen of an amine. An amine is an organic compound with a nitrogen atom with a lone electron pair. While amines are typically considered a base, alkylamines act as an acid, thus disinfecting ballas t water as an oxidizing agent.

Alkylamines can be quantified directly by chemical chromo genic sensors but these are young technologies and will likely require further development and validation for this application. Ballast water treatment systems utilizing alkylamines will also alter pH of treat ed water so pH sensors may also provide indications of system performance.

4.1.2.2 Chlorine Dioxide
Chlorine dioxide is a strong chemical biocide and for balla st water treatment is produce by combining sulfuric acid, sodium chlorate and hydrogen peroxide onboard t he vessel.

Because chlorine dioxide is also an oxidizing chemical trea tment, several of the approaches used for electrochlorination and ozone can be used to quantify ozon e treatment performance, including TRO sensors, ORP sensor. CDOM can indicate the level of remaining or ganic matter, which may be utilized in a feedback

mechanism.

4.1.2.3 Electric Pulse

The application of pulsed electric fields, which transm its energy directly into the water, can cause degradation or decomposition of latent organic materials. Exact mechanisms of action vary greatly, dependant on the wide range of power utilized by different pulses systems.Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

17 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 Operational efficiency can be indicated by active monit oring of installed voltmeters connected to the ballast as well as regular power consumption monitoring.

4.1.2.4 Heat
The utilization of increased temperatures in the ballast water over an extended period of time can result in stressing organisms beyond operational levels or denaturin g necessary proteins, thus resulting in organism death. Many proposed heating systems utilize the waste hea t from the ship’s engines, cooling system, or from other heat exchanges. With the heat from these vessel systems, the ballast water may not be sufficiently heated to a temperature high enough to cause de ath in all organisms, particularly certain types of bacteria. The monitoring of heat ballast water treatm ent systems is fairly straight forward, through the use of

thermistors.

4.1.2.5 Menadione (Vitamin K 3 )
Menadione is a synthetic compound that creates a toxic environment for organisms, inhibiting growth of organisms and causes oxidative damage and chloroplast disruption . To measure treatment operations, doses of menadione can be monitored and samples can be collec ted for laboratory analysis.

4.1.2.6 Plasma Pulse
Similar to electric pulse technology, plasma pulse appli es short burst of electrical energy to entering water in order to degrade or decompose organic materials. Counter to standard electrical pulse, plasma pulse dramatically increases applied energy density by narrowing t he gap between electrodes, causing an intensified electrical arc, simultaneously requiring less treatment duration per effective result.

Due to the higher exothermal release from a pulse resul ting in an arc, successful operation could be monitored by temperature differential across the plasma pu lse zone of activity, as well as installed ballast voltmeters and general ship power consumption. A functiona l issue is the amount of energy required to create a plasma pulse onboard.

4.1.2.7 Shear
BWTS that may utilize shear uses shear stress to cause organism death. The proposed techniques utilize high levels of pressure changes to stress organisms, due to t he rapid change in pressure resulting in the rupture of the cell membrane.

Like cavitation, two approaches can be used to quantify a s hear. Acoustic sensors may be calibrated to estimate shear intensity and pressure sensor before and after the cavitation device could provide indication of the functionality of the system.

4.1.2.8 Ultrasound

Like cavitation, ultrasound treatment creates microbubbl es to rupture cell membranes that result in organism death. The microbubbles are created via high frequency vib ration. The efficacy of ultrasound varies based on the intensity of vibration and length of exposure. To measure functionality, acoustic sensing can measure the output of an ultrasound system and operational effic iency can be monitored by power consumption monitoring.Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

18 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 

### 4.2 Compliance Level 2 – Indirect Measures of Exceedance

As a relatively quick estimate of whether discharge fr om a vessel is significantly above required limits, compliance level 2 would focus on bulk estimates of bioma ss. Exceedance can be assessed using various biological, chemical, and optical measures, currently a vailable or in development, that are related to live organism abundances. Some of these measures might only pr ovide indications of gross exceedance, while others may provide finer resolution. These measures can a lso be collected autonomously or by inspectors. However, the various indirect measure thresholds, at w hich the discharge from a vessel is considered out of compliance, will take additional consideration and study to establish.

Table 6 provides a high level summary of: discharge standa rd categories and associated estimate and indicators; various measurements for the different cat egories; instruments and analyses that can be employed as measures of exceedance; and their estimated T RL. TRL levels also consider sample collection and processing as part of the analysis. See Appendix D an d E for further descriptions of instrument and analyses.

Table 6: Compliance Level 2: Indirect Measures of Exceed ance Discharge Standard Category Measurement Instrument or Analysis TRL

Biomass Estimates Enzymatic activity FDA + fluorometer kit 5

ATP Luminometer reagent kit 6 Chlorophyll fluorescence Chlorophyll fluorometer 8

Variable fluorescence PAM fluorometer 8

DNA/RNA Lab analysis 4

Respiration rate Biochemical Oxygen Demand/DO

sensor Electron Transport Systems 4

Membrane potential 3,3’

- dihexyloxacarbocyanine (DiOC6(3)) 5

Live/Dead Indicators Variable fluorescence PAM fluorometer 8

Dead stain Green nucleic acid stain 8

Live stain FDA/CMFDA 8

General stain Neutral red 8

Movement Visual inspection 8

Movement Video 7 Metabolic activity, enzyme CTC 4 Live organisms > 50 μ m Swimming organisms in sample Visual inspection 8

