The project "Research and Development of the Special Pipe System for Ballast Water Treatment" implemented by the Japan Association of Marine Safety with the support of Japan Foundation has two components: 1) improvement the special pipe system to achieve better effectiveness in the termination of zooplankton and phytoplankton, and 2) development of the procedure and standard for evaluation of the effectiveness of the treatment system. This paper describes the second component, and the first one is also explained in another article recorded in the same proceedings.

　

　

The test procedure was first designed to evaluate the special pipe system, one of the mechanical treatments. But its concept and the procedure itself can be applied to the analysis of the effectiveness of any other method.

　

　

The project has commenced in April 1999 and still on going.

　

　

The objective of this study is to develop a specific test procedure for evaluation of a ballast water treatment system to terminate and eliminate harmful aquatic organisms contaminated in ballast water based on biological and ecological nature of the organisms in coastal waters.

　

　

In order to establish an appropriate test procedure, it is essential to analyze the biological and ecological features of organisms in port areas where ballast water has been taken. Seasonal change and regional difference of composition and numbers of plankton in Japanese waters were observed using several references such as Nomura and Yoshida (1997). Special attention was paid to high phytoplankton numbers occurring at red tides.

Based on data obtained by the analysis of plankton nature, necessity of selection of test organisms for evaluation of ballast water treatment system was assessed. For the selection, following criteria were considered; 1) the test organisms should be available in a certain amount easily anytime and anywhere to put enough concentration in test water to evaluate the result; 2) the organisms must be found in both near-shore and off-shore waters easily, as the evaluation experiment includes a test bed test on land and a onboard test in ship; 3) the organisms should be easily differentiated its survival or fatality with high accuracy for evaluation of effectiveness of treatments. A test procedure and a standard for ballast water treatment were also designed using results of above mentioned analysis.

　

Ballast water has not only planktonic organisms, but also small benthic ones living in bottom sediment and being re-suspended by water flow, if water is charged at shallow ports. But it is appropriate to use only planktonic organisms at first for the materials of the present study in order to simply the way of discussion. Introduction of benthic organisms such as mussel and seaweeds may be made not by transport of benthic adult organisms, but by planktonic eggs and larvae, of which numbers are usually larger more than several thousand times.

　

　

　

　

Tokyo Metropolitan Government monitors red tide occurrence in Tokyo Bay regularly and reports phytoplankton number as one of the parameters observed. In 1999 and 2000, the highest, lowest and average cell numbers were 188,860, 76 and 16,260 cells/ml, respectively, among 312 samples (Tokyo Metropolitan Governement 2002). Their methods of sampling and observation were not described in details. But the cell numbers must be based on quantitative analysis of live samples collected by a bucket and kept without using any fixative reagent under a regular compound microscope, as commonly applied for red tide research.

　

Seasonal fluctuation of plankton number is wide in eutrophic temperate areas such as Tokyo Bay. Because high nutrient concentration can keep high number of plankton individuals. But sometimes other environmental parameters disturb the increase of plankton number, and therefore range of individual number becomes wider. Nomura and Yoshida (1997) reported the change of phytoplankton composition and cell numbers of 35 monthly samples collected by a bucket from surface and preserved by formalin at Tokyo Bay during 1991 and 1993. Summary of their results is as follows;

　

Nomura (1998) reviewed historical phytoplankton records in Tokyo Bay between 1907 and 1997 using more than 45 publications and summarized the number of species reported in certain duration. In the whole years occurrence of more than 273 species, of which 78, 66, 59, 187 and 119 species were reported in 1900-1940s, 1950-1960s, 1970s, 1980s and 1990s, respectively, were recorded. The number of species varied depending on environmental condition of the bay and techniques of sampling and observation used for the study.

　

Red tide is defined as discolored water caused by high concentration of microscopic unicellular organisms. It is one of the ultimate high concentrations of plankton. Japan Fisheries Agency issues an annual report on red tide occurred in Seto Inland Sea in the western Japan from 1973. Following is a summary of data collected between 1992 and 2000 (Japan Fisheries Agency 1993-2001).

　

　

Most of research on zooplankton composition analysis used a plankton net, of which mesh size was more than 80 μm, as a sampling tool. Quite few data are useful to analyze the change of individual number of whole zooplankton, i.e. plankton community of all size ranges.

　

Tokyo Metropolitan Government observes zooplankton number simultaneously at red tide monitoring research in Tokyo Bay regularly and reports the number as one of the parameters observed. In 1999 and 2000, the highest, lowest and average individual numbers of zooplankton were 667,140, 90 and 34,299 ind./l, respectively, among 305 samples (Tokyo Metropolitan Government 2002). The numbers were based on quantitative analysis of live samples collected by a bucket and kept without using any fixative reagent. The large part of community was occupied by unicellular protozooplankton, and large-sized zooplankton such as copepods were usually minor member in individual number.

　

Shizuoka Prefecture (1999) reported seasonal change of plankton composition at the central part of Sagami Bay, which has good water circulation influenced by Kuroshio Current. Microzooplankton smaller than 22 μm was dominated by unicellular protozoa such as ciliates and appeared several hundred individuals per liter. Zooplankton larger than the size was organized various group of animals such as variety of copepods (Maxillopoda), arrow worms (Sagittoidea) and planktonic sea worms (Polychaeta). Among them copepods is common and dominant organism, and individual number is about 100 ind./l.

　

　

Several ecological characters on plankton community in Japanese coastal waters become clearer from the analysis described above.

