Algicidal composition
This invention relates to an algicidal composition. More particularly, it relates to an algicidal composition comprising higher fatty acid or salts thereof. 
     A composition containing germanium compound is suggested as an aquatic algae-controlling agent [Japanese publication (Kokai) No. 60-255706]. 
     However, such composition is subjected to restriction in an amount to be applied and an embodiment of use from a view-point of contamination to aquatic environment. This is why exploitation of a new algicidal composition causes been desired which has little contamination to the environment and has much algicidal activity. 
     The present inventors studied biologically active substances in marine organisms and found strong algicidal activity of 6Z, 9Z, 12Z, 15Z-octadecatetraenoic acid against marine dinoflagellates (Dinophyceae), which is an unsaturated higher fatty acid isolated from an edible brown alga &#34;Mozuku&#34; (Cladosiphon okumuranus). They have further found at last growth inhibition activity as well as algicidal activity of the higher fatty acid and salts thereof against algae which do great harm to fish- and shellfish-aquaculture and against those including sea weeds which adhere to and damage artificial products in sea 
     The higher fatty acid which is an active ingredient for the present composition is obtained, for example, by hydrolysis of fish oil and has 12-30,  preferably 16-24 carbon atoms, from an economical point. The presence of C-C unsaturated bond is not necessary but preferable. More preferable is the presence of two or more unsaturated bonds conjugated or not conjugated in the molecule. Salts of the acid include alkali metal salts, alkaline earth metal salts, ammonium salts, organic quaternary ammonium salts and phosphonium salts 
     Algae to which the present composition is applied are genuses: Haptophyceae, Cryptophyceae, Raphydophyceae, Bacillariophyceae, Chlorophyceae, Prasinophyceae, Euglenophyceae, Dinophyceae, Cyanophyceae, etc. 
     More specifically, mention may be made of the following species: Cric-osphaera roscoffensis, Cryptomonas sp., Chattonella antiqua, Chattonella marina, Olisthodiscus luteus, Heterosigma akashiwo, Chaetoceros debile, Skeletonema costatum, Stephanopyris palmeriana, Chlamydomonas sp., Oltmannsiella sp., Hafniomonas reticulata, Nephroselmis sp., Pteosperma cristatum, Pyramimonas sp., tetraselmis cordiformis, Eutreptia sp., Gymnodinium nagasakiense, Gymnodinium sanguineum, Gymnodinium breve (Ptychodiscus brevis), Gonyaulax monilata, Gonyaulax excavata, Gonyaulax tamarensis, Gyrodinium aureolum, Prorocentrum mariae lelouriae, Noctiluca scintillans, Noctiluca miliaris, Pyrodinium bahamense var compressa, Gymnodinium catenatum, Cochlodinium cartenutum, Protogouyaulax catenella, Protogouyaulux tamavensis, Protogouyaulax acatenella , Cochlodinium catenatum, Microcystis aeruginosa, Aphanizomenon flos-aquae, Oscillatoria agordhii, Cyanodictyon imperfectum, Gonyaulax catenella, Gonyaulax polyedra, Gonyaulax polygramma, Pyrodinium bahamense, etc. 
     The present controlling agent for algae has activity against various algae as mentioned above and inhibits algae from growth on glass surface of a water tank revise, algae from growth in a water-circulating apparatus, or marine animals and/o plants from adherence to a bottom of a ship when the agent is incorporated in paint. The present controlling agent is also superior in preventing red tide from blooming or outbreak which give heavy damage to a fish preserve of, for example, yellow tail, since the agent is remarkably active against dinoflagellates (Dinophyceae) which are planktons which cause red tide. The red tide is recently drawing public attention. Furthermore, the present controlling agent is able to serve as a treating agent for cultivation of layer such as a culturing net or a rope, since reversible retardation effect on cysts of laver readily disappears by dipping them again in sea water free from the present agent. 
     Higher fatty acid, the active ingredient of the present controlling agent, is used in the form of free acid or salts thereof such as alkali metal salts, alkaline earth metal salts, ammonium salts, organic quaternary ammonium salts or phosphonium salts. Alternatively, the acid or salts thereof may be used in the form of emulsifiable concentrate, powder, wettable powder, granule, soluble powder or solution in organic solvent such as ethanol. In the formulations above, various adjuvants such as a surfactant, a dispersant, a stabilizer etc. may be added. 
     An amount of the active ingredient in the composition is usually 1-99% by weight. 
     The effective concentration of the present controlling agent varies depending on the higher unsaturated fatty acid employed and varieties and growing density of algae to be applied to, but it is usually 0.01-1000 ppm, preferably 0.1-500 ppm, more preferably 0.1-100 ppm. 
     The emulsifiable concentrate, wettable powder or the like as above is distributed, after dilution with water, around or inside of a fish preserve when the present controlling agent is used for prevention of red tide. Alternatively, the active ingredient is coated on or impregnated in a net or a fence for a fish preserve. The net or fence may be made from a material in which the active ingredient is previously blended. 
     Experiment 1 
     Effect of the several unsaturated higher fatty acids on viability of Heterosigma akashiwo. 
     Test Organism 
     Heterosigma akashiwo was incubated in PES medium under 14 hr photoperiod (3000 lux) condition at 20° C. The effects of test compounds were examined on the plankton at exponential growth phase and the medium at the phase generally contained 7×10 4  cells/ml. 
     Preparation of Test Solution 
     The unsaturated higher fatty acid was accurately weighed and dissolved in benzene. An aliquot of the solution was transferred in a 10 ml volumetric flask. The benzene was removed in vacuo and the residue was dissolved in 0.2 ml of ethanol. A PES medium was added bit by bit to make a 10 ml solution. 
     Examination of Effects 
     Each of 1 ml of the plankton medium was transferred to the wells of a multi-dish at the cell number of 7×10 4 . After good conditions of the plankton were confirmed under an inverted microscope, 1 ml of graded levels of the test solution was mixed. At specified intervals, morphological examination was conducted under the microscope, and effect concentration (EC 100  ) was defined as the minimum concentration at which all of the plankton tested were cytolyzed 30 min after introduction of a test material. 
     Results 
     EC 100  values to the plankton are summarized in Table 1. 
     