Direct count Flow cytometers (imaging) 8

Direct count Coulter counter 8

Direct count Laser Optical Plankton Counter 7 Live organisms 10

- 50 μ m Variable fluorescence PAM fluorometer 8 Chlorophyll fluorescence Chlorophyll fluorometer 8

Total Live Bacteria CFU Plate counts 9

E. coli CFU Selective substrate and most probable number 9

Enterococci CFU Selective substrate and most probable number 9

V. cholerae (toxigenic) CFU Colorimetric immunoassay kit 8Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

19 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 Based on the lists developed in Table 6, the SME generate d Table 7 that considers the usefulness of the various measurements in compliance monitoring of ballast water discharge and the time require until it has reached true operational readiness for this specific appli cation (i.e., TRL 9). The results of Table 7 can be used to prioritize approaches to compliance level 2.

Column A – Discharge Standard Category

• Biomass estimates are quantifications of biological mat erial from living organisms, which are in turn correlated to numbers of planktonic organisms for estima tes of abundance.

• Live/dead indicators are instruments, assays, stains, an d analyses that distinguish between live and dead organisms.

• Column B – Measurement, the quantification/determination o f physical, chemical or biological parameters as an estimate of organism abundances.

Column C – Instrument of Analysis, observation, instrum ent, sensor, sample, analytical kit, etc. used to measure or estimates of organism abundances.

Column D – Signal Relevance, describes how pertinent, conn ected, or applicable the measurement is in determining live organism abundances.

Column E - Compliance Operational Readiness, estimates of time required for the Instrument or Analysis to be operational (including specific shipboard ballast water Standard Operating Procedures), validated, and implemented.Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

20 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 Table 7: Compliance Level 2: Recommendations Discharge

Standard Measurement Instrument or Analysis Signal

Relevance Compliance

Operational Readiness Category Immediate (< 1 yr), medium (2-3 yr), long (> 4yr)

Biomass estimates Enzymatic activity FDA fluorometer kit high long

ATP Luminometer reagent kit high medium

Chlorophyll fluorescence Chlorophyll fluorometer medium (specific to subset) medium

Variable fluorescence PAM fluorometer high (specific to subset) immediate/medium

DNA/RNA Lab analysis medium long

Respiration rate Biochemical Oxygen

Demand/DO sensor Electron

Transport Systems medium long

Membrane potential 3,3’-dihexyloxacarbocyanine (DiOC6(3)) low long

Live/dead

indicators Variable fluorescence PAM fluorometer high (specific to subset) immediate/medium

Dead stain Green nucleic acid stain (Sytox) low medium

Live stain FDA/CMFDA high (specific to subset) immediate/medium

General stain Neutral red medium immediate/medium

Movement Visual high immediate/medium

Movement Video high medium Metabolic activity,

enzyme CTC medium long Live organisms

> 50 μ m Swimming organisms in small volume Visual high immediate/medium

Direct count flow cytometers (imaging) high (with live stain) long

Direct count Coulter counter low medium

Direct count Laser Optical Plankton Counter low long

Variable fluorescence PAM fluorometer high (specific to subset) immediate/medium Live organisms

10 - 50 μ m Chlorophyll fluorescence Chlorophyll fluorometer medium (specific to subset) medium

Total live bacteria CFU Plate counts medium immediate/medium

E. coli CFU Selective substrate most probable number and other

EPA methods high immediate

Enterococci CFU Selective substrate most probable number and other

EPA methods high immediate
V. cholerae (toxigenic) CFU Colorimetric immunoassay kit high medium
Viruses Virus-like particles Direct count Electron microscopy high* (phase II)Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

21 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 

### 4.3 Compliance Level 3 – Direct Measures of Compliance

If it is suspected that a vessel discharge will exceed the discharge standards, compliance level 3 could be used to make direct measurements of the various regulated biological categories. Compliance level 3 would involve detailed biological testing for live organisms, simi lar to evaluations currently conducted under treatment system certification testing, and provide the highest level of scientific certainty and statistical confidence. While several of the methods and approaches for level 3 compliance monitoring currently exist and are employed in full scale shipboard testing for type approval, several logistic challenges remain to be resolved prior to implementation for assessment of ve ssel compliance.

Table 8 provides a high level summary of: discharge standa rd categories; various measurement for the different categories; instruments and analyses that ca n be employed as measures of compliance; and their estimated TRL. TRL levels also consider sample collect ion and processing as part of the analysis. See Appendix D and E for further descriptions of instrument and a nalyses.

