JP4228074B2 - Culture system containing Daphnia and Earthworm - Google Patents

Description

  The present invention relates to a culture system containing daphnia and earthworm. More specifically, it relates to environmental protection technology research, culture systems and experimental systems useful for long-term exposure and testing of toxicity of harmful factors and harmful substances to organisms. The present invention relates to a novel culture system that can be applied. The present invention further relates to a method for maintaining the culture system and a method for evaluating the influence of a test substance on the environment using the culture system.

When industrial waste, which is expected to have harmful effects from substances contained in industrial wastewater, is released into the environment, it is necessary to test its toxicity to living organisms in order to predict its impact on the environment. In these tests, tests using living organisms that actually live in water are conducted. The ecological impact test method in the OECD is a method developed for such a purpose, and its introduction is examined in Japan as a method for evaluating the toxicity of various harmful substances (see Non-Patent Documents 1 and 2).
Among them, the daphnia acute swimming inhibition test is a simple test method using the fact that Daphnia is highly sensitive to poisons, and its test kit is commercially available. When a toxicity test is conducted by this method, toxicity is evaluated from the observation results after 24 hours and 48 hours after the addition of the poison. There is another known daphnids reproduction test method, which is a method for observing the life and death and status of parent daphnia and the number and status of daphnia, and the test period is up to 21 days.
However, in the method described above, effects on fertility are observed only when poisonous animals that kill daphnia in a few days or when effects appear in a few weeks. On the other hand, to observe the effects of low-concentration harmful substances or low-dose radiation, which are unlikely to be affected by short-term exposure, and the generational succession of those effects, continue for several months or longer. It is necessary to raise them.

OECD GUIDELINES FOR THE TESTING OF CHEMICALS (http://www1.oecd.org/ehs/test/Biotec.htm) OECD (1999): Environmental Health & Safety News, No. 9

Accordingly, an object of the present invention is to provide a culture system capable of breeding daphnia continuously for a period of several months or longer.
Another object of the present invention is to provide a culture system and an experimental system that are useful for studying environmental protection technologies and for testing long-term exposure to toxicity of harmful factors and harmful substances to organisms.
Still another object of the present invention is to provide a novel culture system applicable to environmental science, agriculture, and ecology.
Still another object of the present invention is to provide a method for maintaining the above culture system.
Still another object of the present invention is to provide a method for evaluating the influence of chemical and / or physical stimuli on the environment using the above culture system.

The present invention provides the following culture system, a method for maintaining this culture system, and a method for evaluating the influence of a test substance on the environment using this culture system .
1. Culture system containing daphnia, earthworm, zooplankton and / or phytoplankton, decaying organic matter and water.
2. A method for maintaining a culture system, comprising maintaining the culture system according to 1 above at 15 to 30 ° C. under illumination.
3. Chemical culture and / or physical stimulation is given to the culture system described in 1 above, and it is kept at 15 to 30 ° C. under illumination. After a predetermined period of time, the state of Daphnia and / or earthworm is observed, A method to evaluate the environmental impact of physical or physical stimuli.

Put the deposits and decaying organic matter at the bottom of the aquarium formed by the accumulation of aquatic plants, fish droppings, plankton carcasses, etc. When cultivated under illumination (artificial lighting such as a fluorescent lamp or natural light), the earthworm will enter the sediment and the daphnia will live in the water above the sediment. In an equilibrium state, the number of individuals will not increase excessively and will not coexist and will continue to coexist. If you make up for the evaporated water, you can observe both in the same state for over a year.
When this is exposed to various harmful factors and harmful substances, changes such as the death or decrease of the organisms in the culture system can be observed. At the same time, it is possible to observe the movement, chemical change and behavior of substances in the culture system for a long period of time.
As described above, the culture system of the present invention is a system for simultaneously breeding a plurality of organisms, Daphnia and Otoworm, so that a population of organisms can be maintained for a long time. Therefore, this culture system is capable of observing biological communities composed of multiple species over a long period of time, and clarifying the effects of harmful factors that could not be known in conventional culture experiments and toxicological exposure experiments. Can be expected.

