JPS5920204A - Controlling agent against water mold for marine raising - Google Patents

Abstract

PURPOSE:The titled controlling agent, containing a benzothiazolylazo type dyestuff as an active constituent, usable safely for fishes, and capable of treating water molds infecting fishes in a transcutan bath, etc. in a low concentration. CONSTITUTION:A controlling agent containing a benzothiazolylazo type dyestuff of formula I (R1 is H, lower alkyl or lower alkoxyl; R2 is a residue having an aromatic ring or heterocyclic aromatic ring linked directly or, through a hydrocarbon group to N) or a quaternary salt, e.g. a compound of formula II or III, etc., negative to the rec. assay (recombination assay) with Bacillus subtilis and mutagenicity tests with Salmonella typhimurium, having low toxicity to fishes in a transcutan bath or oral administration, and capable of treating water molds infecting fishes in the transcutan bath as an active constituent. The above-mentioned compound may be directly used or dissolved in a nontoxic solvent, and dissolved in water for use in oral administration. The oral dose of the active constituent is 1-100mg/kg body weight.

Description

DETAILED DESCRIPTION OF THE INVENTION In recent years, farming and farming of various fish, crustaceans, shellfish, etc., has been widely practiced worldwide for various purposes. The targets are a wide variety of seafood and crustaceans, including trout, salmon, sweetfish, eel, yellowtail, carp, flounder, sea bream, prawns, goldfish, and scallops. In some cases, the purpose is to collect fish eggs such as salmon and trout, evaluate them, and release them into rivers as young fish to wait for migration, and to raise ornamental fish such as carp and goldfish.

These farmed fish vary depending on the species, breeding method such as freshwater breeding or brine breeding, optimal temperature for growth, etc.
An extremely wide variety of diseases occur, causing great damage to fish and shellfish farming. There are various causes of diseases, such as those caused by viruses, bacteria, and protozoa, but various diseases caused by fungi have also been recognized, and they cause great damage, especially in the cultivation of freshwater fish.

The most prominent of these water mold diseases are those caused by molds belonging to the oomycete, such as Pythium, Zaprolegnia, Aculia, Aphanomyces, Leptregnia, Deictiux, and Isoacria. All of them, except for the genus Pythium, belong to the water molds (Sabrolegniaceae), and among these, the genus Sabrolegnia in particular accounts for the majority of diseases caused by water molds.

The main diseases caused by these water molds and 1-
For example, when salmon and trout are harvested, they first occur and develop, causing great damage by infecting healthy eggs, and cases in which salmon, trout, argo, and young or adult salmon are infected Water mold diseases that cause death, particularly in rainbow trout, are known to infect the broodstock after egg collection and semen collection, as well as eel, carp, and sweetfish. For example, in the case of eels, from around 1956 there was an explosive outbreak of bollworm disease in Shizuoka Prefecture.
Mass mortality occurred and has continued to occur every year since then. In addition, water mold disease has been observed in the lesser trout of Lake Shibo since 1974.

To prevent these diseases, malachite green, methylene blue, or formalin have all been used to prevent water mold infection through medicated baths, and among these, malachite green is most frequently used. For example, malachite green can be administered to aquaculture ponds at a concentration of around 0.2 ppm to treat eel snail disease, or it can be used in a medicated bath for about 1 to 2 hours in places where water can be exchanged. In addition, for disinfecting salmon and trout roe,
A method of repeating treatment for 1 hour at 5 ppm twice a week has been used.

However, the use of these drugs has some major drawbacks: That is, all three types of drugs are highly toxic, and their safety, especially for the human body, has hardly been confirmed. It has been pointed out that malachite green may be carcinogenic, and in a mutagenicity test using microorganisms, the Lekku assay using Bacillus subtilis was positive. The safety of methylene blue is also extremely questionable; for example, not only has the leukyu test caused by Bacillus subtilis been positive, but when used on eels, etc., it remains in the fish body for an extremely long period of time, and the amount remains in the fish body even after the number of doses is administered. It is permitted to remain in the The same is true for formalin; it has not only been tested positive for mutagenicity by bacteria, but has also been shown to be carcinogenic.