Table 8: Compliance Level 3: Direct Measures of Dischar ge Standard Discharge Standard Category Measurement approach Instrument or Analysis TRL

Live organisms > 50 μ m Visual count Microscope 8

Automated analysis 5 Stain (neutral red) 7 Live organisms 10

- 50 μ m PAM fluorometer 8 FDA and/or Sytox plus chlorophyll

autofluorescence Flow cytometer 8

FDA/CMFDA and/or Sytox Epifluorescent Microscope 8 Rose Bengal (heterotrophs) Microscope 8

Total live bacteria Plate counts CFU 8

Stains Epifluorescent Microscope 8

Stains Flow cytometry 8

E. coli CFU Selective substrate and most probable number 8

mTEC 6 qPCR 6

Enterococci Defined media, Most Probable

Number 8

qPCR 6

V. cholerae (toxigenic) CFU Colorimetric immunoassay kit 8

Direct count Fluorescent antibodies 7

Viruses and Virus

- like Particles Nucleic acid Stains Epifluoren s cent microscope 7

Nucleic acid Stains Electron microscopy 7

Nucleic acid stains Flow cytometry 8Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

22 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 Based on the lists developed in Table 8, the SME generate d Table 9 that considers the usefulness of the various measurements in compliance monitoring of ballast water discharge and the time require until it has reached true operational readiness for this specific appli cation (i.e., TRL 9). The results of Table 9 can be used to prioritize approaches to compliance level 2.

Several of these analyses are likely going to be conducte d in a laboratory setting. Issues with how to sample, who will sample, standardization, laboratory verifica tion, and procedural issues of testing and sampling are issues that were raised during the workshop, however would n eed to be addressed in a different venue.

Column A – Discharge Standard Category

Column B – Measurement, the quantification/determination o f physical, chemical or biological parameters as a direct indication of organism abundances.

Column C – Instrument of Analysis, observation, instrum ent, sensor, sample, analytical kit, etc. used to measure organism abundances.

Column D – Signal Relevance, describes how pertinent, conn ected, or applicable the measurement is in determining live organism abundances.

Column E - Compliance Operational Readiness, estimates of time required for the Instrument or Analysis to be operational (including specific shipboard ballast water Standard Operating Procedures), validated, and implemented.Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

23 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 Table 9: Compliance Level 3: Recommendations Discharge

Standard Category Measurement Instrument or Analysis Signal

Relevance Compliance

Operational

Readiness
Live organisms Direct count and movement Microscope high immediate

> 50 μ m Direct count and movement Automated video analysis high medium

Direct count Live stain and flow
cytometry high medium

Live organisms Variable fluorescence PAM fluorometer low medium

10

- 50 μ m

Live stain direct count FDA/CMFDA epifluorescent microscopy or flow
cytometry high medium

Live and dead

stain direct count Green nucleic acid stain +

FDA/CMFDA epifluorescent microscopy or flow
cytometry high medium

Total live bacteria CFU Plate counts low immediate

live stains Epifluorescent Microscope high immediate

live stains Flow cytometry high immediate

E. coli CFU Selective substrate most probable number kit high immediate

CFU m-TEC high immediate

Direct count Other EPA

- approved

methods high immediate

Abundance estimates qPCR high long

Enterococci Selective substrate most probable number Selective substrate

most probable number kit high immediate

CFU Other EPA

- approved

methods high immediate

Abundance estimates qPCR high long

V. cholerae (toxigenic) Direct counts Colorimetric immunoassay

kit high immediate

Direct counts Lab fluorescent antibodies high immediate

Viruses

Virus-like

Particles Direct count Nucleic acid stains and epifluorescence microscope

Direct count Electron microscopy

Nucleic acid stains Nucleic acid stains and flow
cytometryProceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

24 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 

### 5 RECOMMENDATIONS AND CONCLUSIONS

At the conclusion of the workshop, the SMEs proposed a host of specific recommendations to the USCG for a ballast water discharge compliance and enforcement fr amework. The chief recommendation was that treatment performance should be built into ballast wate r treatment systems. This recommendation is tied directly to the approaches outlined in level 1 complianc e monitoring measures. By building in treatment performance into the actual ballast water systems, rea dily available technology can be leveraged quickly, and comparatively cost effectively. Any data collected by the installed performance measure can then be provided to the USCG for verification. It was clear to the SMEs that of the three levels of compliance monitoring, treatment performance measures (level 1) are considered the highest priority for USCG to take

action on.

However, the SMEs did propose additional recommendations t hat were given lower priority than the level 1 compliance monitoring measures. One of the lower priori ty recommendations is for further development and validation of bulk biomass estimates. In particular , FDA stains with hand-held fluorometers, commercial ATP kits, and in situ PAM fluorometry were approaches and technology earmarked as requiring further consideration through research, development and t esting. Another lower priority recommendation is for increased investments in transitioning/maturation ac tivities for those level 1 and level 2 indicator instruments with TRL rankings of 6 – 8. The goal of these investments is to bring those indicator instruments from a TRL 6-8 to a TRL 9 where they are va lidated and truly operational for this specific application. TRL 9 instruments must be developed specifica lly for shipboard, ballast water discharge compliance monitoring. Therefore, related to the need fo r investments in sub 9 TRL instruments is also need

for clear criterion to dictate method and/or instrumen t validation and recognition.

