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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 to 

U.S. Government Agencies and their contractors (Administrative/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 reconstruction of the document. 

August 2011
Proceedings of Ballast Water Discharge Standards Compliance Subject 

Matter Expert Workshop 

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N O T I C E 

This document is disseminated under the sponsorship of the Department of 

Homeland Security in the interest of information exchange. The United 

States Government assumes no liability for its contents or use thereof. 

The United States Government does not endorse products 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, specification, or regulation. 

 

 

United States Coast Guard 

Research & Development Center 

1 Chelsea Street 

New London, CT 06320 
Proceedings of Ballast Water Discharge Standards Compliance Subject 

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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 Discharge 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 Roderick, (860) 271-2658, Gail.E.Roderick@uscg.mil 

16. Abstract (MAXIMUM 200 WORDS) 

This report describes the results of inquiries and analyses in support of HSCG23-08-Q-MSR068, Study of Current Ballasting 

Operations Conducted in U.S. Waters. In keeping with the Coast Guard’s mission, the report supports a range of Coast Guard 

decisions to manage the risk of biological invasions, focusing primarily on the existing ballasting operations. 

The USCG is charged with developing protocols and regulations for verifying compliance with both Phase One and Phase 

Two ballast water discharge standards. It has proposed a two phase approach for implementing regulation of ballast water, 

with the Phase One a set of standards similar to standards in the IMO Ballast Water Convention and Phase Two standards 

potentially 1,000 times stricter than the Phase One standards. In this work, a compliance framework will be developed and 

refined with the input from research and from SMEs address any technologies or concepts that address compliance 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/Operational Use) (25 

May 2011). Other requests shall be referred to the Technical 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. Price 
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EXECUTIVE SUMMARY 

This report describes the proceedings of the Ballast Water Discharge Standards Compliance Subject Matter 

Expert (SME) Workshop held 28 – 30 June 2011 at the ABSG Consulting office in New London, CT in 

support of HSCG32-11-J-200082, Assessment of Approaches for Compliance and Enforcement of Ballast 

Water Discharge Regulations. In keeping with the Coast Guard’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 measures 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 for implementing regulation of ballast water, 

with Phase One including a set of discharge standards similar to those found in the International Maritime 

Organization (IMO) Ballast Water Convention and potential 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 framework will seek to address any methods, 

technologies or concepts 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. 

The ABSG Consulting team has developed a compliance monitoring framework to identify the requisite 

methods, instruments and approaches for determining compliance 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 standard; and 

3. Direct measures of compliance with the discharge standard. 

Compliance level 1 would utilize indirect or indicative system performance and/or water quality measures 

that demonstrate appropriate treatment operations, or conditions have been met, for various ballast water 

treatment approaches. While level 1 measures may not directly establish compliance with numerical 

discharge standards, they can determine if equipment is operating as certified or per manufacturer’s 

specifications. While these measures can be collected independently by inspectors, the majority should be 

integrated into the ballast water treatment systems themselves, and allow for inspectors to review instrument 

outputs. Some level 1 parameters might also be measured independently by inspectors in appropriately 

collected samples of water before or during discharge. 

As a relatively quick estimate of whether discharge from a vessel is significantly above required limits, 

compliance level 2 would focus on bulk estimates of biomass. Exceedance can be assessed using various 

biological, chemical, and optical measures, currently available or in development, that are related to live 

organism abundances. Some of these measures might only provide indications of gross exceedance, while 

others may provide finer resolution. These measures can also be collected autonomously or by inspectors. 
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However, the various indirect measure thresholds, at which 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, similar 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 vessel compliance. 

To further refine the compliance monitoring framework, the 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 bodies like the USCG, Environmental Protection 

Agency (EPA), Maritime Administration (MARAD), and Naval Research Laboratory (NRL) to provide 

input on the compliance verification framework and other insights on the subject. For each compliance 

level, the recommendations are based on either the treatment 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 listed the discharge category and list the measures 

to be utilized for each level and has captured the method, instrument or analysis necessary. For each 

instrument, the participants in the workshop indicated the level of deployment readiness for each technology 

and the signal relevance of each measurement. In addition, the advantages, disadvantages, and requirements 

for operational utilization were discussed for each level and for each instrument. 
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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-1
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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 Performance ........................................................... 10

Table 4: Compliance Level 1: Measures of Treatment Performance (Potential) .......................................... 11

Table 5: Compliance Level 1: Recommendations ....................................................................................... 12

Table 6: Compliance Level 2: Indirect Measures of Exceedance ................................................................ 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 ....................................................................................... 23
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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 Oxidant 
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Acronym Definition 

U.S. United States 

USCG United States Coast Guard 

UV Ultraviolet 

UV Ultraviolet 

VHF Very High Frequency 

VOC Volatile Organic Compound 
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1 BACKGROUND 

Ballasting is a necessary function of vessels. Vessels take on ballast water for a variety of reasons, 

including, but not limited to, adjusting trim and reversing the effects of discharged cargo on stability. 

