Comparison of Indicative Testing Tools for Ballast Water Compliance Assessment Christopher Brown California State Lands Commission Marine Invasive Species Program PBWG - April 16, 2014

Another Performance Standard Refresher CSLC’s Development of Compliance Protocols Compliance Assessment Indicative Tools Sampling Methods Results Conclusions/Next Steps Presentation Outline 2

Potential End-Users of Handheld Compliance Analysis Tools: Vessel Owners/Crew Port State Control/Regulators BWTS Vendors BWTS Testing Laboratories Scientists Before We Begin... 3

Potential End-Users of Handheld Compliance Analysis Tools: Vessel Owners/Crew Port State Control/Regulators BWTS Vendors BWTS Testing Laboratories Scientists Before We Begin... 4

Presentation is designed to keep tools and manufacturers anonymous All data in analyses have been normalized to allow for comparison between different measurement types and to keep anonymity We are not comparing the 2 tools to endorse one over the other, but to allow us to provide independent feedback to the manufacturers and understand their capabilities more thoroughly  A couple caveats… Dinoflag-ellate? 5

2006 Coastal Ecosystems Protection Act Implement Performance Standards for the discharge of ballast water Assess currently available ballast water technologies Why Are We Here? 6

Organism Size ClassCaliforniaIMO Regulation D-2/ U.S. Federal Organisms greater than 50 µm in minimum dimension No detectable living organisms < 10 viable organisms per cubic meter Organisms 10 – 50 µm in minimum dimension < 0.01 living organisms per ml < 10 viable organisms per ml Living organisms less than 10 µm in minimum dimension Escherichia coli Intestinal enterococci Toxicogenic Vibrio cholerae (O1 & O139) < 10 3 bacteria/100 ml < 10 4 viruses/100 ml < 126 cfu/100 ml < 33 cfu/100 ml < 1cfu/100 ml or < 1cfu/gram wet weight zoological samples < 250 cfu/100 ml < 100 cfu/100 ml < 1 cfu/100 ml or < 1 cfu/gram wet weight zooplankton samples Performance Standards 7

Implementation Schedule Ballast Water Capacity of VesselStandards apply to new vessels in this size class constructed on or after Standards apply to all other vessels in this size class beginning in < 1500 metric tons – 5000 metric tons2016 > 5000 metric tons Ballast Water Capacity of VesselStandards apply to new vessels in this size class constructed on or after Standards apply to all other vessels in this size class beginning in < 1500 metric tons12/1/ – 5000 metric tons12/1/ > 5000 metric tons12/1/ US Federal California 12 months after ratification by more than 35 countries representing over 35% of world tonnage. Currently: 38 countries representing 30.8% of the world's tonnage. IMO 8

Implementation Schedule Ballast Water Capacity of VesselStandards apply to new vessels in this size class constructed on or after Standards apply to all other vessels in this size class beginning in < 1500 metric tons – 5000 metric tons2016 > 5000 metric tons Ballast Water Capacity of VesselStandards apply to new vessels in this size class constructed on or after Standards apply to all other vessels in this size class beginning in < 1500 metric tons12/1/ – 5000 metric tons12/1/ > 5000 metric tons12/1/ US Federal California 12 months after ratification by more than 35 countries representing over 35% of world tonnage. Currently: 38 countries representing 30.8% of the world's tonnage. IMO 9

To assess compliance with performance standards Specific protocols for the collection, handling, & analysis of ballast water samples Provide transparency to the vessel owners, operators, and treatment vendors Based on ETV protocols IMO G8 shipboard testing protocols CA’s Compliance Assessment Protocols 10

Technical Advisory Group Established TAG of scientists and engineers familiar with technology evaluation Met several times in 2011 and again in 2012 Included industry, state agency, environmental organization representatives Draft regulations currently undergoing peer review. Rulemaking process anticipated to begin in late 2014 Compliance Assessment Protocols 11

Levels of Testing 1.Land-based, Type Approval – not appropriate for shipboard use, volumes of water not practical 2.Compliance Verification - simple, practical tests for each size class so that Commission inspectors (and vendors) can verify compliance from shipboard samples using CA protocols 3.Indicative Testing (red light/green light) Allow vessels/ship owners to self-check for compliance Detection of gross exceedance Compliance Assessment Protocols 12