The SMEs strongly recommend that a second workshop shou ld be held at some point in the future. The purpose of the second workshop would be to take the items di scussed in the first workshop and shape them into components that contribute to a practical complianc e and enforcement framework or strategy for the

USCG to consider. Particular needs for discussion include b ut are not limited to: defining key terminology (i.e.: “gross exceedance”) and identifying appropriate approach es and methodologies for employment.Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

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### (This page intentionally left blank.)Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

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### APPENDIX A. SUBJECT MATTER EXPERT WORKSHOP AGENDA

Tuesday, 28 June 2011

USCG R&D Center

Part 1: Overview of Current Regulatory Practices

8:30 a.m. – 9:00 a.m. Welcome, Workshop Outline and Objectives

, Participant Introductions (Roderick, Schnappinger USCG)

9:00 a.m. – 9:30 a.m. Review of the USCG (Proposed) ballast water discharge regulations (Everett USCG)

9:30 a.m. – 10:00 a.m. Mandatory Reporting: Review of EPA General Vessel Permit in relation to ballast water discharge (Albert EPA)

10:00 a.m. – 10:10 a.m. Break

10:10 a.m. – 10:30 a.m. Onboard Inspections: Regulatory case study – USCG compliance and enforcement of oily water discharge and separators (Allain/F luke USCG)

10:30 a.m. – 10:50 a.m. Onboard Inspections: Regulatory case study – USCG compliance and enforcement of Marine Sanitation Devices (MSD) (Allain/Fl uke USCG)

Part 2: Overview of Current Technology and Verification of Systems

10:50 a.m. – 11:10 a.m. Review of currently available ballast water t reatment systems (Tamburri UMCES)

11:10 a.m. – 11:30 a.m. Overview of the ETV Generic Protocols fo r the Verification of Ballast Water Treatment Technology (Drake NRL)

11:30 a.m. – 11:45 a.m. Review of International Testing Efforts (V eldhuis NIOZ)

11:45 a.m. – 1:15 p.m. Lunch 1:15 p.m. – 2:00 p.m. Overview of EPA water quality testing in regards to the Clean Water and Safe Drinking Water Acts and related system verification (Dan esi EPA) Part 3: Overview of Measurable Water Quality Parameters and the Avai lability of Tools and Methods

2:00 p.m. – 3:30 p.m. Measures of treatment performance – Discussions of indirect or indicative water quality measures, collected autonomously or by inspec tors, that could demonstrate appropriate treatment conditions have been met for various ballast water treatment approaches.

3:30 p.m. – 3:45 p.m. Break

3:45 p.m. – 4:45 p.m. Measures of treatment performance – Discussions of availability, requirements, confidence, limitations, logistic constr aints of instrumentation for indirect measures of treatment performance.

4:45 p.m. – 5:00 p.m. Summary (Schnappinger USCG)Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

A-2 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 Wednesday, 29 June 2011

USCG R&D Center

8:30 a.m. – 8:45 a.m. Introduction (Schnappinger USCG)

8:45 a.m. – 10:30 a.m. Indirect measures of non-compliance – Discussions of indirect biological, chemical, optical measures of live organism abundances, collected autonomously or by inspectors, for indications of clear/ gross non-compliance.

10:30 a.m. – 10:45 a.m. Break

10:45 a.m. – 12:00 p.m. Indirect measures of non-compliance – Discussion of availability, requirements, confidence, limitations, logistic constr aints of instrumentation for indirect measures of non-compliance, and discussio ns of emerging or promising novel approaches/technologies.

12:00 p.m. – 1:15 p.m. Lunch

1:15 p.m. – 1:30 p.m. Travel reimbursement procedures – (Murp hy, USCG)

1:30 p.m. – 3:30 p.m. Direct measures of discharge standard – Discussions of direct measures for concentration of live organisms in the various regulated c ategories – adapting current certification testing methods to compliance monitoring.

3:30 p.m. – 3:45 p.m. Break

3:45 p.m. – 4:45 p.m. Direct measures of discharge standard - Discussions of requirements, confidence, limitations, and logistic constraints for direct measures of concentration of live organisms in the various regulated c ategories. 4:45 p.m. – 5:00 p.m. Summary (Schnappinger USCG)

Thursday, 30 June 2011

USCG R&D Center

8:30 a.m. – 8:45 a.m. Introduction (Schnappinger USCG)

8:45 a.m. – 10:30 a.m. Consensus on most promising approaches and r equired specifications:

Measures of treatment performance Indirect measures of non-compliance Direct measures of discharge standard

10:30 a.m. – 10:45 a.m. Break

10:45 a.m. – 11:30 p.m. Consensus on future work/research need ed

11:30 a.m. – 12:00 p.m. Recommendations for a ballast water discha rge compliance and enforcement framework (Schnappinger USCG) 12:00 p.m. Lunch and adjourn workshopProceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

B-1 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 

### APPENDIX B. SUBJECT MATTER EXPERT WORKSHOP ATTENDEES

On 28-30 June 2011, an SME meeting was held in the ABSG Consul ting office in New London, CT, with 32 in attendance. The table below lists the workshop atte ndees and their affiliation or organization.

Name Affiliation/Organization Ryan Albert, Ph.D. EPA LCDR Ryan Allain CG-5431/5224 Sarah A. Bailey Fisheries and Oceans Canada
Alex Balsley R&D Center Allegra Cangelosi Great Ships Initiative
Robin Danesi EPA Office of Wastewater Management
Charles Dorchak American Bureau Of Shipping Lisa A. Drake Naval Research Laboratory Key West, C ode 6136

Rich Everett CG-5224 Ray Finley Sandia National Laboratories
Michele Fitzpatrick PMG/ATSC

Jim Fletcher R&D Center

LCDR Chuck Fluke CG-5432
Ray Fredrick EPA

John Griffith Southern California Coastal Water Res earch Project
Thomas H. Johengen University of Michigan Carolyn E. Junemann MARAD Sue Kim ABS Consulting
Dennis M. King University of Maryland Brandon Lee ABS Consulting Edward Lemieux Naval Research Laboratory, Code 6130