Vessels also discharge ballast water for similar reasons. 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 compromise 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 dangers 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 comprised of one or more sea chests, from which one or 

more pumps draw water, typically via filters. These pumps are connected to tanks used for ballast via one or 

more manifolds, which allow the direction of flow to and 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), sometimes 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 of 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 proposed a two phase approach for 

implementing regulation of ballast water, with the Phase One a set of standards similar to standards in the 

IMO Ballast Water Convention and Phase Two standards potentially 1,000 times stricter than the Phase One 

standards (Table 1). To comply with the standards, vessels have begun installing ballast water treatment 

systems to treat ballast water to comply with the accepted 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 m3per 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 10 100 
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Diverse ballast water treatment systems are being developed to meet the IMO and USCG discharge standard 

(see Efficacy of Ballast Water Treatment Systems: a Report by the EPA Science Advisory Board, 2011). Prior 

to use on vessels, each treatment system must go through extensive laboratory, land-based and shipboard 

testing. Only after the treatment systems has be demonstrated to safely, effectively and reliably meet the 

discharge standards is it provided with a Type Approval certificate 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 developed and refined, with input from research and 

SMEs. The framework addressed methods, technologies or concepts 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 and 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 Vessel General Permit (VGP); 

2. The current state of ballast water treatment systems; 

3. Current ballast water treatment certification testing methods and approaches; 

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

5. Aggregation of measurements, methods, instruments and analyses 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 evaluations, and a report. Highlights included 

the following: 

• Coordinating with the Contracting Officer’s Technical Representative (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, instruments 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 ABSG Consulting office to elicit 

feedback on the framework and input into factors and methods. The workshop agenda is included as 

Appendix A. 
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3.1 Literature Review 

The initial step in the methodology was to obtain and review 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 Shipping, 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 technologies. 

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

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

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

verification framework to evaluate the identified technologies and compliance. 

3.2 Compliance Monitoring Framework 

The ABSG Consulting team has developed a compliance monitoring framework to identify the requisite 

methods, instruments and approaches for determining compliance 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 standard; and 

3. Direct measures of compliance with the discharge standard. 

Compliance level 1 would utilize indirect or indicative system performance and/or water quality measures 

that demonstrate appropriate treatment operations, or conditions have been met, for various ballast water 

treatment approaches. While level 1 measures may not directly establish compliance with numerical 

discharge standards, they can determine if equipment is operating as certified or per manufacturer’s 

specifications. While these measures can be collected independently by inspectors, the majority should be 

integrated into the ballast water treatment systems themselves, and allow for inspectors to review instrument 

outputs. Some level 1 parameters might also be measured independently by inspectors in appropriately 

collected samples of water before or during discharge. 

As a relatively quick estimate of whether discharge from a vessel is significantly above required limits, 

compliance level 2 would focus on bulk estimates of biomass. Exceedance can be assessed using various 

biological, chemical, and optical measures, currently available or in development, that are related to live 

organism abundances. Some of these measures might only provide indications of gross exceedance, while 

others may provide finer resolution. These measures can also be collected autonomously or by inspectors. 

However, the various indirect measure thresholds, at which 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, similar to evaluations currently conducted under 
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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 vessel 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 included 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 related disciplines such as wastewater and drinking 

water treatment; aquatic toxicology; water quality instrumentation; 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 Operating and Environmental Standards discussed the 

current status of the ongoing process towards developing a federal 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 category and list the measures to be utilized for each 

level and has captured the instrument or analysis necessary. For each instrument, the participants in the 

workshop indicated the level of readiness for each technology 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 utilization were discussed for each level and for each 

instrument. 

A goal of the workshop was to discuss what realistic approaches, methods and instruments are available, or 

in development, that could measure vessel compliance for the Phase One standard. Additional discussion 

focused on whether Phase One methodology and tools for measuring compliance would be translatable to 

the standards established for Phase Two. By discussing the 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. 
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Additional material on the first day of the workshop included a presentation by an EPA Water Permits 

Division representative on the current and upcoming modifications 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 technology based (driven by best management and best 

professional judgment practices) and water quality based. The precedence established by EPA was 

discussed relative to the methodology for establishing compliance and appropriate measurement technology 

and methodologies. 

Representatives from the USCG gave a presentation outlining vessel onboard inspection procedure. Two 

regulatory case studies were highlighted: oily water separators and marine sanitation devices. The practice 

of port state control inspection was brought up as a means to remove substandard shipping practices in 

domestic waters. Applicability of port state control inspections to ensure ballasting compliance with 

international standards was discussed. Also of interest was the USCG hierarchical approach to compliance 

characterized by oily water separator shipboard inspection.

