2024-18597. Consideration for Acceptance of One or More Viability Testing Methods for Type Approval of Ballast Water Management Systems

Table 1—Potential Viability Testing Methods for Coast Guard Evaluation

Viability test method	Target organisms	Brief description
Serial dilution culture most probable number (SDC-MPN) assay 1	10-50 µm size class (Autotrophs)	SDC-MPN is a growth-based method that relies on the use of serial dilutions to track population growth over time, particularly in organisms with defined growth characteristics, like microalgae. The MPN culturing technique for protists was adapted from food microbiology laboratories for use in evaluating phytoplankton communities. It uses a 14-day grow-out period to quantify reproductively viable phytoplankton.
Most Probable Number Dilution Culture + Motility 1	10-50 µm size (Heterotrophs)	The Heterotroph Method uses epifluorescence microscopy to identify and exclude from enumeration organisms that display the red fluorescence of chlorophyll, a diagnostic of photosynthetic autotrophs. The remaining organisms—those without detectable chlorophyll—are defined as heterotrophs. Heterotrophs that are observed to move are classified as living.
Microscopy 2 (Standard, Epifluorescence, Variable fluorescence, Chlorophyll a fluorescence, Raman spectroscopy, Confocal Raman spectroscopy with near infrared excitation)	10-50 µm and greater than or equal to 50 µm size classes	Direct observation of ballast water samples under magnification using stereo or compound microscopes. Specific types of microscopies use various light sources and sensors to enhance detection of cells and cell processes. Several indicative tests using variable fluorescence are commercially available.
Motility and fluorescence assay 3	10-50 µm and greater than or equal to 50 µm size classes	Automated system that counts motile and fluorescent organisms; indicative tests are available and under development.
Adenosine triphosphate (ATP) assay 3	10-50 µm size class	1st Generation methods measure active ATP in the presence of luciferase enzyme. 2nd Generation ATP test method measures all living bacteria, culturable and non-culturable as well as autotrophs and heterotrophs. ATP assays are commercially available for indicative shipboard commissioning testing using the IMO D-2 standard.
( print page 67650)
Polymerase chain reaction (PCR) 3		Several kinds of molecular methods based on PCR are used to detect live cells, including a molecular activity test and viability PCR, Messenger ribonucleic acid (mRNA) is a type of single-stranded RNA involved in protein synthesis. Because mRNA is synthesized by living cells during the process of transcription, the MVT measures changes in concentration of mRNA as an indicator of the cell's vitality. Reverse transcription PCR allows the use of RNA as a template to generate complementary deoxyribonucleic acid (cDNA). Using the reverse transcriptase enzyme, a single-stranded copy of cDNA is generated. This can then be amplified by a DNA polymerase, generating double-stranded cDNA, feeding into a standard PCR-based amplification process. Quantitative PCR (qPCR) is used to detect unwanted microbes ( e.g., pathogens) and to identify DNA sequences used to classify the organisms in real time; qPCR methods generally use a fluorescent probe to quantify the DNA.
mRNA 3	bacteria (less than 10 µm size class) and	Viable bacteria can be identified through mRNA, which exists only in molecularly active organisms. This tool is used in conjunction with reverse transcriptase PCR to reduce the time required to culture bacteria in food products.
mRNA 3	10-50 µm and greater than or equal to 50 µm size classes	In principle, an mRNA primer can be created for any organism for which adequate genetic Information is available, including chordates such as tunicates.
Notes:
1  Test may be used for IMO type approval of all BWMS technologies (BWM.2/Circ.61/Rev.1 Annex).
2  Various types of microscopies are currently used to identify living organisms in these size classes; reproductive viability cannot be determined through microscopy alone.
3  Theoretically suitable for viability testing method; research and development are ongoing.