M. J. Lewandowski R&D Center
Gary Logsdon Black & Veatch

Gail Roderick R&D Center Craig Schnappinger ABS Consulting
Mike Sprague ABS Consulting

Tom Stevens NSFI Lynn Takata California State Lands Commission Mario Tamburri ABS Consulting/University of Maryland

Marcel J.W. Veldhuis Royal Netherlands Institute for Sea Research
Nick Welschmeyer Moss Landing Marine Laboratories Greg Ziegler UMD Wye Research and Education CenterProceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

C-1 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 

### APPENDIX C. TECHNOLOGY READINESS LEVELS

NASA TRL Levels 3

3 “Technology Readiness Levels.” National Aeronautics and Space Administration, www.hq.nasa.gov/office/codeq/trl/trl chrt.pdf.Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

C-2 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 TRL Description

1. Basic principles observed and reported Lowest level of technology readiness. Scientific res earch begins to be translated into applied research and development. Exam ple might include paper studies of a technology's basic propert ies.

2. Technology concept and/or application formulated Invention begins. Once basic principles are observed, prac tical applications can be invented. The application is speculat ive and there is no proof or detailed analysis to support the assumpt ion. Examples are still limited to paper studies.

3. Analytical and experimental

critical function and/or characteristic proof of concept Active research and development is initiated. This include s analytical studies and laboratory studies to physically v alidate analytical predictions of separate elements of the tec hnology. Examples include components that are not yet integrated or representative.

4. Component and/or

breadboard validation in laboratory environment Basic technological components are integrated to establi sh that the pieces will work together. This is relatively "low fide lity" compared to the eventual system. Examples include integra tion of 'ad hoc' hardware in a laboratory.

5. Component and/or

breadboard validation in

relevant environment Fidelity of breadboard technology increases significant ly. The basic technological components are integrated with rea sonably realistic supporting elements so that the technology can be tested in a simulated environment. Examples include 'high fidelity' laboratory integration of components.

6. System/subsystem model or prototype demonstration in a

relevant environment Representative model or prototype system, which is well be yond the breadboard tested for TRL 5, is tested in a relevan t environment. Represents a major step up in a technology's demonstrated readiness. Examples include testing a prototype in a high fidelity laboratory environment or in simulated opera tional

environment.

7. System prototype

demonstration in an operational environment Prototype near or at planned operational system. Represents a major step up from TRL 6, requiring the demonstration of an actual system prototype in an operational environment, such as in an aircraft, vehicle or space. Examples include testing th e prototype in a test bed aircraft.

8. Actual system completed and 'flight qualified' through

test and demonstration Technology has been proven to work in its final form an d under expected conditions. In almost all cases, this TRL repr esents the end of true system development. Examples include developm ental test and evaluation of the system in its intended weapo n system to determine if it meets design specifications.

9. Actual system 'flight proven' through successful mission operations Actual application of the technology in its final form and under mission conditions, such as those encountered in operat ional test and evaluation. In almost all cases, this is the end of the last "bug fixing" aspects of true system development. Examples inc lude using the system under operational mission conditions. (Source: DOD (2006), Defense Acquisition Guidebook )Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

D-1 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 

### APPENDIX D. MEASUREMENTS

For Compliance Levels 2 and 3, the count or estimation of organisms is done to measure whether the vessel is in compliance with the specific discharge standard. These measurements are listed below.

Abundance estimates Abundance estimates are calculated quantities of biomass based on indicators and measurements taken. Total tank biomass is often inferred from smaller sampl e populations taken, and then extrapolated using statistics. Population quantities can either be directly counted or inferred from detected amounts of biological activity characteristic of population sizes.

ATP A well developed system for monitoring general biological a ctivity is monitoring of Adenosine Triphosphate (ATP). This molecule is utilized virtually across all domains of life, thereby providing a universal indicator of the presence or absence of biologic al activity. A unique characteristic of ATP is the immediate degradation (within minutes) following cell death, thereby lowering risk of false positives post

sterilization. Dormant forms of life such as bacteri al spore or viral capsides in the absence of host cel ls may not be detected by this method. ATP is commonly measured re latively cheaply using a combination of chemical reagent kits, and a photometer (i.e. luminomet er). ATP must be extracted from living cells using a combination strong acid with ATP optimized buffer solutio n. Once extracted, the product is mixed with an ATP reactive chemical which emits light in the presen ce of ATP. The light is measured by the photometer in relation to the calibrated biomass to produce a vali dation of presence or absence of biological activity. Modern photometers are generally palm sized, portable, and inexpensive.

CFU Colony forming Bacteria and Fungi are often reported using Colony Forming Units (CFUs). A sample containing these organisms is incubated on a culture plate, which form small visible clusters of genetically identical specimens. Due to localized nutrient restriction these colonies generally grow to approximately the same size, before spreading elsewhere on the plate to f orm additional colonies. The advantage of organisms growing this way is that from a given dilution of sampl e, the counted CFUs can be used to infer their

concentration in tested ballast tank. The two main di sadvantages of this measure, are the incubation time (hours to days) and the limited detectable species. Cultur e plates work for detecting commonly cultured bacteria such as Escherichia coli (E. Coli); however, it is estimated that less than 0.2% of all microbes h ave been successfully grown on culture plates.