A representative from the Maritime Environmental Resource 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 type 

approved systems, were specified. The main questions that arose during this presentation were: how to 

monitor compliance for ballast water management systems; the issue of detecting gross exceedance with 

discharge regulations; and, how to directly quantify the discharge regulations. 

The EPA Environmental Technology Verification (ETV) Program generic protocols for verifying ballast 

water treatment technology was presented by a representative from the Naval Research Laboratory, Key 

West. The main discussion was centered on possibility of adapting methods and approaches in the ETV 

protocols, to verify performance of ballast water treatment systems are land-based test facilities, for use in 

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 given by a representative from the Royal Netherlands 

Institute for Sea Research. In this review, the following topics were introduced with an international 

perspective: common standards and uniform implementation; involvement of public and private 

stakeholders; encouragement of technological innovation; and practical constraints to compliance 

enforcements. 

The first day of workshop presentations concluded with the 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 regulatory priorities and to develop limitations or 

standards. The USCG may elect to use an analytical method at the same robust level as the EPA to establish 

ballast water standards and the framework to monitor and determine compliance. USCG may be able to use 

EPA methods that are currently in place for wastewater 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. 
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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 most, if not all, measurements of treatment system 

performance should be made directly by the treatment systems 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 technology, but may need some modification and 

validation for shipboard use and ballast system application. 

On the second day of the workshop, June 29th 2011, discussions began by focusing on indirect measures of 

gross exceedance. Specifically, biological, chemical, optical estimates of live organism abundances, 

collected autonomously or by inspectors, for detecting vessels in gross-exceedance of the discharge 

standards. The discussion also covered the availability, requirements, confidence, limitations, logistic 

constraints of instrumentation for indirect measures of gross exceedance, and discussions of emerging or 

promising novel approaches/technologies. 

The first presentation of that day was given by a representative of Moss Landing Marine Laboratories as 

examples of a potential new approach to detect gross exceedance using proxies such as FDA stains plus 

fluorometry, ATP kits and in situ / in vivo chlorophyll fluorometers. The presentation emphasized that 

analytical techniques are needed that: (a) shows a reliable 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 indirect measures of gross exceedance concluded 

that analytical kits and instruments (e.g., PAM Fluorometers) have the potential to detect gross exceedance, 

however, they need additional development for this specific application and rigorous validation before it can 

be universally employed. 

The afternoon of day two focused on direct measures of discharge standards. Specifically, on direct 

measures of live organism abundances in the various regulated categories, collected autonomously or by 

inspectors. The discussion also covered the availability, 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 confidence 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 required 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 operational use in ballast water compliance. It was agreed that 

the DoD TRL definitions and scale of 1 through 9 is beneficial for this workshop. In general, there was 

consensus that future work and research is needed to establish the ballast water compliance measurement 
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framework. Though, there was general agreement that existing 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 systematic 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 between 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 new technology is subjected to experimentation, 

refinement, and increasingly realistic testing. Once the technology has past enough tests and is proven safe, 

reliable, and functions by itself in its planned application, it can be incorporated into a system or subsystem. 

Each TRL represents a specific timeframe within the overall development process of new technology 

depicted in Table 2. For the purpose of this workshop, we have taken these TRLs and measured the 

readiness of the technologies that may be utilized to measure 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 technologies were market ready and available. Many of the 

technologies were market ready, but have not been utilized 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. 

 
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Table 2: Department of Defense TRL Levels2

TRL Description 

1. Basic principles observed 

and reported 

Lowest level of technology readiness. Scientific research 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, practical applications can be invented. 

The application is speculative and there is no proof or 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 includes analytical studies and laboratory 

studies to physically validate analytical predictions of 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 establish that the pieces will work together. 

This is relatively "low fidelity" compared to the eventual 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 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 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 prototype 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. 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 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 and 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 intended weapon 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 operational test and evaluation. In almost all cases, this is the end of the 

last "bug fixing" aspects of true system development. Examples include using the system 

under operational mission conditions. 

 

2

 DOD (2006), Defense Acquisition Guidebook
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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 shall include a thorough compilation of the 

technologies and the measurements, instruments or analyses and their associated TRL score to ensure 

compliance with the ballast water discharge standard (BWDS). 

4 FINDINGS 

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

sections are summaries of SME discussions and conclusions 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 conditions have been met, for various ballast water 

treatment approaches. While level 1 measures may not directly establish compliance with numerical 

discharge standards, they can determine if equipment is operating as certified or per manufacturer’s 

specifications. While these measures can be collected independently by inspectors, the majority should be 

integrated into the ballast water treatment systems themselves, and allow for inspectors to review instrument 

outputs. Some level 1 parameters might also be measured 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. Instead of addressing each BWTS and their resulting 

combinations, this report breaks down the treatment systems into the various treatment components. Most 

BWTS utilizes one or a combination of the treatment components found in Table 3 below. For Compliance 

level 1, the measurements of each component are measured, instead of the different combinations of each 

BWTS. 