Furthermore, although malachite green, which is most commonly used, does exhibit a growth inhibiting or bactericidal effect on water mold, its major drawbacks in practical use are as shown in Table 1.

Table 1 Fish Species TLM value (48 hours) (ppm) Goldfish 0.18 Eel
0.16 Himedaka 0.25 Rainbow Trout 0.69 Brown Trout 0.64 Brook Trout 0.26' Rainbow Trout Juvenile 0.10 Malachite Green is approximately 0.1 depending on the species of fish and the type of disease.
It is used at a concentration of ~5 ppm, but even the slightest mistake in use can result in the death of fish due to the toxicity of the drug. In fact, many cases of death due to incorrect use have been reported.

In addition, malachite green and methylene blue do not actually have the effect of treating serious conditions caused by water mold infections on fish bodies. It is only possible to prevent infection by acting as a preventive measure.

In order to solve these various problems, the present inventors
After searching for a drug that has low toxicity to both the human body and fish, but has a strong bactericidal effect, and which can treat fish as completely as possible even if they are infected with hydrochloric acid, which would lead to death if left untreated, we found this drug. invention has been achieved.

The present invention provides a water mold inhibitor for aquaculture containing a benzothiazolylazo dye or its salt as an active ingredient.

Preferable compounds as the water mold control agent of the present invention have the general formula (wherein R1 is a hydrogen atom, a lower alkyl group, or a lower alkoxy group, and R2 is an aromatic ring bonded to a nitrogen atom directly or via a hydrocarbon group, or It is a benzothiazolylazo compound represented by (representing a residue having a heteroaromatic ring) or a quaternary salt thereof.

As the lower alkyl group or lower alkoxy group for R2,
Examples include straight-chain or branched carbon atoms having 1 to 4 carbon atoms. R1 is preferably a hydrogen atom or a methoxy group. Examples of residue R2 include the following.

cH3 cH.

Examples of the salts of benzothiazolylazo compounds include hydrochloride, sulfate, nitrate, lactate, acetate, monomethyl sulfate, and monoethyl sulfate. Preferred salts are quaternary salts, such as lower alkyl- or carbamoyl lower alkylbenzothiazolium salts. These compounds are commercially available as basic dyes and are known under trade names such as Basacryl Yellow and Basacryl Blue.

All of the compounds used in the present invention were negative in the retox assay using Bacillus subtilis and the mutagenicity test using Salmonella typhimurium, and the acute toxicity value (Ll)5o) using mice was also negative. 20
00 m9 or more/body weight 1 kg, which is extremely low compared to its predecessor. The mutagenicity is further confirmed by rat liver homosyne (S9).
It does not appear even if m1x is added, and the possibility that mutagenicity appears due to metabolism is extremely low. In addition, the compound used in the present invention has significantly lower toxicity to fish than malachite green and the like both in a medicinal bath and in oral administration. Furthermore, since it is effective even at extremely low concentrations in a medicated bath, there is a large difference between the toxic level and the effective level, making it extremely safe to use for fish. A further feature of the compounds used in the present invention is that they can be used to treat water mold diseases that actually occur in fish by medicinal bathing or the like. This is an important feature that is not available at all with malachite green or methylene blue.

The above-mentioned compounds are effective in preventing and treating infectious diseases caused by water molds that occur in the cultivation of marine and freshwater fish and shellfish and crustaceans. Examples include water mold infection of eggs of salmon and trout, water mold infection of salmon, trout, amago, and yamame trout, water mold infection of rainbow trout eggs, post-semen collection, eel, carp, etc.
These include cotton capri disease of sweetfish. In addition, it is also effective against, for example, Fusarium disease in shrimp, fungal granulomatosis in trout, visceral mycosis in juvenile trout, ichthyophunosis, thermocystidiosis, franciomycosis, and the like.