Chlorophyll fluorescence Chlorophyll fluorescence refers to the measurement by fluorom eter whereby a calibrated wavelength of light specific to chlorophyll is directed into a given sampl e. If chlorophyll is contacted by the test beam, then the emitted fluorescent light of reaction is dete cted by the fluorometer and reported as a positive test result. Modern fluorometers are typically able to dete ct Chlorophyll as biological indicators in extremely low concentrations, often orders of magnitude lower tha n other test.

Dead stain Stains are often utilized to enhance visualization during mi croscopy analysis. Dead staining agents such as Green SYTO further enhance this process due to the inability to strongly stain living cells due to their intactProceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

D-2 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 cell membranes. As a result dead stains allow for dire ct measure of deceased cells in a given sample, which is often compared to a live stain for a ratio of living t o deceased cells.

Direct count Direct counting refers to any test where organisms are vis ually counted in a given sample as a means to statistically derive their concentration in a larger po pulation area (such as a ballast tank). Examples include counting of cell colonies on culture plates, or direct cel l counts on microscope slides.

DNA/RNA Deoxy- or Ribo- Nucleic Acids (DNA/RNA) refers to genet ic material found in all forms of life. Some general test such as nucleic acid dyes can assist in di rect microcopy analysis, while more advanced laboratory analysis, match specific DNA sequences to iden tify contaminants to a high degree of certainty. Typically the results of these tests are used as final verification of other testing protocols.

Enzymatic activity Enzymatic activity testing refers to a technique used in mic robe analysis as an identifying characteristic filter. In its most basic test forms, small samples are directly exposed to an enzymatic substrate capable of

reaction with cells of interest. If there is a visib le reaction (i.e. bubbling), then the test is recorded po sitive for the presence of this group of applicable organisms. More modern forms of this test combine use of a fluorometer with this test, in order to quantify the che mical energy released from the chemical reaction as a direct measure of the quantity of cells in solution. De pending on the specific enzyme reaction selected for testing, the protocol can be targeted at broad or specific ranges of microbial species.

General stain General stain refers to a broad category of test util izing chemical stains/dyes for microscopy analysis to enhance visualization of transparent microbes. While ther e are broad catalogues of available staining agents, each one typically is good for a spectrum of organisms a nd test should further designed with staining agents relevant to the contaminating species of interest.

Live stain In microscopy analysis live stains is a division within general cell stains, wherein the test is designed to show the number of living cells separate from deceased ce lls. Live stain techniques often utilize a broad scope stain such as SYTO red, to visualize all cells liv ing or dead while a secondary dead stain such as SYTO green is used to differentiate dead cells. As a resu lt the cells positive for SYTO red, but negative for SYTO green are inferred to be living cells.

Live + dead stain direct count Live and dead stains are commonly done simultaneously on the same sample in order to calculate the amount of viable living cells versus total biomass. Each sta in only provides additional information, but also provides a counter stain to the other, which is enhances th e visualization of each stain.

Membrane potential Tests utilizing membrane potential rely on a nearly universa l biological principle that living cells require a proton gradient across a membrane in order to facilitate biological activity. As such variable fluorescent dye kits have been designed in order to visibly color living an d dead cells differently. Once treated with theseProceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

D-3 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 membrane potential reagent kits, the samples can be analy zed using either microscopy or high through-put flow cytometry.

Metabolic activity, enzyme Enzymatic activity test are often used to quantify presen t living biomass via metabolic activity. FDA kits coupled with measure by a flow cytometer are effective m eans to detect metabolic activity via enzyme activity.

Movement Movement is a detectible indicator of living organisms in test samples. Detection methods include manual microscopy, or computerized analysis of video from microsc opy. Accurate counting is often difficult when done manually, due to the nature of constantly moving cells . Computerized optical recognition can be programmed for applicable characteristic patterns of mic robial movement and used to take living cell counts versus total particulate background. It should be noted though, that while movement is an affirmative indicator, the absence of movement does not conclude the absence of living organisms due to the nature of many microbes lacking the capability of active movement. Algae, for example, rely on buoyancy for its position in the environment, but cannot control its own horizontal movement.

Nucleic acid stains Virtually all forms of life current known contain a nuc leic acid, either in the form of DNA or RNA. Nucleic acid stains are specifically designed to bind to these mole cules. An example of a common nucleic acid stain is SYTOX-green, which is also a dead stain.

Respiration rates Respiration rate refers to rate at which a given livi ng biomass consumes a predictable amount oxygen. Tests measuring changes in oxygen concentration over a set tim e can be used to quantify the presence of biological activity in a closed ballast water tank.

Selective substrate most probable number Selective substrates such as FDA are used to measure enzym atic activity in living organisms. Measured enzyme activity is then adjusted for variance between di fferent sized cells, before finally estimating the most probable number of living cells.

Swimming organisms in small volume Swimming organisms readily stand out to visual analysis un der microscopy. For microorganisms, the space between the microscope slide and the cover slip often pr ovides a small volume of water with a height of less than a millimeter, but plenty of room for microbial move ment.