In Table 3 below, ballast water treatment components are listed, followed by the indicative measurement to 

show the proper functioning/operations of the equipment, and 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 remove, kill or inactivate organisms in 

ballast water 

• Column B – Indicative Parameter: the quantification/determination 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) for this specific application 

 
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Table 3: Compliance Level 1: Measures of Treatment Performance 

Treatment 

Component Indicative Parameter Instrument or Method TRL 

Cavitation 

Acoustic Passive acoustic sensor or acoustic interferometry 7 

Pressure Pressure sensors (before/after) 9 

Coagulation 

(flocculent) 

Dose of coagulant used System diagnostics 9 

Turbidity Turbidity sensor 8 

Deoxygenation 

pH (if CO2 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 sensor 8 

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

Back flush frequency System diagnostics 9 

Hydrocyclone 

Water clarity of treated water Sight glass, water sample, 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 sample, 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 6 
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Table 4 lists other proposed treatment components, which are in development. 

Table 4: Compliance Level 1: Measures of Treatment Performance (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 Passive acoustic sensor or acoustic interferometry 7

Pressure Pressure sensors (before/after) 9

Ultrasound 

Power consumption System diagnostics of voltage and current 9

Acoustic sensing Passive acoustic sensor or acoustic interferometry 7

For each of these technologies, the indicators for each of the treatment types can be incorporated into the 

BWTS and/or be an inline process for validation and verification of functionality as per manufacturer’s 

specifications. If a BWTS has multiple components, each component would optimally utilize a measure to 

ensure functionality. 

Based on the lists developed in Tables 3 and 4, the SME generated Table 5 that considers the usefulness of 

the various measurements in compliance monitoring of ballast water discharge and the time required until it 

has reached true operational readiness for this specific application (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, kill or inactivate organisms in ballast water. 

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

Column C – BWMS internal (should be replaced with “Instrument or Analysis”), observation, instrument, 

sensor, sample, analytical kit, etc. used to make a measurement. 

Column D – Signal Relevance, describes how pertinent, connected, or applicable the measurement is in 

determining ballast water management systems (BWMS) effective 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. 

 
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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 long 
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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 components 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 ballast 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 integrated into fundamental BWMS diagnostics; and 

demonstrates effective and reliable system operations and/or appropriate treatment conditions have been 

met. 
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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 water to high velocities, followed by the subsequent 

implosion following the sudden decrease in pressure. Equipment 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 that 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 pressure 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. They 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 are multivalent cations such as aluminum, iron, calcium, or 

magnesium. Flocculants are also manufactured by creating long-chain polymer flocculants like modified 

polyacrylamides. 

Two indicators can be used to indicate treatment performance: dose of coagulant used and water clarity. A 

dosage meter and a turbidity sensor can be incorporated into 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 CO2. Removing oxygen creates lethal hypoxic conditions; however, typically requires holding 

times of three of four days for the reduction of organisms to reach discharge standard levels. 

To quantify changes produced by deoxygenation systems would simply require the use of dissolved oxygen 

(DO) sensors. DO sensors are currently available, widely used in water quality and oceanography 

measurements, and may require only minimal additional development and validation. If the deoxygenation 

systems utilize CO2 as one of the inert gases, pH sensors can also estimate treatment effectiveness. 

Similarly pH sensors are currently available, widely used in water quality and oceanography measurements, 

but will likely require significant additional development and validation. 

4.1.1.3 Electrochlorination (NaOCl) 

Electrochlorination, or electrolysis, utilizes an electrical current through ballast water in an electrolytic 

chamber in a manner that creates free chlorine, sodium hypochlorite, hydroxyl radicals, and potentially 

other unwanted residual compounds. This process is limited to brackish 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 hypochlorite produced, organic content of treated 

water and holding time, neutralization of treated water prior to discharge may also be required. 
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For systems that utilize electrochlorination, the following indicators can be used to measure system 

functionality: Total Residual Oxidant (TRO), Oxidation Reaction Potential (ORP), chlorine levels, power 

use (voltage and current), water conductivity/salinity, and 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 likely require some additional development and 

validation. CDOM can indicate the level of remaining organic matter, which may be utilized in a feedback 

mechanism. 

4.1.1.4 Filters 

Filters capture and physically remove organisms and particles. 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 utilized as the first stage of ballast water treatment,

typically upon ballast water intake. Filters often create pressure gradients and reduce the flow rate 

accordingly. 

To quantify operations during ballasting, pressure sensors before 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. Finally, a variety of water clarity estimates or measures 

(from a simply sight glass to a turbidity sensor) could provide qualitative estimate of filter effectiveness. 

4.1.1.5 Hydrocyclone (Cyclonic Separation) 

Cyclonic separation utilizes centrifugal forces to separate particles from the water. Particles with a specific 

gravity greater than water will be separated and removed from the mater. Particles and organisms with 

similar density to water are not easily removed. 

