MARINE ENVIRONMENT PROTECTION

COMMITTEE

52nd session

Agenda item 2

　

　

　

　

1 At MEPC 51 the Ballast Water Working Group decided to develop 13 guidelines for the uniform implementation of the International Convention for the Control and Management of Ships' Ballast Water and Sediments. One of these guidelines, the Guidelines for Approval of Ballast Water Management Systems (G8), needs submissions from delegations on quantitative analytical methods of organisms used in influent water for test-bed tests.

　

2 This document presents a first draft of the analytical methods. The draft, which can be found in the annex to this document, has to be further developed. Japan wishes to finalize the Guidelines for Approval of Ballast Water Management Systems (G8) by MEPC 52 and to start type approval process using standardized procedure by the Organization.

　

　

3 The regulation D-2 of the Convention shows the maximum allowable number of organisms in discharging ballast water. Ballast water treatment systems have to decrease organisms in influent water to the same level. Therefore the guideline needs to provide following methods to evaluate the treatment efficacy:

　

　

4 The Committee is invited to note the proposals in this document, including the text in the annex, and take action as appropriate.

　

　

　

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1.1 This draft text is intended to clarify the quantitative analytical methods of organisms larger than or equal to 50μm in their minimum dimension (referred to as "L size group" hereafter) and those smaller than 50μm and larger than or equal to 10μm (referred to as "S size group" hereafter) used for a test-bed test which is a part of procedures for approval and certification of a single piece of equipment, or a series of equipment (Type Approval) of the Guidelines for Approval of Ballast Water Management Systems (G8).

　

1.2 The regulation D-2 of the International Convention for the Control and Management of Ships' Ballast Water defines allowable maximum concentration in discharging water. Ballast water management systems have to decrease organisms in influent water to the same level concentration. Therefore the guidelines need to provide sampling and counting methods of the organisms to evaluate the treatment efficacy.

　

1.3 Live organisms in processed samples by a prototype treatment system shall be measured by the following methods. Organisms in effluent water and control samples also shall be measured by the same methods with slight modification such as strength of concentration, if necessary.

　

　

2.1 For measurement of organisms of L size group, water of 1m³ more shall be used. The sample water shall be concentrated using a plankton net or net cloth with a mesh size of 50μm (Figure 1) gently in order to avoid giving damage by compaction. The volume of sample water shall be measured as accurate as possible to the units of 100 litres and that of the concentrated sample water to units of 1 litre to calculate concentration factor.

　

2.2 For measurement of organisms of S size group, water of 1 litre or more shall be used. Sample water that has passed through plankton net or net cloth with a mesh size of 50μm shall be concentrated using a plankton net or mesh with a pore size of 10μm (Figure 1). The concentration step must be processed carefully and gently not to give any damage to organisms by compaction or clogging on the mesh. The volume of sample water shall be measured as accurate as possible to the units of 10 ml and that of the concentrated sample water to units of 1 ml.

　

2.3 The concentration step is necessary to facilitate microscopic observation. Degree of concentration varies depending on type of treatment systems. Filtration treatment system may have few organisms in treated water, and high level of concentration is possible. But some systems such as chemical treatment may have a lot of dead organisms after processing, and only low level of concentration may be possible. Possible level of concentration is also different depending on composition of test organisms. Therefore strength of concentration must be decided in case by case principle, considering compaction damage and necessary time for microscopic observation.

　

2.4 Concentrated sample water shall be transfered into a wide mouthed container such as a beaker, using GF/F filtered water. A small portion of the sample will then be taken by a pipette from the container and put onto a small chamber for microscopic observation. Care should be taken not to put excess number of organisms, either live or dead, in a container. If there are many dead bodies of organisms after treatment, it could affect the viability of remaining surviving organisms and disturb counting procedures.

　

　

 It is a most important and fundamental step for evaluation of efficacy of treatment systems to count number of viable organisms with measuring their minimum dimension. Only viable organisms in either L or S size group must be counted. For the purpose, it is necessary to count them with measuring their dimension and observing their viability under microscope. It is not necessary to identify organisms, but if it is possible, it facilitates the observation, especially judgement of size group. As viable unicellular organisms in samples often complete binary cell division within a few hours under optimum condition, quick processing and observation of samples for counting is necessary. If condition does not allow immediate observation, samples should be kept under low temperature. Preservation of samples using chemicals must be avoided, because preservation causes loss of fragile organisms and also loss of important characters of organisms useful for judgement of viability.

　

　

3.1.1 Criteria for judgement of viability of organisms are 1) morphological change, 2) mobility, 3) changes of color of organisms after using staining dyes utilized for differentiation of viability, and 4) re-growth after inoculation into optimum environment. As every method has been developed to work effectively to certain specific taxonomic groups of organisms and eventual uncertainity of effectiveness remains in some groups of organisms that are outside the original target of the method. Considering test water for a treatment system may contain more than two different organisms, measurement by a single criteria cannot avoid uncertainity. Therefore viability of organisms shall be evaluated by using at least [one/two different] criteria among the four.

　

3.1.2 Morphological change, one of the criteria, shall include 1) damage to body of organisms, 2) loss of flagellum and 3) discoloration of pigments (Figure 2).

　

3.1.3 Mobility, a criterion applicable to mobile organisms, shall be judged by the presence or absence of swimming movement. Some flagellates are known to make immobile temporary resting cells by environmental stress and therefore careful observation of recoverance of mobility is needed. If necessary, re-growth experiment described in paragraph 3.3 shall be made.

　

3.1.4 If the dye staining method is used, documents explaining the scientific basis for dyes used, test methods, and judgment criteria shall be attached to reports of results on treatment effectiveness.

　

3.1.5 For confirmation of re-growth of organisms needs laboratory culture experiment using individual organisms in doubt. Details of the experiment design is shown in paragraph 3.4.2.

　

　

3.2.1 Minimum dimension is the only criterion to subdivide organisms in samples into the L or S size group or outside of them. Every organism has three dimensions, i.e. body length, body width and body depth which exclude appendages, and the minimum dimension is the shortest one among the three.

　

3.2.2 At microscopic observation of samples for L size group analysis, which are prepared by a sampling method described in the paragraph 2.2, most of organisms in the samples are expected to belong to L size group. But some may belong to S size group because of their specific body shape which prevent passing through a mesh of 50μm size. If such organisms are detected, their minimum dimension must be measured under microscope and an appropriate size group must be determined.

　

3.2.3 It would be useful to prepare a list of organisms that might appear in test water with a table of their minimum dimensions (Table 1). If taxonomic identification of organisms in samples is easy, their classification of L or S size groups can be determined by observing the list during counting.

　

　

3.3.1 Many organisms in aquatic environment make colonies to increase buoyancy. Their individual unit is often smaller than 10μm but the colony becomes larger than several hundred micrometers. If we strictly apply to the criterion of minimum dimension, organisms smaller than 10μm are outside of the target, and then all the large colonies of the organisms can be neglected for counting, even a lot of them are found in samples. In order to avoid such conflict, the following arrangement for counting can be set.

　

3.3.2 For species that form colonies, the colony itself instead of its components shall be treated as an individual, and L or S size groups are determined by the minimum dimension of the colony (Figure 3).