Daphnia to be used in the culture system of the present invention includes Daphnia, Harina Daphnia, Uskawa Harina Daphnia, Kabuto Daphnia, Camry Harina Daphnia, Kawari Hara Daphnia, Daphnia magna, Red-tailed Daphnia, Red-tailed Daphnia, Togeoka Daphnia, Examples include Togario daphnia, Amida catfish, Kireo catfish, Himeneko dameko, Nekozemujinko and the like.
Daphnia usually lays eggs or pups about once a week and has a lifespan of about 3 months.

Examples of the earthworms used in the culture system of the present invention include earthworms, lily earthworms, earthworm earthworms, era earthworms, earthworm earthworms, and white earthworms.
The earthworm lays eggs in a timely manner and grows gradually depending on the culture conditions. The lifetime is usually about 6 months to 1 year.

  As the decaying organic substances used in the culture system of the present invention, fish, for example, sediment deposited on the bottom of ornamental fish breeding tanks such as guppy and goldfish, activated sludge, sewage sludge, water aquatic plants such as water hyacinth and chromo Examples include decayed ones. If necessary, chicken dung and the like may be added to replenish nitrogen and phosphorus.

In order to configure the culture system, if the scale of the culture system is about 5 liters relative to water (when using tap water, leave it for about a day to remove chlorine) Add about 0.2-1.0% by weight in dry mass (the water content of the decayed organic substance and the aquarium sediment is about 98%), add 20-30 worms and 20-30 daphnids to this, 15-30 What is necessary is just to maintain under illumination of degreeC, Preferably 22-24 degreeC. The lighting may be artificial lighting or natural light. For example, a single fluorescent lamp for home use is sufficient. The illuminance is 2000 to 50000 lux, preferably about 3000 to 5000 lux. Depending on the environment, organisms can survive even under 2000 lux or over 50000 lux. The lighting may be continuous day or night, but it is desirable to turn off the lights at night and have a day / night cycle. However, it is generally better to avoid direct sunlight.
When cultured for about 1 to 6 months under these conditions, the number of daphnia initially increases and reaches equilibrium. Subsequently, the number of worms is almost constant, but it takes a longer time than Daphnia. Usually, if the amount is about 5 liters as described above, a culture system of 200 to 500 worms and 300 to 1000 daphnids is established in an equilibrium state. In this case, daphnids reach a certain number within a month, while worms grow slowly over a period of several months. However, the effect of lighting on Daphnia depends on the depth of the culture system and the transparency of the water, and the effect on the earthworm also depends on the thickness of the sediment at the bottom of the aquarium. Therefore, when culturing is started under a predetermined culture system, the environment can be adjusted by observing living organisms for a while and adjusting the illuminance if it does not reach an equilibrium state.

In order to maintain the culture system, the culture system is opened to the air, water that is reduced by natural evaporation is appropriately supplemented, and the above temperature is maintained under appropriate illumination. Even if the temperature is lower than the above temperature, there is no problem in maintaining the culture system for a short period.
In the culture system of the present invention, animal and plant plankton grows using an organic decay substance as a nutrient source. It is thought that the ecosystem where animals and phytoplankton grow is circulated because the body and carcass become part of the organic decay material.

The present invention also applies chemical and / or physical stimulation to the culture system, holds the light at 15 to 30 ° C. under illumination, and observes the state of daphnia and / or earthworms after a predetermined period of time, A method for evaluating the influence of the chemical or physical stimulus on the environment is provided.
Examples of chemical and / or physical stimuli include various harmful factors such as radiation, ultraviolet rays, electromagnetic waves, and harmful substances such as environmental disturbing substances, heavy metals, and agricultural chemicals.