Like filters, the operations hydrocyclones can be examined 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 with an organism. Ozone will also interact with 

chemicals present in sea water to create bromine compounds, creating additional biocides. Ozone generators 

utilize ambient air through an electric discharge field to 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 treatment performance, including TRO sensors (also 

called ozone sensors), ORP sensor, power use (voltage and 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 (CH3CO3H) that acts as an oxidizing agent. Peracetic acid can be 

created in multiple methods, but sold in solution to maintain stability of the chemical. The cell membrane of 

organisms is disrupted by the hydroxyl radical (HO-). 
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Sensors and inline monitoring systems (membrane and polarographic sensor) are available to quantify 

concentrations of peracetic acid and while in wide use would likely require some additional development 

and validation for this specific application. 

4.1.1.8 Ultraviolet (UV) Light and UV+TiO2

Ultraviolet radiation changes and breaks down cellular units 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 TiO2 as a catalyst increases the oxidation rate by creating hydroxyl radicals and oxide ions. 

Efficacy is dependent upon variables like the distance from 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 sensors placed in the treatment chamber and by 

quantifying system power use. Additionally, routine bulb replacements can be utilized to ensure 

performance as per manufacturer specifications. 

4.1.2 Potential Treatment Components 

In this section, the treatment components listed are those that are in utilized in a BWTS but have not yet 

received Type Approval Certification from an Administration (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 ballast water as an oxidizing agent. 

Alkylamines can be quantified directly by chemical chromogenic 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 treated 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 ballast water treatment is produce by combining 

sulfuric acid, sodium chlorate and hydrogen peroxide onboard the vessel. 

Because chlorine dioxide is also an oxidizing chemical treatment, several of the approaches used for 

electrochlorination and ozone can be used to quantify ozone treatment performance, including TRO sensors, 

ORP sensor. CDOM can indicate the level of remaining organic matter, which may be utilized in a feedback 

mechanism. 

4.1.2.3 Electric Pulse 

The application of pulsed electric fields, which transmits 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. 
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Operational efficiency can be indicated by active monitoring 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 denaturing necessary proteins, thus resulting in organism 

death. Many proposed heating systems utilize the waste heat 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 death in all organisms, particularly certain types of 

bacteria. The monitoring of heat ballast water treatment systems is fairly straight forward, through the use of 

thermistors. 

4.1.2.5 Menadione (Vitamin K3) 

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 collected for laboratory analysis. 

4.1.2.6 Plasma Pulse 

Similar to electric pulse technology, plasma pulse applies 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 the 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 resulting in an arc, successful operation could be 

monitored by temperature differential across the plasma pulse zone of activity, as well as installed ballast 

voltmeters and general ship power consumption. A functional 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 the rapid change in pressure resulting in the 

rupture of the cell membrane. 

Like cavitation, two approaches can be used to quantify a shear. 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 microbubbles to rupture cell membranes that result in organism 

death. The microbubbles are created via high frequency vibration. 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 efficiency can be monitored by power consumption 

monitoring. 
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4.2 Compliance Level 2 – Indirect Measures of Exceedance 

As a relatively quick estimate of whether discharge from a vessel is significantly above required limits, 

compliance level 2 would focus on bulk estimates of biomass. Exceedance can be assessed using various 

biological, chemical, and optical measures, currently available or in development, that are related to live 

organism abundances. Some of these measures might only provide indications of gross exceedance, while 

others may provide finer resolution. These measures can also be collected autonomously or by inspectors. 

However, the various indirect measure thresholds, at which 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 standard categories and associated estimate and 

indicators; various measurements for the different categories; instruments and analyses that can be 

employed as measures of exceedance; and their estimated TRL. TRL levels also consider sample collection 

and processing as part of the analysis. See Appendix D and E for further descriptions of instrument and 

analyses. 

Table 6: Compliance Level 2: Indirect Measures of Exceedance 

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 μm 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 μm 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 8
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Based on the lists developed in Table 6, the SME generated 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 application (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 material from living organisms, which are in turn 

correlated to numbers of planktonic organisms for estimates of abundance. 

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

dead organisms. 

•

Column B – Measurement, the quantification/determination of physical, chemical or biological parameters 

as an estimate of organism abundances. 

Column C – Instrument of Analysis, observation, instrument, sensor, sample, analytical kit, etc. used to 

measure or estimates of organism abundances. 

Column D – Signal Relevance, describes how pertinent, connected, 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. 

 
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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 μm 

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 μm 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) 
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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, similar 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 vessel compliance. 

Table 8 provides a high level summary of: discharge standard categories; various measurement for the 

different categories; instruments and analyses that can be employed as measures of compliance; and their 

estimated TRL. TRL levels also consider sample collection and processing as part of the analysis. See 

Appendix D and E for further descriptions of instrument and analyses. 