Variable fluorescence Techniques utilizing variable fluorescence involve fluorescent agents capable of producing more than one color depending on exposed conditions. An example would b e with membrane potential reagent where the reagent molecule fluoresces different colors depending on its location in the cell where different pH zones

appear.Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

E-1 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 

### APPENDIX E. INSTRUMENT OR ANALYSIS OF MEASUREMENT

3,3’-dihexyloxacarbocyanine (DiOC6(3)) DiOC6(3) is a lipophilic stain specific to mitochonrdrial ce ll components within eukaryotic cells (nonbacteria). Membrane potential test kits often utilize DiOC6(3) as a fluorescent stain because its emitted spectrum is ideal for reading by flow cytometers.

Automated video analysis Optic recognition programs can be combined with video of visual analysis in order to automate visual analysis processes such as microscopy. Software packages can be programmed to look for general morphological profiles, movement profiles, or specific test fluorescence.

Biochemical oxygen demand/dissolved oxygen (DO) sensor, Elect ron Transport Systems Living biomass is commonly detected by aerobic respiration and consumption of oxygen. Cellular consumption of oxygen by their electron transport can be detected either by dissolved oxygen monitoring of a closed system or by specialized electron transport ch ain fluorescent stains. In the case of electron transpo rt chain fluorescent stains, the consumption of oxygen excites the electron transport chain complex, releasing energy which causes uniquely detectible fluorescence of the respiring cells. This provides real-time quantification of living biomass in a sample.

Chlorophyll fluorometer Chlorophyll fluorometers are utilized in detection of plan ts, algae and cyanobacteria via the presence their green pigment, chlorophyll. The fluorometer technology takes advantage from chlorophyll’s chemical property to react to the presence of light, and can read t he excitation energy released by the pigment. As such the benefits are greatly increased detection limits by orders of magnitude.

Colorimetric immunoassay kit Immunoassay kits refer to any test where antibodies a re used to screen for and attach markers to specific molecules of interest. In the past such test were lim ited to lab testing, however modern commercial suppliers have incorporated this technology into effective field tes t strips. Colorimetric markers are often utilized by field test because they lack specialized equipment require ments in order to be read.

Coulter counter A Coulter counter is a device used to count cells in a give n solution, often times with the addition capability to differentiate between cell sizes. The primary techno logy relies on its ability to draw the solution through

micro channels and measure the electrical resistance acr oss these microchannels. As cells are drawn through each microchannel, they increase the electrical resis tance in a predictable way relative to their cell siz e.

CTC CTC refers to a bacterial live stain reagent also known as 5-cyano-2,3-ditolyl tetrazolium chloride. This reagent reacts within cells undergoing aerobic respiratio n and fluoresces Red in their presence.

Electron microscopy Electron microscopy is a form of microscopy utilizing el ectrons in place of visible light. The advantage of this substitution is electrons have a drastically shorte r wavelength, allowing for high resolution, up to 5,000Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

E-2 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 times greater than a light microscope. This is particul arly useful for the smaller organisms like certain

bacteria and viruses.

Epifluorescent microscope An epifluoresecent microscope is one that controls le vels of background light, in order to maximize visualization of fluorescent light emitted by fluorescen t dyes. This microscope is used in quantitative and qualitative lab test.

FDA fluorometer kit A Fluorescein diacetate (FDA) kit is a pack of necessa ry reagents for performing an enzymatic activity test on a given sample. The test is an indirect measure of biological activity, which in combination with statistical models can derive quantities of living biomass versus dead biomass.

FDA/CMFDA Fluorescein diacetate (FDA) and Chloromethylfluorescein diacetate (CMFDA) are similar variable fluorescent live stains used to differentiate viable living ce lls from dead cells via intracellular esterase activity. In living cells, naturally present enzymes will chemically convert these dyes changing the spectrum of fluorescent light emitted.

Flow cytometry Flow cytometry is a high through-put version of automat ed microscopy whereby the machine analyzes a stream of cells in solution, and automatically counts c ells satisfying programmable optic or conductive criteria. In many cases a flow cytometer can be progra mmed in combination to a fluorescent live or dead stain in order to differentiate living cells versus total biomass in solution.

Flow cytometers (imaging) Flow Cytometers are designed to automate various evaluati on task previously examined manually via microscopy. Flow Cytometers measure the difference in electrical resistance of a solution as a test sampl e is drawn through micro-channels. The process is sensitive en ough to detect individual cellular bodies as they flow through the channel gates, allowing for automated counti ng of cells in a given sample. Additional advances in technology can further filter the counted res ults, by size or programmed spectrum of fluorescence given off by a stain administered prior to the flow cytometer.

Green nucleic acid stain (Sytox) Green sytox is a common dead stain, which specifically binds to nucleic acids within the cell. Due to the internal nature of its binding activity, green sytox is o nly able to bind with dead cells with a permeated cell

membrane.

Lab analysis Lab analysis is any analysis that cannot be conducted in t he field. Typically tests that require lab analysis will require specialized personnel and will have lower re sults.

Lab fluorescent antibodies

Lab fluorescent antibodies are biochemical substances w ith uniquely targeted binding properties for a targeted substance (substrate) of interest. Once bound, th e attached fluorescent molecules allow theProceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

E-3 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 substrates to be detected and quantified. The lab designation separates this class of reagents from some newer age test strips able to be utilized in the field.

Laser optical plankton counter (LOPC) Laser optical plankton counters track the flow of plan kton cell bodies through a test stream, and provide quantitative analysis of plankton concentrations. Advan ced instruments are able to further filter counted results via basic morphological shape readings specific to species of interest.

