Evaluation Of Past Shipboard Testing

Evaluation of Past Shipboard Testing Brian L. Howes1 & Craig D. Taylor2 School for Marine Science & Technology

1

University of Massachusetts

2

Biology Department

Woods Hole Oceanographic Institution

Background The U.S. Coast Guard review programs of shipboard testing of BWTS effectiveness: – – –

Shipboard Testing Audit Program (2000-2002) Advanced Approval Program (2003) Shipboard Technology & Evaluation Program (2004- )

*All Program Review by DOT Volpe Center Technical Team

USCG Shipboard BWTS U.S. Coast Guard & Technical Review Teams: Regulatory/Policy – Richard Everett, USCG Environmental Standards Division Penny Herring & Gail Roderick USCG R&D Center Naval Engineering – Mike Dyer & Chris Murray DOT Volpe Tech Team Leads BWTS Engineering – Ed Conde, DOT Volpe Marine Biology -- B. Howes, UMass, C. Taylor, WHOI

Test Program Challenge How to design a realistic Shipboard Test Program that captures the goal of reducing the risk of ANS invasions to the “Policy” level (USCG (BWE), IMO,etc) Given: – Shipboard constraints – Biological & Chemical comparisons must be quantitative – Metric needs to be removal/inactivation based – Biological diversity

BWTS & Testing Challenge

Phylogenetic Tree Illustrating the Complex Diversity of Ballast Water-borne Organisms

Groups that have been found in ballast water.

Findings of USCG Shipboard Review Program (2000-5) Vessel Owner

BWTS Vendor

BWTS Test Team

Test Goal

USCG: Technical Review & Regulatory / Policy Role

Encountered Issues: Experimental Design Need for suitable “source water” Experimental Design: – Biological Controls Dose-Response experiments Time-course of response Sufficient replication for statistical rigor Internal vs External replication

– Chemical (residuals) Time-course of breakdown Holding time issues

Need for Analytical QA/QC

(Quality Assurance Project Plans)

Example of Sampling Locations within a Shipboard BWTS

Generalized Experimental Design for BWT Test Programs

Major Issues: - under replication of “samples”, over replication of analyses - individual vessel test programs will pseudoreplicate the Technology

Encountered Issues Assays of BWTS Effectiveness* Use of direct counts or numbers (without time-course, no viability) – Example: Bacterial direct counts

Minor Species – Example: zooplankton viability assay (statistics)

Biomass or bulk measures – Examples: Chlorophyll a, ATP, Protein

* Need for species specific analysis

Complication of using Chlorophyll Bulk Measurements for Estimating Phytoplankton Viability Total Chl a

Total Chl a

Sensitive Dominant Low Growth Minor Resistant Sp. Sensitive Dominant High Growth Minor Resistant Sp. Time (hrs)

Time (hrs)

Chl Decay Org 1

Chl Decay Org 2

Growth of Org 1

Growth of Org 2

C1(t) = c1(0)*N1*f1*exp(-k1*t)

C2(t) = c2(0)*N1*f2*exp(-k2*t)

C1(t) = c1(0)*N1*(1-f1)*exp(u1*t)

C2(t) = c2(0)*N2*(1-f2)*exp(u2*t)

Testing Approaches Implemented by Six Test Programs 1

Bulk measures of Chla.

6 1

6 1

Integrated “viability” physiological measures (e.g., ATP, PAM). 2

Autofluorescence enumeration. 3

4

1 0

Bulk measures of protein.

Development of resting stages in culture. 5

5 --

(A) Dyer et. al., 2003; (B) Sutherland et. al., 2001; (C) Waite

1

Examples of Biological Assays of BWTS Effectiveness Functional Group

Assays

Comments

Zooplankton & large animals

Species-specific counts with viability scoring

Viability as organism movement (motility, heart, cilia, etc), response to stimulation.

Phytoplankton

Species-specific counts immediately after treatment

Standard cell counts do not indicate viability

Species-specific counts during grow out incubations

Change in numbers over time indicates viability

Species-specific counts during grow out incubations;

Change in numbers over time indicates viability

Species-specific counts with viability scoring

Viability as organism movement (motility, cilia, flagella, etc),

Bacteria

Viable Plate Counts

Dilution series and growth into colonies

Viruses

Viable Plate Counts Phage Methods

Dilution series and growth determined by plaque formation

Cysts & Dormant Stages*

Quantitative microscopic assessment of germination in controlled incubations

Determination of fraction of dormant stages that are able to germinate

Protozoa

Evolving Viability Methods Potential for Automation: Optical/Photographic & Image Analysis primarily for Zooplankton Vital Stains (e.g., SYTOX)

Manual Approach: Species Specific MPN for Phytoplankton & Protozoans

USCG Shipboard BWTS Moving Forward Review of Status of Scope and Scale of emerging BWTS & Test Programs USCG Acceptance of Vessels with evidence adequate BWTS performance into its Shipboard Technology & Evaluation Program (STEP).

– Attempts to gauge a technology’s biological effectiveness as the removal of organisms or treatment effects on organism viability.

Questions & Discussion

Questions & Discussion

Problem with Chlorophyll Bulk Measurements for Estimating Phytoplankton Viability

Total Chl a

Total Chl a

Sensitive Dominant Low Growth Minor Resistant Sp. High Growth Minor Resistant Sp.

Chl Decay Org 1

Chl Decay Org 2

Growth of Org 1

Growth of Org 2

C1(t) = c1(0)*N1*f1*exp(-k1*t)

C2(t) = c2(0)*N1*f2*exp(-k2*t)

C1(t) = c1(0)*N1*(1-f1)*exp(u1*t)

C2(t) = c2(0)*N2*(1-f2)*exp(u2*t)