Table 8: Compliance Level 3: Direct Measures of Discharge Standard 

 

Discharge Standard 

Category Measurement approach Instrument or Analysis TRL 

Live organisms > 50 μm Visual count Microscope 8

Automated analysis 5

Stain (neutral red) 7

Live organisms 10 - 50 μm 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 Epifluorenscent microscope 7

Nucleic acid Stains Electron microscopy 7

Nucleic acid stains Flow cytometry 8
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Based on the lists developed in Table 8, the SME generated 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 application (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 conducted in a laboratory setting. Issues with how to sample, 

who will sample, standardization, laboratory verification, and procedural issues of testing and sampling are 

issues that were raised during the workshop, however would need to be addressed in a different venue. 

Column A – Discharge Standard Category 

Column B – Measurement, the quantification/determination of physical, chemical or biological parameters 

as a direct indication of organism abundances. 

Column C – Instrument of Analysis, observation, instrument, sensor, sample, analytical kit, etc. used to 

measure organism abundances. 

Column D – Signal Relevance, describes how pertinent, connected, 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. 

 
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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 μm 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 μm

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 

cytometry 

 
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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 framework. The chief recommendation was that 

treatment performance should be built into ballast water treatment systems. This recommendation is tied 

directly to the approaches outlined in level 1 compliance monitoring measures. By building in treatment 

performance into the actual ballast water systems, readily 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 that were given lower priority than the level 1 

compliance monitoring measures. One of the lower priority 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 testing. Another lower priority recommendation is 

for increased investments in transitioning/maturation activities 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 validated and truly operational for this specific 

application. TRL 9 instruments must be developed specifically for shipboard, ballast water discharge 

compliance monitoring. Therefore, related to the need for investments in sub 9 TRL instruments is also need 

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

The SMEs strongly recommend that a second workshop should be held at some point in the future. The 

purpose of the second workshop would be to take the items discussed in the first workshop and shape them 

into components that contribute to a practical compliance and enforcement framework or strategy for the 

USCG to consider. Particular needs for discussion include but are not limited to: defining key terminology 

(i.e.: “gross exceedance”) and identifying appropriate approaches and methodologies for employment. 
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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/Fluke USCG) 

10:30 a.m. – 10:50 a.m. Onboard Inspections: Regulatory case study – USCG compliance and 

enforcement of Marine Sanitation Devices (MSD) (Allain/Fluke 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 treatment systems (Tamburri 

UMCES) 

11:10 a.m. – 11:30 a.m. Overview of the ETV Generic Protocols for the Verification of Ballast 

Water Treatment Technology (Drake NRL) 

11:30 a.m. – 11:45 a.m. Review of International Testing Efforts (Veldhuis 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 (Danesi EPA) 

Part 3: Overview of Measurable Water Quality Parameters and the Availability 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 inspectors, 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 constraints of instrumentation 

for indirect measures of treatment performance. 

4:45 p.m. – 5:00 p.m. Summary (Schnappinger USCG)
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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 constraints of instrumentation 

for indirect measures of non-compliance, and discussions 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 – (Murphy, 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 categories – 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 categories.

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 required 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 needed 

11:30 a.m. – 12:00 p.m. Recommendations for a ballast water discharge compliance and 

enforcement framework (Schnappinger USCG) 

12:00 p.m. Lunch and adjourn workshop
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APPENDIX B. SUBJECT MATTER EXPERT WORKSHOP ATTENDEES 

On 28-30 June 2011, an SME meeting was held in the ABSG Consulting office in New London, CT, with 

32 in attendance. The table below lists the workshop attendees 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, Code 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 Research 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 Center 
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APPENDIX C. TECHNOLOGY READINESS LEVELS 

NASA TRL Levels3

 

 

3

 “Technology Readiness Levels.” National Aeronautics and Space Administration, www.hq.nasa.gov/office/codeq/trl/trlchrt.pdf. 
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TRL Description 

1. Basic principles observed 

and reported 

Lowest level of technology readiness. Scientific research 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, practical 

applications can be invented. The application is speculative and 

there is no proof or 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 includes 

analytical studies and laboratory studies to physically validate 

analytical predictions of 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 establish that the 

pieces will work together. This is relatively "low fidelity" 

compared to the eventual 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 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 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 prototype 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. 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 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 and 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 intended weapon 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 operational test 

and evaluation. In almost all cases, this is the end of the last "bug 

fixing" aspects of true system development. Examples include 

using the system under operational mission conditions. 

(Source: DOD (2006), Defense Acquisition Guidebook) 
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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 sample 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 activity 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 biological 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 bacterial spore or viral capsides in the absence of host cells may 

not be detected by this method. ATP is commonly measured relatively cheaply using a combination of 

chemical reagent kits, and a photometer (i.e. luminometer). ATP must be extracted from living cells using a 

combination strong acid with ATP optimized buffer solution. Once extracted, the product is mixed with an 

ATP reactive chemical which emits light in the presence of ATP. The light is measured by the photometer 

in relation to the calibrated biomass to produce a validation 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 form additional colonies. The advantage of organisms 

growing this way is that from a given dilution of sample, the counted CFUs can be used to infer their 

concentration in tested ballast tank. The two main disadvantages of this measure, are the incubation time 

(hours to days) and the limited detectable species. Culture 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 have 

been successfully grown on culture plates. 