Measuring Compliance Full Scale Testing Pros Accurate counts of discharge organisms Enforceable Cons Time Consuming Expensive Requires additional personnel with special training 13

Measuring Compliance IMO Ballast Water Sampling Guideline G2, paragraph 6.3: “Prior to testing for compliance with the D-2 standard, it is recommended that, as a first step, an indicative analysis of ballast water discharge may be undertaken to establish whether a ship is potentially compliant or non- compliant. Such a test could help the Party identify immediate mitigation measures, within their existing powers, to avoid any additional impact from a possible non-compliant ballast water discharge from the ship.” Unfortunately, there is no explicit determination of how an indicative sample is to be taken. 14

Indicative Testing Pros Fast Relatively Simple Can be used by existing inspection personnel Cons Confidence in numeric correlations Definition of “gross exceedence” Enforceability Grey areas – “Medium risk” or “yellow light” Only measuring one organism type Measuring Compliance 15

Indicative Tests In-Line – Good for monitoring while vessel is under way or BWTS is operating. Maybe of limited use to PSC? Hand held – Mobile, relatively inexpensive, PSC officers and vessel crew can be easily trained to operate. 16

Chlorophyll Fluorescence 17

Active Chlorophyll Fluorescence Measurement FOFO FMFM FVFV Time Fm = maximum fluorescence Fo = initial fluorescence Fv = Fm – Fo = variable fluorescence Fv/Fm = photosynthetic yield (health) Measurement Techniques Fast Repetition Rate Fluorescence (FFR) Pulse-amplitude Modulation (PAM) Fluorescein Diacetate (FDA) 18

“Ship” Sampling – to test for variability between replicates Collect samples directly from ballast tanks 3 replicate samples for each tool Data normalized Coefficient of Variation Methods 19

Results – Variability Testing 8 samples collected: 2 freshwater (American River) 3 vessels with coastal (~50nm) exchange 3 vessels with open ocean (>200nm) exchange Data normalized to preserve anonymity Coefficient of Variation (CV) used to measure relative sample variation. CV = standard deviation/mean 20

Fresh Water 0.05 Tool 1Tool 2Tool 1Tool 2 Results – Variability Testing 21

Coastal Exchange Tool 1Tool 2 * Samples had 1 or more non-detect/zero reading Tool 1Tool 2 Tool 1Tool 2 Results – Variability Testing 22

Open Ocean Exchange Tool 1Tool 2Tool 1Tool 2 * Samples had 1 or more non-detect/zero reading Tool 1Tool 2 Results – Variability Testing 23

MinMedianMax Tool 1 Metric Metric Tool 2 Metric Metric Coefficient of Variation Samples with 1 or more non-detects/zero readings not included in table (n = 6) Results – Variability Testing 24

Organism Counts/Dilution Series Water collected from 4 salinities (0, 10, 25, 35 ppt) Organism counts done on raw water samples using epifluorescence and FDA staining Diluted raw water samples into 10x, 100x, 1000x concentrations 3 replicate samples from each volume Methods “Ship” Sampling – to test for variability between replicates Collect samples directly from ballast tanks 3 replicate samples for each tool Data normalized Coefficient of Variation 25

Results – Cell Counts SampleSalinity#organisms/ml 1 – American River 04,700 2 – Carquinez Strait 1017,633 3 – Central SF Bay 265,880 4 – Long Beach 3513,540 26

Tool 1 – Metric 1 Results – Cell Counts 27

Tool 1 – Metric 2 Results – Cell Counts 28

Tool 2 – Metric 1 Results – Cell Counts 29

Federal/IMO Standard Is a fold increase in organism number acceptable for gross exceedence determination? 0.01/mL ~2,000-5,000 cells/ML 30

Conclusions Both units showed good consistency between replicate samples. Both units show limitations of sensitivity with lower organism densities fold difference in organism numbers. These data could help in defining what gross exceedence means in terms of actual numbers of organisms/ml. D-2 = 10 organisms/ml CA = 0.01 organisms/ml More data… 31

Recently acquired a 3 rd indicative testing tool and will be conducting similar tests. Focus on vessels discharging water treated with AMS. Reports of all data collected will be submitted to manufacturers along with our feedback. Should be receiving Nick’s FDA unit soon …right, Nick? Finalize Compliance Assessment Protocols and begin Rulemaking procedures. Next Steps 32

Thank You For More information: 33