                       TABLE 1                                                     
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Effects of the several unsaturated                                        
higher fatty acid on viability of                                         
Heterosigma akashiwo                                                      
Compound          EC.sub.100 (ppm)                                        
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6Z, 9Z, 12Z, 15Z- 2.5                                                     
octadecatetraenoic acid                                                   
arachidonic acid  2.5                                                     
γ-linolenic acid                                                    
                  25                                                      
linoleic acid     25                                                      
oleic acid        70-80                                                   
5, 8, 11, 14, 17- 2.5                                                     
eicosapentaenoic acid                                                     
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     Experiment 2 
     Effect of 6Z, 9Z, 12Z, 15Z-octadecateraeonic acid (OTA) on viability of several plankton species. 
     The test system was the same as in Experiment 1, except test concentration of 5 and 25 ppm and test duration of 48 hr. 
     The summarized results are shown in Table 2. 
     
                       TABLE 2                                                     
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Effects of OTA on viability of several plankton species.                  
                   Effect*                                                
Planktons            5 ppm   25 ppm                                       
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Dinophyceae                                                               
Gymnodinium nagasakiense                                                  
                     +       +                                            
Gymnodinium sanguineum                                                    
                     +       +                                            
Heterocapsa triguetra                                                     
                     +       +                                            
Prorocentrum micans  +       +                                            
Haptophyceae                                                              
Cricosphaera roscoffensis                                                 
                     ±    ±                                         
Cryptophyceae                                                             
Cryptomonas sp.      +       +                                            
Raphydophyceae                                                            
Chattonella antiqua  +       +                                            
Chattonella marina   +       +                                            
Olisthodiscus luteus +       +                                            
Heterosigma akashiwo +       +                                            
Euglenophyceae                                                            
Eutreptia sp.        +       +                                            
Prasinophyceae                                                            
Hafniomonas reticulata                                                    
                     +       +                                            
Nephroselmis sp.     +       +                                            
Pterosperma cristatum                                                     
                     +       +                                            
Pyraminonas sp.      +       +                                            
Tetraselmis cordiformis                                                   
                     ±    ±                                         
Chlorophyceae                                                             
Chlamydomonas sp.    +       +                                            
Oltmannsiella sp.    +       +                                            
Bacillariophyceae                                                         
Chaetoceros debile   +       +                                            
Skeletonema costatum +       +                                            
Stephanopyris palmeriana                                                  
                     +       +                                            
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 *+: 100% cytolysis of planktons after 2 hr                               
 ±: No cytolysis but no movement                                       
 -: No effect                                                             
 