　

Quite wide diversity of plankton, both phyto- and zooplankton, is obvious in terms of organism number and species variety. Phytoplankton cell number differs about 5,000 times, and species number 3 times by sampling times. Zooplankton individual number also varies about 7,000 times. Consequently it is fundamentally necessary to define organism(s) to use as test materials for evaluation of ballast water treatment system. Result using low plankton concentration is not comparable to those using 7,000 times high concentration.

　

　

Among phytoplankton diatoms and dinoflagellates are two major components. Zooplankton has two groups, i.e., small unicellular protozooplankton has ciliates, and larger microzooplankton has copepods as constant major members. For evaluation of effectiveness of treatment the judgment of live or dead of the test organisms is crucial. Change of shape and mobility is indicative character useful for evaluation.

　

Diatoms and copepods do not change their shape by preservation using chemicals. But almost all ciliates burst and disappear by sudden change of temperature or salinity and also by fixative reagents. Dinoflagellates have both groups. One half has thick cellulose plates on cell surface and then do not change their shape by fixation, but the other has no plate and change shape or disappear by bursting by fixation.

　

Differentiation of live or dead is easy in organisms that have mobility. All zooplankton actively move and some phytoplankton such as dinoflagellates also can move by their flagella. But diatoms cannot move because of lack of any organ for movement and therefore mobility cannot is not indicating character for them to judge live or dead.

　

Photosynthetic members of phytoplankton, i.e. diatoms and a half of dinoflagellates, have color of photosynthetic pigments. After death of cells, the color disappears gradually, but it remains sometimes more than a day. Damaged cells also have less color, but potential of recovery cannot be judged by appearance. Therefore color of organisms, either by organic specific pigment or stained by chemicals, is not adequate to use for the judgment. Many cells show faint color after treatment and it makes judgment of effectiveness very difficult..

　

Test organisms cannot be preserved by fixative chemical to observe live or dead. It means that organisms in samples have potential to grow even after treatment. Unicellular organisms can make cell division often once to several times a day. Diatoms often have short doubling time of several hours. But dinoflagellates can make cell division once a day at maximum. Therefore samples after treatment should be analyzed within a few hours to avoid the change of cell number.

　

　

In phytoplankton group, diatoms and dinoflagellates are two major members available everywhere almost always. Judgment of effectiveness using diatoms must be impossible, because they are immobile and do not change their shape. Dinoflagellates (Dinophyceae) have advantage, as they stop moving and about a half of them change shape after death. Therefore Dinophyceae have better indicative feature for a test organisms.

　

In zooplankton group, protozooplankton smaller than 20μm are abundant near shore, but rare in off shore waters. Copepods (Maxillopoda) is major member and appears everywhere any time, but other zooplankton such as arrow worms occur at certain time in a year.

　

Based on criteria described in the Methods and data described in the Section 1 of the Results, the testing organisms could be selected Dinophyceae from phytoplankton and Maxillopoda（Copepoda）from zooplankton. These individuals with 20μm or more in size may be used for experiments.

　

　

Plankton number varies in very wide range. High concentration is several thousand times larger than low concentration. Results of treatment using high concentration of plankton must be very different from those using low concentration.

　

 The observation of performance and the judgment of effectiveness of the treatment may be conducted in the following steps. Observation of all samples collected should be conducted within one day after the sampling of the water, thus avoiding, as far as practical, the change of conditions of targeted organisms under storage. The environmental parameters of the waters before and after the treatment should be observed, including temperature, salinity and pH.

　

　

　

According to the analysis of plankton community and its ecological characters, such as wide variation of cell density, described above, following standards for type approval is suggested.

　

 95% of Dinophyceae and Maxillopoda more than 20μm in size should be removed, rendering harmless, inactivated through the process from inlet to outlet of the system.

　

The percentage looks small, but it should be thought as the starting point of system development. Higher percentage, i.e. higher efficacy, should be applied after certain period.

　

　

As the experiment to evaluate treatment systems will be conducted at various places throughout the world under various circumstances by both test-bed and on-board tests, the procedure of the experiment should be clearly defined with special consideration to the reproductivity and reliability of the result. Use of whole planktonic organisms occurring in the areas of the experiment as test organisms for the evaluation increases difficulty of experiments themselves and evaluation of results of the experiments. Because analysis of plankton composition before and after the experiment by counting only live individuals is thought to be essential and inevitable, but it is practically impossible to conduct it with scientific accuracy. Diatoms, one of the major components of phytoplankton, are immobile and the change of diatom cell color may not occur in a short time, even in case the cells died completely.

　

The conclusion of the present study is:

1. The testing organisms for evaluation of ballast water treatment system are Dinophyceae from phytoplankton and Maxillopoda（Copepoda）from zooplankton. These individuals with 20μm or more in size can be used for experiments.

2. Evaluation of efficacy should be based on termination rate of the test organisms before and after treatment. Live or dead can be distinguished by shape and mobility of the test organisms.

3. In order to keep reproductivity and accuracy of the evaluation, number of test organisms in test water should be counted no less than three times.

4. Standard for treatment approval is termination rate of test organisms more than 95 %. The rate should be set higher along with the development of techniques.

　

Concerning the cost of experiments, it is difficult to calculate it, because it varies depending on scale of experiments. Quantitative analysis (triplicate observation) of phytoplankton and zooplankton with judgment of live or dead costs 200 US$ per sample.