Chlorophyll fluorescence 

Chlorophyll fluorescence refers to the measurement by fluorometer whereby a calibrated wavelength of 

light specific to chlorophyll is directed into a given sample. If chlorophyll is contacted by the test beam, 

then the emitted fluorescent light of reaction is detected by the fluorometer and reported as a positive test 

result. Modern fluorometers are typically able to detect Chlorophyll as biological indicators in extremely 

low concentrations, often orders of magnitude lower than other test. 

Dead stain 

Stains are often utilized to enhance visualization during microscopy analysis. Dead staining agents such as 

Green SYTO further enhance this process due to the inability to strongly stain living cells due to their intact 
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cell membranes. As a result dead stains allow for direct measure of deceased cells in a given sample, which 

is often compared to a live stain for a ratio of living to deceased cells. 

Direct count 

Direct counting refers to any test where organisms are visually counted in a given sample as a means to 

statistically derive their concentration in a larger population area (such as a ballast tank). Examples include 

counting of cell colonies on culture plates, or direct cell counts on microscope slides. 

DNA/RNA 

Deoxy- or Ribo- Nucleic Acids (DNA/RNA) refers to genetic material found in all forms of life. Some 

general test such as nucleic acid dyes can assist in direct microcopy analysis, while more advanced 

laboratory analysis, match specific DNA sequences to identify 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 microbe 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 visible reaction (i.e. bubbling), then the test is recorded positive 

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 chemical energy released from the chemical reaction as a 

direct measure of the quantity of cells in solution. Depending 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 utilizing chemical stains/dyes for microscopy analysis to 

enhance visualization of transparent microbes. While there are broad catalogues of available staining agents, 

each one typically is good for a spectrum of organisms and 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 cells. Live stain techniques often utilize a broad 

scope stain such as SYTO red, to visualize all cells living or dead while a secondary dead stain such as 

SYTO green is used to differentiate dead cells. As a result 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 stain only provides additional information, but also 

provides a counter stain to the other, which is enhances the visualization of each stain. 

Membrane potential 

Tests utilizing membrane potential rely on a nearly universal 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 and dead cells differently. Once treated with these 
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membrane potential reagent kits, the samples can be analyzed using either microscopy or high through-put 

flow cytometry. 

Metabolic activity, enzyme 

Enzymatic activity test are often used to quantify present living biomass via metabolic activity. FDA kits 

coupled with measure by a flow cytometer are effective means 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 microscopy. 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 microbial 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 nucleic acid, either in the form of DNA or RNA. Nucleic 

acid stains are specifically designed to bind to these molecules. 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 living biomass consumes a predictable amount oxygen. Tests 

measuring changes in oxygen concentration over a set time 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 enzymatic activity in living organisms. Measured 

enzyme activity is then adjusted for variance between different 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 under microscopy. For microorganisms, the space 

between the microscope slide and the cover slip often provides a small volume of water with a height of less 

than a millimeter, but plenty of room for microbial movement. 

Variable fluorescence 

Techniques utilizing variable fluorescence involve fluorescent agents capable of producing more than one 

color depending on exposed conditions. An example would be with membrane potential reagent where the 

reagent molecule fluoresces different colors depending on its location in the cell where different pH zones 

appear. 
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APPENDIX E. INSTRUMENT OR ANALYSIS OF MEASUREMENT 

3,3’-dihexyloxacarbocyanine (DiOC6(3)) 

DiOC6(3) is a lipophilic stain specific to mitochonrdrial cell 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, Electron 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 chain fluorescent stains. In the case of electron transport 

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 plants, 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 the 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 are used to screen for and attach markers to specific 

molecules of interest. In the past such test were limited to lab testing, however modern commercial suppliers

have incorporated this technology into effective field test strips. Colorimetric markers are often utilized by 

field test because they lack specialized equipment requirements in order to be read. 

Coulter counter 

A Coulter counter is a device used to count cells in a given solution, often times with the addition capability 

to differentiate between cell sizes. The primary technology relies on its ability to draw the solution through 

micro channels and measure the electrical resistance across these microchannels. As cells are drawn through 

each microchannel, they increase the electrical resistance in a predictable way relative to their cell size. 

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 respiration and fluoresces Red in their presence. 

Electron microscopy 

Electron microscopy is a form of microscopy utilizing electrons in place of visible light. The advantage of 

this substitution is electrons have a drastically shorter wavelength, allowing for high resolution, up to 5,000 
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times greater than a light microscope. This is particularly useful for the smaller organisms like certain 

bacteria and viruses. 

