4 Methodologies for Compliance Testing Under the BWM Convention

4.1 Table 2: Analysis methods that may provide an indication of compliance with the D-1 standard^footnote

Indicator	General approach	Standard method	Notes	Level of confidence or detection limit and citation for validation studies
Salinity	Conductivity meter to monitor salinity.	No international standard for ballast water analysis at this time although standard methods for measuring salinity do exist.	External elements can affect the salinity.	To be determined.
Salinity	Refractometer to monitor salinity.	No international standard for ballast water analysis at this time although standard methods for measuring salinity do exist.	Temperature can affect the readings.	To be determined.
Types of organisms in discharge – oceanic, coastal, estuarine or fresh water	Visual identification.	No international standard for ballast water analysis at this time.	Expensive, time-consuming, needs extensively trained personnel; may produce false results if encysted organisms from previous ballasting operations hatch.	To be determined.
Turbidity	Portable turbidity sensors.	No international standard for ballast water analysis at this time.	Requires understanding of turbidity characteristics in relation to the distance from shore.	To be determined.
Dissolved Inorganic and Organic constituents (Nutrients, metals coloured dissolved organic matter (CDOM))	Portable nutrient sensors.	No international standard for ballast water analysis at this time.	Requires understanding of inorganic or organic constituent characteristics in relation to the distance from shore.	To be determined.

4.2 Table 3: Indicative analysis methods for use when testing for potential compliance with the D-2 standard^footnote

Indicator	General approach	Standard method	Notes	Level of confidence or detection limit and citation for validation studies
Viable organisms ≥ 50 μm	Visual counts or stereo-microscopy.	No international standard for ballast water analysis at this time.	Can be expensive and time-consuming, needs moderately trained personnel. (Note that OECD Test Guideline for Testing of Chemicals 202, "Daphnia sp. Acute immobilization test and reproduction test" could be used as basis for standard methodology.)	To be determined.
Viable organisms ≥ 50 μm	Visual inspection.	No international standard for ballast water analysis at this time.	Visual inspection is likely to only register organisms bigger than 1,000 micro-metres in minimum dimension.	To be determined.
Viable organisms ≥ 10 μm and < 50 μm	Variable fluorometry.	No international standard for ballast water analysis at this time.	Only monitors photosynthetic phytoplankton and thus may significantly underestimate other planktonic organisms in this size fraction.	To be determined.
Viable organisms ≥ 50 μm and ≥ 10 μm and < 50 μm	Photometry, nucleic acid, ATP, bulk fluorescein diacetate (FDA), chlorophyll a.	No international standard for ballast water analysis at this time.	Semi-quantitative results can be obtained. However, some of these organic compounds can survive for various lengths of time in aqueous solution outside the cell, potentially leading to false positives. Welschmeyer and Maurer (2012). The reference to organic compound survival does not refer to CV6; further information on CV6 can be found in documents MEPC 74/INF.17 and PPR 7/INF.5.	To be determined.
Viable organisms ≥ 50 μm and ≥ 10 μm and < 50 μm	Flow cytometry.	No international standard for ballast water analysis at this time.	Very expensive.	To be determined.
Enterococci	Fluorometric diagnostic kit.	No international standard for ballast water analysis at this time.	Minimum incubation time 6 h. Semi-quantitative results from portable methods (see paragraph 2.2.2 of annex 1).	To be determined.
Escherichia coli	Fluorometric diagnostic kit.	No international standard for ballast water analysis at this time.	Minimum incubation time 6 h. Semi-quantitative results from portable methods (see paragraph 2.2.2 of annex 1).	To be determined.
Vibrio cholerae (O1 and O139)	Test kits.	No international standard for ballast water analysis at this time.	Relatively rapid indicative test methods are available.	To be determined.
Viable organisms ≥ 50 μm and ≥ 10 μm and < 50 μm	Pulse counting fluorescein diacetate (FDA).	No international standard for ballast water analysis at this time.	Sampling kit can be larger than that for bulk fluorescein diacetate (FDA).	To be determined.
Total living bacteria including Enterococci, Escherichia coli, Vibrio cholerae	Second-generation ATP	No international standard for ballast water analysis at this time.	Semi-quantitative results can be obtained	PPR 7/INF.4