Example 1
Establishment of culture system The present invention will be described more specifically with reference to the following examples.
Sediment deposited on the bottom was collected when fish such as guppy were raised in the aquarium. This is a stack of fish excrement and plankton carcasses that have propagated in the water. This sediment itself becomes the home of bacteria or worms that are decomposers in the culture system. 70 ml of medium (containing 70 g of water in a dry weight of 0.4 g of sediment) was placed in a cylindrical container having a diameter of 60 mm and a depth of 40 mm. Ten Daphnia and 10 earthworms were put into this and cultured at 22 ° C. under fluorescent light (4000 lux). After being exposed to air and cultured for about 1 month, Daphnia magna grew to about 20 animals and almost reached equilibrium. The number of individual worms did not change during this period. This culture system is maintained for a long period of time by gradually replacing generations of water fleas and earthworms by simply replenishing water that is reduced by natural evaporation. There is no need to stir the culture system. In previous experiments, when the culture solution was 5 liters, the culture system was maintained for at least 4 years.
In the above culture system, in the culture system of 100 ml or less, if only the Daphnia magna or the earthworm were added and cultured, in either case, the cells died within a few months. The scale of about 5 liters may survive longer than 100 ml, but never reached one year.

Example 2
Irradiation test When irradiated to the culture system (70 ml) of the present invention using Daphnia magna and damselfish, all individuals died at 1000 Gy within about 2 days. At 500 Gy and 300 Gy, some survived but no longer born and disappeared. However, 200Gy and 100Gy irradiation reduced the number of pups born, and a decrease in the number of Daphnia magna was observed but never died. These small populations continue to recover for a while, but the number of Daphnia magna in the culture system recovers in the same way as before irradiation in about 2 months at 100 Gy, and irradiation occurs after 3 months at 200 Gy. It recovered almost as before.
Of the above-mentioned irradiation doses, the death of earthworms was not observed with 200 Gy and 100 Gy irradiation.

Example 3
Examination of the culture conditions of the culture system The following experiment was also conducted in an environment where it was assumed that a small culture system was transported.
500 ml of the main culture system was placed in a 2 liter PET bottle, and the remaining space was sealed with oxygen gas. This was wrapped in black film to prevent light from entering, and placed in a cardboard box and left in the trunk of the car for 5 days. As a result, daphnia and earthworms did not die even in an environment where the outside air temperature varied between 11 ° C. and 20 ° C. under such vibration conditions. Later, I found that if I put it under bright conditions (14 hours a day, 3000-5000 lux), I could continue to live.
Daphnia and earthworms continued to live for about two months under the environmental conditions of 22 to 24 ° C. with light of 3000 to 5000 lux in the above sealed state.

When this culture system is observed under various conditions, if the water layer on the precipitate is about 1 cm, organisms can survive even under 2000 lux, but the number of daphnia tends to decrease gradually. On the other hand, when the water layer on the sediment is about 10 cm, the number of daphnia is maintained by the natural light of the greenhouse. If the inside of the greenhouse is not heated by direct sunlight, it can survive at 10000 lux or more. Increasing the amount of precipitates was more effective, and Daphnia and Earthworms continued to live for a period of about 8 months in an environment where the room temperature fluctuation range was 15 ° C to 30 ° C and the natural light of daytime was 10,000 to 50,000 lux.

  As described above, the present invention is a small-scale organism community that simulates an actual environment using daphnia and earthworms. Since these organisms change generations in the container, it is possible to continuously observe subsequent changes to various loads. In the conventional technology, the mortality rate of organisms due to load has been used as an index of impact. I can do it.

Claims (3)

A culture system comprising daphnia, worm, zooplankton and / or phytoplankton, decaying organic matter and water,
Add 0.2 to 1.0% by weight of dry organic matter per 5 liters of culture system, add 20 to 30 worms and 20 to 30 daphnids, and illuminate 2000 to 50000 lux at 15 to 30 ° C. Established by culturing for 1 to 6 months, under continuous day or night conditions, or under only daytime irradiation conditions,
In equilibrium, including 200-500 worms, 300-1000 daphnids,
A culture system that is open to the air, replenished with water that is reduced by natural evaporation, and maintained at the above temperature under the illumination, so that daphnids and earthworms are maintained while changing generations. A method for maintaining a culture system, comprising maintaining the culture system according to claim 1 at 15 to 30 ° C. and illuminating 2000 to 50000 lux continuously day or night or under the condition of irradiating only in the daytime . A chemical and / or physical stimulus is applied to the culture system according to claim 1, and illumination of 2000 to 50000 lux is maintained at 15 to 30 ° C. under conditions of continuous day and night or only in the daytime. A method for observing the habitat of Daphnia and / or earthworm after a period of time and evaluating the influence of the chemical or physical stimulus on the environment.