    
    
    
     Reference Example 1 
     Acute toxicity of the several unsaturated higher fatty acids. 
     Test Organism 
     Killifish (Oryzias latipes) at adult stage were acclimated to laboratory conditions in dechlorinated water. Fish were withheld from food for 24 hr prior to the test. 
     Preparation of Test Solution 
     Graded levels of the compounds in 0.5 ml ethanol were added to glass vessels, and then mixed with 100 ml of dechlorinated fresh water. Five fish were introduced to the vessel and maintained for 48 hr at 25° C. 
     As a solvent control, 0.5% ethanol solution was used. Mortality and behavior were observed for 48 hr. 
     Results 
     The results are shown in Table 3. 
     
                       TABLE 3                                                     
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Acute toxicity of the unsaturated higher fatty acids to killfish          
                 Maximum no                                               
                 effect                                                   
                 concentration                                            
                            LC.sub.100                                    
Compound         (ppm)      (ppm)                                         
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6Z, 9Z, 12Z, 15Z-                                                         
                 &lt;20 ppm    &gt;25 ppm                                       
octadecatetraenoic acid                                                   
5, 8, 11, 14, 17-                                                         
                 &lt;25 ppm    &gt;30 ppm                                       
eicosapentaenoic acid                                                     
Na salt of       &lt;30 ppm    &gt;50 ppm                                       
5, 8, 11, 14, 17-                                                         
eicosapentaenoic acid                                                     
γ-linolenic acid                                                    
                 &lt;40 ppm    &gt;50 ppm                                       
sodium linolenate                                                         
                 &lt;100 ppm   &gt;150 ppm                                      
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     Reference Example 2 
     Isolation and identification of 6Z, 9Z, 12Z, 15Z-octadecatetraenoic acid from Mozuku (Cladosiphon okamuranus) 
     10 kg of salted Mozuku (Cladosiphon okamuranus) was extracted with methanol at room temperature for 2 weeks. A solid material was removed by filtration and the filtrate was concentrated in vacuo at a temperature below 30° C. 2% of ethanol was added to the concentrate and the precipitated solid material was removed by filtration. This process was repeated twice more to remove inorganic salts and sugars. The filtrate was concentrated in vacuo. To the concentrated syrup, was added 1l of water and the mixture was extracted three times with 1l of ether. The ether layers were combined and dried over anhydrous sodium sulfate. The ether layer was concentrated in vacuo to give 20 g of crude extract. The crude extract was chromatographed over a silica-gel column eluted with CH 2  Cl 2  /MeOH gradient solvent system. Each fractions were monitored by the bioassay against Heterosigma akashiwo described in Example 1. The algicidal activity was concentrated in the fraction eluted with CH 2  Cl 2  MeOH (95/5), and the fraction was evaporated in vacuo to give 5 g of an oily material. 
     Similar purifications with silica-gel column chromatography were repeated and the active component was purified finally by preparative medium pressure reversed phase column chromatography (Lobar RP-8, E. Merck, eluted with dioxane-H 2  O solvent system) twice to give 150 mg of 6Z, 9Z, 12Z, 15Z-octadecatetraenoic acid as an oily material. Since 6Z, 9Z, 12Z, 15Z-octadecatetraenoic acid was reported in Biochemistry Journal Vol 68, 695 (1958) by M. M. Matic et al, identification was made by comparison of IR, &#39;HNMR,  13  CNMR and Mass spectrum of the corresponding methyl ester of the present compound against the reported data.