4.3 Table 4: Detailed analysis methods for use when testing for compliance with the D-2 standard

Indicator	General approach	Standard method	IMO citation	Notes	Level of confidence or detection limit and citation for validation studies
Viable organisms ≥ 50 μm and ≥ 10 μm and < 50 μm	Visual counts or stereo-microscopy examination. May be used with vital stains in conjunction with fluorescence + movement.	No international standard for ballast water analysis at this time, but see US EPA ETV Protocol, v. 5.1	BLG 15/5/5 and BLG 15/5/6 BLG 15/INF.6	Can be expensive and time-consuming, needs trained personnel. (Note that OECD Test Guideline for Testing of Chemicals 202, "Daphnia sp. Acute immobilization test and reproduction test" could be used as basis for standard methodology.)	To be determined.
Viable organisms ≥ 10 μm and < 50 μm	Visual counts with use of vital stains.	No international standard for ballast water analysis at this time, but see US EPA ETV Protocol, v. 5.1	BLG 15/5/10 (method) BLG 15/5/5 and BLG 15/5/6 (approach) MEPC 58 /INF.10	Requires specific knowledge to operate them. It should be noted that there may be limitations using vital stains with certain technologies.	To be determined. Steinberg et al., 2011
Viable organisms ≥ 10 μm and < 50 μm	Flow cytometers (based on chlorophyll a and vital stains).	No international standard for ballast water analysis at this time.	BLG 15/5/5 and BLG 15/5/6	Expensive and require specific knowledge to operate them. It should be noted that there may be limitation using vital stains with certain technologies.	To be determined
Viable organisms ≥ 50 μm and Viable organisms ≥ 10 μm and < 50 μm	Flow cameras (based on chlorophyll a and vital stains).	No international standard for ballast water analysis at this time.	BLG 15/5/5 and BLG 15/5/6	Expensive and require specific knowledge to operate them. It should be noted that there may be limitations using vital stains with certain ballast water management systems.	To be determined
Viable organisms ≥ 50 μm and Viable organisms ≥ 10 μm and < 50 μm	Culture methods for recovery, regrowth and maturation.	No international standard for ballast water analysis at this time.	BLG 15/5/5, BLG 15/5/6 and PPR 7/INF.10	Densities are expressed as the sum of cultivable autotrophs after a 2-week incubation time and motile heterotrophs as determined by epifluorescence microscopy.	Validation available in Cullen (2019).
Enterococci	Culture methods.	ISO 7899-1 or ISO 7899-2	BLG 15/5/5 and BLG 15/5/6	Requires specific knowledge to conduct them. At least 44-h incubation time. EPA Standard Method 9230	To be determined.
Escherichia coli	Culture methods.	ISO 9308-3 or ISO 9308-1	BLG 15/5/5 and BLG 15/5/6	Requires specific knowledge to conduct them. At least 24-h incubation time. EPA Standard Method 9213D	To be determined.
Vibrio cholerae (O1 and O139)	Culture and molecular biological or fluorescence methods.	ISO/TS 21872-1/13/	BLG 15/5/5 and BLG 15/5/6	Requires specific knowledge to conduct them. 24-48 h incubation time. US EPA ETV Fykse et al., 2012 (semi-quantitative pass/fail-test) Samples should only be cultured in a specialized laboratory.	To be determined.
Enterococci, Escherichia coli, Vibrio cholerae (O1 and O139)	Culture with 11holera11ence-in-situ hybridization (FISH)	No international standard for ballast water analysis at this time.		Requires specific knowledge to conduct them. Quantitative and qualitative results after 8 h. Samples should only be cultured in a specialized laboratory.	To be determined.
Viable organisms ≥ 50 μm and viable organisms ≥ 10 μm and < 50 μm	Visual counts using stereo-microscopy examination and flow cytometry.	No international Standard for ballast water analysis at this time.	BLG 17/INF.15	A Sampling Protocol that identifies whether a system is broken or not working and producing a discharge that is significantly above the D-2 standard. Designed to detect gross non-compliance with 99.9% confidence. Needs to be Validated.	To be determined.

4.4 Table 5: General approaches for sampling use when testing for compliance with the BWM Convention

General approaches for sampling	Discharge line or BW tank	Citation for validation study or use	Sample error and detection limit	Relative sample error amongst approaches
Filter skid + isokinetic sampling	Discharge line	Drake et al., 201First et al., 2012 (land-based testing); shipboard validation underway, Prototype 01, SGS	To be determined	Lower
Cylinder containing plankton net + isokinetic sampling	Discharge line	MEPC 57/INF.17	To be determined	Lower
Sampling tub containing plankton net + isokinetic sampling	Discharge line	Gollasch, 2006 and Gollasch et al., 2007 Cangelosi et al., 2011	To be determined	Lower
Continuous drip sampler + isokinetic sampling	Discharge line	Gollasch and David, 2010, 2013	To be determined	Lower
Grab sample	BW tank	David and Perkovic, 2004; David et al. 2007, BLG14/INF.6	To be determined	Higher

4.5 Table 6: Sampling and analysis methods/approaches for use when testing compliance with the BWM Convention. A checkmark indicates an appropriate combination of sampling and analysis.

