MARINE ENVIRONMENT PROTECTION

COMMITTEE

52nd session

Agenda item 2

　

　

　

1 This document describes a proposal for the minimum concentration of indicator organisms in the two size ranges larger than or equal to 10 pm in influent water for test-bed tests, including data of extreme high concentration of organisms found in natural environment which were used to find the minimum concentration.

　

2 It was agreed at MEPC 51 to refer such extreme high concentration of organisms in natural environment to decide the minimum concentration of indicator organisms.

　

3 Data used for the analysis of the extreme high concentration found in natural environment were taken from reports of regulatory red tide monitoring program implemented in the inner part of Tokyo Bay. As the data were collected at one of the highly eutrophicated areas, Tokyo Bay, where phytoplankton blooms almost always using high amount of nutrients and zooplankton also grows well supported by high number of food phytoplankton, the data can be used to enumerate the extreme high organism numbers in natural environment.

　

4 The data of the organisms were collected by conventional methods adequate for regulatory red tide monitoring. It is necessary to add re-evaluation and re-calculation to modify the data, in order to find the extreme high organisms number of the two size categories of D-2 standard, i.e. size larger than or equal to 50μm (referred to as "L size group" hereafter) and size smaller than 50μm and larger than or equal to 10μm (referred to as "S size group" hereafter), considering the minimum dimension as the size of organisms.

　

5 There are several factors which make the extreme high number of organism of the S size group overestimate and also underestimate. The former factors are 1) counting method of colony forming organisms and 2) package capacity of wild small blooming organisms vs. large culture indicator organisms. The latter factor is disappearance of fragile cells by preservative chemicals.

　

6 After analysis of these factors, Japan proposes the minimum concentration of indicator organisms the L and S size groups necessary to prepare in influent water for test-bed tests are 10⁷ ind./m³ and 10³ ind./ml, respectively.

　

　

7 Data used for the analysis were 320 sets of plankton composition observed in red tide monitoring activities implemented by the Tokyo Metropolitan Government at the inner part of Tokyo Bay during April 1999 and March 2001. The inner part of Tokyo Bay was selected as the study site, because the area has almost always high organism number due to its eutrophicated condition, and also water of the area was often loaded as ballast water by big vessels used for international trades departing from ports such as Yokohama, Tokyo and Chiba. All the data is open to public in reports published by the Metropolitan Government in March 2002.

　

8 The data of quantitative analysis of organisms were taken by conventional methods adequate for regulatory red tide monitoring. Samples were collected by a bottle sampler at the monitoring, preserved by adding glutaraldehyde, and then observed under microscope for quantitative analysis, i.e. identification and enumeration of all organisms.

　

　

9 (Step 1) It is necessary to add re-evaluation and re-calculation to modify the data, because the D-2 standard uses a new concept, i.e. minimum dimension, which has never been used for subdivision of organisms in marine science.

　

10 (Step 2) It is also necessary to observe several factors which make the extreme high concentration of organism overestimate and also underestimate. The former factors are 1) counting method of colony forming organisms and 2) Cell volume effect between wild small blooming organisms vs. large culture indicator organisms. The latter factor is disappearance of fragile cells by preservative chemicals.

　

　

11 In order to find the highest number of organisms of the L and S size groups, following steps of modification were given to the original data.

　

12 Figures 1-1 and 1-2 show the change of the three months average (January to March, April to June, July to September, and October to December) of the high concentration of organisms in the L and S size groups, respecitively. The extreme high concentration of the L and S size groups in Tokyo Bay are 10 ind./m³ and 10⁴ ind./ml, respectively.

　

　

　

13 In the case of colony forming species, their minimum dimension were decided based on a size of colony, not individual unit organism.

　

14 Tiny planktonic organisms tend to form colonies to increase buoyancy. Individual cells of most of colony forming species such as diatoms and dinoflagellates are smaller than 10μm in their minimum dimension, and naturally becomes outside of the L and S size groups. But colonies themselves are larger than 10μm in their minimum dimension. Therefore these colonies can be counted as one individual, and must be included in L or S size group, depending on its colony size in minimum dimension.

　

15 The original data used for this analysis contained all individual number, not colony number. Re-evaluation of data following above mentioned procedure may make the high concentration 10 to 10² times smaller. Because many colonies are consisted of 10 to 10² individual cells.

　

　

16 Volume of cells of the several representative organisms of S size group producing extreme high concentration in natural environment is often more than 10 times smaller than that of several representative indictor organisms expected to be used in influent water in test-bed tests, regardless of species variation. Under extreme high concentration organisms become smaller because of high cell division rate and nutrient depletion in ambient waters. Under artificial culture condition indicator organisms do not become small.

　

17 It is practically impossible to reproduce extreme high concentration made possible by organisms gotton smaller in wild environment using largere indicator organisms in artificial environment.

　

18 The difference of the cell volume may induce practicable minimum concentration of indicator organisms 10 times smaller than the high extreme concentration of wild organisms.

　

　

19 It is known that preservative chemicals such as glutaraldehyde often break fragile organisms. Broken individuals cannot be recognized as organisms and eventually dropped from the data of the quantitative analysis. It should be noted that the data contains such kind of inevitable errors and tends to be underestimated. The degree of underestimation cannot be taken into account because of the lack of scientific knowledge, but it is thought that the number of disappeared organisms are usually smaller than that of remaining organisms after preservation.

　

　

20 Based on the extreme high concentration of aquatic organisms in Tokyo Bay, i.e. 10⁷ ind./m³ and 10⁴ ind./ml for the L and S size groups, respectively, and considering several above mentioned factors, Japan considers that 10⁷ ind./m³ and 10³ ind./ml for the L and S size groups, respectively, as an appropriate minimum concentration of aquatic organisms in the influent water.

　

 With reference to L size group, it may be noted that ISO 14669: 1999 (Water quality - Determination of actual lethal toxicity to marine copedods (copepoda, Crustacea)) recommends that the density of copepods does not exceed 1 per 0.5 ml of solution and for Acartia tonsa, 1 copepod per 5 ml solution. This translates to 10⁵〜10⁶ ind./m³.