Live stain and flow cytometry Fluorescent live stains when coupled with flow cytometry provide an automated means to quickly count the number of live organisms in a sample. Sample concentrati ons can but utilized to extrapolate the concentration within a larger system such as a ballast water tank.

Luminometer reagent kit Luminometers are used to measure the intensity of fluore scent test, and as such their associated reagent kits typically contain cell fluorescent agents along with ne cessary reaction buffers and cell permeability enhancing chemicals.

Microscope Microscopes are devices often utilizing lenses to enhance an d magnify visualization of tiny objects normally too small for the human eye to see. Advanced microscope te chnologies such as electron microscopes may utilize backscatter reading technology similar to airport security scanners, in order to further magnify beyond the physical limits of lens technology.

m-TEC Modified membrane-Thermotolerant Escherichia coli (m-TE C) agar is a specialized culture medium, used to enhance plate counts of E. coli, a common biological contaminant species in water samples. m-TEC agar includes an extra selective enzyme substrate, 5-bromo-6-chl oro-3-indolyl-$-D-glucuronide, which is broken down by enzymes in E. Coli. The resulting chemical product p roduces a bright magenta color for each applicable colony on the test plate. The test increases visualization of colonies, and lowers the required expertise needed by the test technician.

Neutral red Neutral red is a general stain, which non-discriminately attempts to dye all particles in solution, red. This general stain is commonly used in combination with dead sta ins to calculate the ratio of live to dead

biomass.

Nucleic acid stains and epifluorescence microscope

Fluorescent acid stains are often used in combination wit h epifluorescence microscopy to enhance visualization in the display of the microscope. With fluorescence, special microscopes such as epifluorescence types are needed in order to block out backgr ound light that would overwhelm the weak fluorescent signals. Nucleic acid stains and epifluoresc ence micscropy can be used to quantify dead biomass in a sample.Proceedings of Ballast Water Discharge Standards Co mpliance Subject Matter Expert Workshop

E-4 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 Nucleic acid stains and flow cytometry Flow cytometry can be utilized in junction with nuclei c acid stains in order to automate manual processes such as microscopy which quantify dead biomass in sample s olutions.

PAM fluorometer Pulsed Amplitude Modification (PAM) fluorometers are used in chlorophyll quantification test. The test laser is pulsed quickly on the sample chlorophyll in order to increase signal strength of detection.

Plate counts When measuring microbes, culture plates are commonly use d to grow and magnify bacteria within samples up to detectible limits. Colony forming microbes are partic ularly convenient to measure using plate counting techniques because the number of visible colonies can be e asily counted without aided visual enhancement, and the be used to derive the original bacterial concentrat ion in a given sample.

qPCR Quantitative Polymerase Chain Reaction (qPCR) is an am plified detection method for measurement of low concentrations of DNA. PCR is normally a method for r eplicating DNA in order increase a sample size. The rate of replication is controlled closely by limiting r eagent and controlled reaction temperatures. In qPCR the initial quantity of DNA before replication is calc ulated using the known rate of replication and the measured duration. qPCR is a laboratory technique requiring spec ialized personnel.

Selective substrate most probable number kit S Selective substrates are test molecules designed to in teract with specific target enzymes as a measure of living biomass. Due to variance in individual cells containi ng the target enzymes, statistical models have been designed to predict the most probable number (MPN) of c ells in a given sample. These MPN models are calibrated to unique sample compositions using MPN kits.

Video Video recordings of visual test are often created for repea tability, or for automated video analysis techniques.

Visual Visual counting techniques generally refer to any technique wher e the test facilitator looks for visual indicators of biological contaminants.F-1 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 

### APPENDIX F. SUBJECT MATTER EXPERT WORKSHOP SPEAKERS

The following is a list of speakers followed by their res pective presentations as part of the SME workshop. This list is included to assist researchers and an alysts for any follow-on projects.

1. Everett, Richard. U.S. Coast Guard: Review of the USCG proposed ballast water discharge regulations

2. Albert, Ryan. Environmental Protection Agency: Mandatory Re porting: Review of EPA General Vessel Permit in relation to ballast water discharge

3. Allain, Ryan and Fluke, Chuck. U.S. Coast Guard: Onboard In spections: Regulatory case study – USCG compliance and enforcement of oily water discharge and se parators
4. Allain, Ryan and Fluke, Chuck. U.S. Coast Guard: Onboard In spections: Regulatory case study – USCG compliance and enforcement of Marine Sanitation Devices

5. Tamburri, Mario. University of Maryland, Center for Envi ronmental Science: Review of currently available ballast water treatment systems

6. Drake, Lisa. Naval Research Laboratory Key West: Over view of the ETV Generic Protocols for the Verification of Ballast Water Treatment Technology

7. Veldhuis, Marcel. Royal Netherlands Institute for Se a Research: Review of International Testing Efforts

8. Danesi, Robin. Environmental Protection Agency: Overview of EPA water quality testing with regards to the Clean Water and Safe Drinking Water Acts and rela ted system verification

9. Welschmeyer, Nick. Moss Landing Marine Laboratories: I ndirect measures of non-complianceG-1 UNCLAS | R&DC | G. E. Roderick | RDC | August 2011 

### APPENDIX G. SUBJECT MATTER EXPERT WORKSHOP PRESENTATIONS