The water mold control agent of the present invention may be used as it is or by dissolving the active ingredient in a non-toxic solvent in water, or may be used after being diluted with a non-toxic medium such as sugar or salt. . The water mold control agent is used in ponds and other water for aquaculture, or in water for bathing fish. Furthermore, in some cases, it is also effective to administer the water mold control agent of the present invention orally. The active ingredient has been found to be relatively highly absorbable and is therefore either absorbed transmembranely with feed or in aqueous solutions, suspensions or other dosage forms such as powders, granules, coated tablets, etc. It can also be forcibly administered orally as a pill. The dosage varies depending on the type of disease, species of fish, type of compound, dosage form, etc., but in the case of oral administration, it is generally a membrane force of about 1 to 1 oom9 per gram of body weight, and in the case of medicated baths, it is recommended for prevention. Approximately 0.02~1 pl,
m for treatment at concentrations of about 0.1 to 10 ppn+, which are effective for most diseases.

Inhibitory concentration of the Example drug against Sabrolegnia water mold and bath toxicity for trout.Sabrolegnia water mold, which was isolated from diseased fish affected by water mold disease and confirmed under a microscope, was inoculated on sterilized hemp seeds. 1
After culturing at 5° C. for 6 days and allowing sufficient growth, the cells were suspended in sterile water and used as a seed culture.

On the other hand, five sterile hemp seeds were cultured in a petri dish in a thermostat at 15°C, and the inhibitory concentration was determined based on whether mycelia were observed on the hemp seeds after 4 days.

In order to investigate the toxicity to fish, five juvenile rainbow trout weighing approximately 60 g were placed in each test plot, and their survival and death within 48 hours were observed and expressed as a 50% lethal concentration. The results are shown in Table 2. 1 to 5 in the table are the present invention, 6 to 1
0 is a comparative compound.

As shown in Table 2, it is clear that the water mold control agent of the present invention has stronger antibacterial activity and lower toxicity than the comparative agents. In contrast, malachite green, which has been used in the past, effectively inhibits growth at low concentrations, but it is also recognized to be highly toxic.

Example Chemical bath toxicity (48 hour TLM value) for various fish species.

The breeding water temperature is 20°C for trout and 25°C for others.

The results are shown in Table 6.

Table 3 (ppm) TLM value (ppm) Nishiki goi
28 Goldfish 28 0.18 Eel 30 0.16 Medaka 26 0.25 Angel fish 11 Zebra danio Z2 Platy 20 Rainbow trout (, lS5g) 28
0.14 rainbow trout (2g) 24
o, +2 Sakura Trout (3.5 g) 20 From the results in Table 6, it is recognized that Compound 1 has significantly lower toxicity than Malachite Green.

EXAMPLE Therapeutic effect in water mold infection experiment According to preliminary experiments conducted by the present inventors, when rainbow trout were fed with testosterone at a ratio of 500 m97 to 9 body weight for 10 days, the rainbow trout were able to absorb the hormone. It has been confirmed that if the balance is disrupted and water mold zoospores are inoculated into these animals, they will easily become infected and die. Therefore, an infection experiment was conducted using the rainbow trout preliminarily reared in this way using the method described below. Specifically, 2 mg of testosterone/tail was further intramuscularly injected into pre-reared rainbow trout, a burn of approximately 57nrIL was made on the skin of the caudal peduncle using a soldering iron, and water mold zoospores, which had been previously cultured on hemp seeds and suspended in sterile water, were injected intramuscularly into the tail. Coated. According to this method, 100% of the animals become sick and die within 4 to 5 days of rearing.

Appropriate concentrations of the drugs used were selected from the growth inhibitory concentrations obtained in in vitro tests, and rainbow trout were continuously bathed in the drugs (water temperature 20°C). The results are shown in Table 4. The numbers in the table are the number of dead rainbow trout (10 fish per group, average weight 40I), and the total is the total number of dead rainbow trout over 10 days.

Table 4 As is clear from Table 4, malachite green and methylene blue have almost no infection prevention effect, whereas the water mold control agent of the present invention (compound number is No.
(same as in the table) clearly has an infection-preventing effect.

Furthermore, water mold pathogens (
We obtained 45 strains of Zaprolegnia (genus Zaprolegnia) and calculated the minimum inhibitory concentration of the most effective compound 1 from 0.12 to
o, s ppm, of which 62 strains were 0.25
It was ppm.

