Part 4 – Sample Analysis Methods for the Determination of Biological Constituents in Ballast Water

  4.1 Samples taken during testing of BWMS are likely to contain a wide taxonomic diversity of organisms, varying greatly in size and susceptibilities to damage from sampling and analysis.

  4.2 When available, widely accepted standard methods for the collection, handling (including concentration), storage, and analysis of samples should be used. These methods should be clearly cited and described in test plans and reports. This includes methods for detecting, enumerating, and identifying organisms and for determining viability (as defined in these Guidelines).

  4.3 When standard methods are not available for particular organisms or taxonomic groups, methods that are developed for use should be described in detail in test plans and reports. The descriptive documentation should include any experiments needed to validate the use of the methods.

  4.4 Given the complexity in samples of natural and treated water, the required rarity of organisms in treated samples under regulation D-2, and the expense and time requirements of current standard methods, it is likely that several new approaches will be developed for the analyses of the composition, concentration, and viability of organisms in samples of ballast water. Administrations/Parties are encouraged to share information concerning methods for the analysis of ballast water samples, using existing scientific venues, and papers distributed through the Organization.

  4.5 Sample analysis is meant to determine the species composition and the number of viable organisms in the sample. Different samples may be taken for determination of viability and for species composition.

  4.6 Viability of an organism can be determined through live/dead judgement by appropriate methods including, but not limited to: morphological change, mobility, staining using vital dyes or molecular techniques.

  4.7 A treatment test cycle should be deemed successful if:

• .1 it is valid in accordance with paragraph 2.2.2.5 or 2.3.36 as appropriate;

• .2 the average density of organisms greater than or equal to 50 micrometres in minimum diameter in the replicate samples is less than 10 viable organisms per cubic metre;

• .3 the average density of organisms less than 50 micrometres and greater than or equal to 10 micrometres in minimum diameter in the replicate samples is less than 10 viable organisms per millilitre;

• .4 the average density of Vibrio cholerae (serotypes O1 and O139) is less than 1 cfu per 100 millilitres, or less than 1 cfu per 1 gramme (wet weight) zooplankton samples;

• .5 the average density of E. coli in the replicate samples is less than 250 cfu per 100 millilitres; and

• .6 the average density of intestinal Enterococci in the replicate samples is less than 100 cfu per 100 millilitres.

  4.8 It is recommended that a non-exhaustive list of standard methods and innovative research techniques be considered^footnote.

  4.9 Toxicity tests of the treated water discharge should be conducted in accordance with paragraphs 5.2.3 to 5.2.7 of the Procedure for approval of ballast water management systems that make use of Active Substances, as revised (resolution MEPC.169(57)).