Epifluorescent microscope 

An epifluoresecent microscope is one that controls levels of background light, in order to maximize 

visualization of fluorescent light emitted by fluorescent dyes. This microscope is used in quantitative and 

qualitative lab test. 

FDA fluorometer kit 

A Fluorescein diacetate (FDA) kit is a pack of necessary 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 cells 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 automated microscopy whereby the machine analyzes a 

stream of cells in solution, and automatically counts cells satisfying programmable optic or conductive 

criteria. In many cases a flow cytometer can be programmed 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 evaluation task previously examined manually via 

microscopy. Flow Cytometers measure the difference in electrical resistance of a solution as a test sample is 

drawn through micro-channels. The process is sensitive enough to detect individual cellular bodies as they 

flow through the channel gates, allowing for automated counting of cells in a given sample. Additional 

advances in technology can further filter the counted results, 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 only able to bind with dead cells with a permeated cell 

membrane. 

Lab analysis 

Lab analysis is any analysis that cannot be conducted in the field. Typically tests that require lab analysis 

will require specialized personnel and will have lower results. 

Lab fluorescent antibodies 

Lab fluorescent antibodies are biochemical substances with uniquely targeted binding properties for a 

targeted substance (substrate) of interest. Once bound, the attached fluorescent molecules allow the 
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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 plankton cell bodies through a test stream, and provide 

quantitative analysis of plankton concentrations. Advanced 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 concentrations 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 fluorescent test, and as such their associated reagent kits 

typically contain cell fluorescent agents along with necessary reaction buffers and cell permeability 

enhancing chemicals. 

Microscope 

Microscopes are devices often utilizing lenses to enhance and magnify visualization of tiny objects normally 

too small for the human eye to see. Advanced microscope technologies 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-TEC) 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-chloro-3-indolyl-$-D-glucuronide, which is broken 

down by enzymes in E. Coli. The resulting chemical product produces 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 stains to calculate the ratio of live to dead 

biomass. 

Nucleic acid stains and epifluorescence microscope 

Fluorescent acid stains are often used in combination with 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 background light that would overwhelm the weak 

fluorescent signals. Nucleic acid stains and epifluorescence micscropy can be used to quantify dead biomass 

in a sample. 
Proceedings of Ballast Water Discharge Standards Compliance Subject 

Matter Expert Workshop 

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

Nucleic acid stains and flow cytometry 

Flow cytometry can be utilized in junction with nucleic acid stains in order to automate manual processes 

such as microscopy which quantify dead biomass in sample solutions. 

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 used to grow and magnify bacteria within samples 

up to detectible limits. Colony forming microbes are particularly convenient to measure using plate counting 

techniques because the number of visible colonies can be easily counted without aided visual enhancement, 

and the be used to derive the original bacterial concentration in a given sample. 

qPCR 

Quantitative Polymerase Chain Reaction (qPCR) is an amplified detection method for measurement of low 

concentrations of DNA. PCR is normally a method for replicating DNA in order increase a sample size. The 

rate of replication is controlled closely by limiting reagent and controlled reaction temperatures. In qPCR 

the initial quantity of DNA before replication is calculated using the known rate of replication and the 

measured duration. qPCR is a laboratory technique requiring specialized personnel. 

Selective substrate most probable number kit S 

Selective substrates are test molecules designed to interact with specific target enzymes as a measure of 

living biomass. Due to variance in individual cells containing the target enzymes, statistical models have 

been designed to predict the most probable number (MPN) of cells 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 repeatability, or for automated video analysis 

techniques. 

Visual 

Visual counting techniques generally refer to any technique where the test facilitator looks for visual 

indicators of biological contaminants. 
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APPENDIX F. SUBJECT MATTER EXPERT WORKSHOP SPEAKERS 

The following is a list of speakers followed by their respective presentations as part of the SME 

workshop. This list is included to assist researchers and analysts 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 Reporting: Review of EPA General Vessel 

Permit in relation to ballast water discharge 

3. Allain, Ryan and Fluke, Chuck. U.S. Coast Guard: Onboard Inspections: Regulatory case study – USCG 

compliance and enforcement of oily water discharge and separators 

4. Allain, Ryan and Fluke, Chuck. U.S. Coast Guard: Onboard Inspections: Regulatory case study – USCG 

compliance and enforcement of Marine Sanitation Devices

5. Tamburri, Mario. University of Maryland, Center for Environmental Science: Review of currently 

available ballast water treatment systems 

6. Drake, Lisa. Naval Research Laboratory Key West: Overview of the ETV Generic Protocols for the 

Verification of Ballast Water Treatment Technology 

7. Veldhuis, Marcel. Royal Netherlands Institute for Sea 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 related system verification 

9. Welschmeyer, Nick. Moss Landing Marine Laboratories: Indirect measures of non-compliance 
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APPENDIX G. SUBJECT MATTER EXPERT WORKSHOP PRESENTATIONS