Analysis type size class or indicator microbe analysis method/approach		Filter skid + isokinetic sampling^footnote	Plankton net + isokinetic sampling	Continuous drip sampler + isokinetic sampling	Grab sample
Indicative Analysis ≥ 50 μm Visual inspection Stereomicroscopy counts Flow cytometry Nucleic acid
	ATP Chlorophyll a, Bulk FDA
Indicative Analysis < 50 μm and ≥ 10 μm variable fluorometry Flow cytometry Nucleic acid
	ATP Chlorophyll a, Bulk FDA
Indicative Analysis Enterococci, E. coli
	Fluorometric diagnostics
Indicative Analysis Vibrio cholerae
	Test kits Culture methods + microscopy
Detailed Analysis ≥ 50 μm
	Stereomicroscopy counts Flow cytometry/Flow camera
Detailed Analysis < 50 μm and ≥ 10 μm
	Visual counts + vital stain(s) Flow cytometry/Flow camera Culture methods
Detailed Analysis Enterococci, E. coli
	Culture methods FISH with pre-cultivation
Detailed Analysis Vibrio cholerae
	Culture methods FISH with pre-cultivation

4.6 References

Cullen JJ (2019). The best available science describing type-approval testing methods and protocols for ballast water management systems that render nonviable organisms in ballast water. http://doi.org/10.5281/zenodo.2656597

David M & Perkovic M (2004). Ballast Water Sampling as a Critical Component of Biological Invasions Risk Management, Marine Pollution Bulletin, Vol. 49, 313-318.

David M, Gollasch S, Cabrini M, Perkovič M, Bošnjak D & Virgilio D (2007). Results from the First Ballast Water Sampling Study in the Mediterranean Sea – the Port of Koper Study. Marine Pollution Bulletin 54(1), 53-65.

Drake LA, Moser CS, Robbins-Wamsley SH, Riley SC, Wier TP, Grant JF, Herring PR, First MR (2014). Validation trials of a shipboard filter skid (p3SFS) demonstrate its utility for collecting living zooplankton. Marine Pollution Bulletin 79, 77–86.

First MR, Lemieux EJ, Hyland WB, Grant JF, Moser CS, Riley SC, Robbins-Wamsley SH, Steinberg MK, Wier TP, Drake LA (2012). Validation of a closed-housing filter skid for in-line sampling of aquatic organisms. Journal of Plankton Research 34:321-331.

Fykse EM, Nilsen T, Nielsen AG, Tryland I, Delacroix S, Blatny JM (2012). Real-time PCR and NASBA for rapid and sensitive detection of Vibrio cholerae in ballast water. Marine Pollution Bulletin 64:200-206.

Gollasch S (2006). A new ballast water sampling device for sampling organisms above 50 micron. Aquatic Invasions, Volume 1, Issue 1: 46-50.

Gollasch S, David M, Voigt M, Dragsund E, Hewitt C & Fukuyo Y (2007). Critical review of the IMO International Convention on the Management of Ships' Ballast Water and Sediments. In Hallegraeff, G.M. (ed.): Harmful Algae 6, 585-600.

Gollasch S & David M (2013). Recommendations for Representative Ballast Water Sampling. Final report of research study of the Bundesamt für Seeschifffahrt und Hydrographie (BSH), Hamburg, Germany. Order Number 4500025702. 28 pp.

Gollasch S & David M (2010). Testing Sample Representativeness of a Ballast Water Discharge and developing methods for Indicative Analysis. Final report of research study undertaken for the European Maritime Safety Agency (EMSA), Lisbon, Portugal, 124 pp.

Steinberg MK, Lemieux EJ, Drake LA (2011). Determining the viability of marine protists using a combination of vital, fluorescent stains. Marine Biology 158:1431-1437.

Throndsen, J (1978). Chapter 7.6: The dilution-culture method. In Phytoplankton manual, Ed: Sourina, A., UNESCO, France, p. 218-224.

U.S. Environmental Protection Agency (2010). Environmental Technology Verification Program (ETV) Generic protocol for the verification of ballast water treatment technology, Version 5.1. Report number EPA/600/R-10/146, United States Environmental Protection Agency, Washington, D.C.

Welschmeyer N & Maurer B (2012). A portable, sensitive plankton viability assay for IMO shipboard ballast water compliance testing. In: Proceeding of the Global R and D forum on Compliance Monitoring and Enforcement, Eds. A. Olgun, F.T. Karokoc and F. Haa.