Example Oral Toxicity The water mold control agent of the present invention (compounds are the same as in Table 2) was orally administered by force to 10 rainbow trout (average weight: 35 g) in group 1, and the trout were observed for 48 hours, and the premises where they died were examined. The results are shown in Table 5, and it is clear that the toxicity is extremely low.

Table 5 Example 1 Eggs collected and artificially fertilized at a salmon breeding farm are stored in the breeding farm, and the compound 1 of Table 2 as a water mold control agent of the present invention is added to the running water in the fermentation tank. The fertilized salmon eggs were disinfected by dripping a highly concentrated aqueous solution over an hour to maintain the concentration in the curing tank at 0.5 ppm. This operation was repeated a total of 5 times at intervals of 4 eyes, and the survival rate up to the eye development stage was observed. As a result, due to parasitism of water mold fungi accompanying the death of unfertilized eggs and infection of healthy eggs, 47% of the eggs developed normally in the non-chemical treatment area, and water mold encrustation was found in most of the cases of death. It was done. On the other hand, in the medicated bathing area using this drug, the eye development rate was 87%, and no growth of the deadly athlete's foot fungus was observed, demonstrating an excellent preventive effect.

Example 2 A water mold infection occurs when a burn is made on the caudal peduncle of a rainbow trout with an average weight of 45.9 kg and a water mold zoospore suspension is applied.

The effectiveness was investigated by artificial infection using this method. The area where the medicinal bath was started 2 hours after the artificial infection was designated as the preventive medication area, and the time point was determined when it was confirmed that water mold had grown on the side of the body. The drug used was Compound 1 in Table 2.

The results are shown in Table 6, and in the case of the present invention, the dose is 0.05-0.11 ppm for preventive administration.

Therapeutic dosing was effective at concentrations of 0.11-0.35 ppm. On the other hand, in the case of the comparative drug malachite green, only some effects were seen at a concentration of 0.53 ppm in the preventive administration group, toxicity appeared at higher concentrations, and no effect at lower concentrations. .

Table 6 Note) The numbers in the table indicate the survival rate (%) after 10 days.

Example 6 At a cherry salmon farm in a lake, water mold disease frequently occurred, and many solids with water mold were observed, and the individuals that had spread all over their bodies died one after another. Therefore, at this farm, 400 cherry salmon with an average weight of 54.9
The water level of the breeding tank containing 0 fish was lowered, and Compound 1 shown in Table 2 was dissolved in water at a ratio of 200 g per 100 tons of water, followed by a medicinal bath for 1 hour. During this period, sufficient aeration was carried out, and after 1 hour, the chemical solution was added under running water.
The water mold disease ended within a day. During this time, increase the water temperature to 95-11
It was kept at ℃.

Table 7 *Medical Bath Formulation Example 1 Take 1 part of Compound 1 in Table 2, dissolve it in purified water, and add an appropriate amount of purified water to make a total of 100 parts of an aqueous solution.

Formulation Example 2 1 part of Compound 6 (monomethyl sulfate) in Table 2 is diluted with lactose, and an appropriate amount of lactose is added to form a powder with a total volume of 50 parts.

Formulation Example 6 1 part of Compound 4 (chloride) in Table 2 was diluted with salt,
Add an appropriate amount of salt to make a powder with a total volume of 10 parts.

Applicant: Ueno Pharmaceutical Application Research Institute Co., Ltd. Agent: Patent Attorney Masao Kobayashi

Claims (1)

[Claims] 1. A water mold control agent for aquaculture containing a benzothiazolylazo dye or its salts as an active ingredient. 2. The benzothiazolylazo dye has the general formula (in the formula 1 (
1 means a hydrogen atom, a lower alkyl group or a lower alkoxyl group, R2 means a residue having an aromatic ring or a heteroaromatic ring bonded to a nitrogen atom directly or via a hydrocarbon group, or a compound thereof; The water mold control agent according to claim 1, which is a quaternary salt.