AU2004207481A1 - A method for neutralization of toxin agents, pesticides and their hydrolysates, and a reagent for it - Google Patents

Description

WO 2004/066726 PCT/US2004/001175 A METHOD FOR NEUTRALIZATION OF TOXIN AGENTS, PESTICIDES AND THEIR HYDROLYSATES, AND A REAGENT FOR IT FIELD OF THE INVENTION This invention relates to a technology for processing of ecotoxicants, such as, for example, chemical weapons (toxin agents), pesticides, hydrolysis products of toxin agents and pesticides. In January 1993, representatives from more than 130 nations signed the final draft of the Chemical Weapons Convention. The Convention provisions prohibit the production, use, sale, and stockpiling of all chemical weapons and their means of delivery and calls for the destruction of existing stocks by the year 2005. In accordance with international obligations, by April 25, 2003 Russia shall have destroyed 1 % of Russian chemical weapons stockpiles. This amounts to about 400 tons of toxin agents. BACKGROUND OF THE INVENTION Until the 1970s, chemical weapons were destroyed by the following methods, which are now prohibited: open-pit burning, evaporation and dissolution in atmosphere, sea dumping and land burial. In the 1970s detoxification of toxin agents was conducted by means of chemical reagents and incineration of detoxification products. However, due to difficulties in carrying out these processes, the management of the USA chemical weapons destruction program concentrated on direct incineration of toxin agents at specially equipped plants. At the same time, public concerns and technical failures at toxin agents incineration plants required a search for new technologies for toxin agents detoxification. A method for processing of substances polluting the environment is disclosed in US Patent No. 6,039,882. The method and composition of this patent 1 WO 2004/066726 PCT/US2004/001175 are developed for the remediation of environmental contaminants in soil, sediment, aquifer material, water, or containers in which contaminants were contained. Contaminants are reacted with a remediating composition comprising a metal and a sulfur-containing compound to produce environmentally acceptable, chemically reduced products. The method is useful for treating contaminants such as halogenated hydrocarbons, pesticides, chemical warfare agents and dyes. The remediating composition preferably contains comminuted, commercial grade iron and iron sulfide. Addition of alcohol to the reactants enhances the rate of the remediation reaction, particularly for contaminants of soils and sediments. A method for detoxification of toxin agents by hydrolysis is also disclosed in US Patent No. 5,678,243. The process of this patent comprises adding water to the chemical agent so that a hydrolysis reaction of the chemical agent with water occurs at specified molar ratios. In a preferred embodiment, the detoxification process is carried out in situ within the chemical agent (aminoalkyl phosphonothiolates) storage containers in the field and includes mixing the contents of the container after adding the water. A method to destroy chemical warfare agents by alkaline hydrolysis is disclosed in WO 97/18858. The method of this published application comprises a reaction of chemical warfare agents with a nitrogenous base such as anhydrous liquid ammonia, in which an active metal, for example, sodium, is dissolved. The drawback of the methods described above is their non-conformity to modem requirements of ecological safety for chemical weapons (toxin agents) and pesticides destruction. The US National Research Counsil recommended for further development and implementation an alternative technology for chemical warfare agents detoxification by means of alkaline hydrolysis followed by biodegradation of generated less toxic hydrolysis products. 2 WO 2004/066726 PCT/US2004/001175 Biodegradation of chemicals is a natural process which is not damaging to the environment. Scientists have tried to find microorganisms and enzymes for direct biodegradation of toxin agents. However, due to high toxicity of toxin agents microbe cells can destroy toxin agents, but they themselves are killed. This significantly raises the cost of toxin agents biodegradation and makes it problematic from ecological point of view. Selective enzymes, (an enzyme that is for each type of toxin agent are unpredictable and expensive for practical employment to directly destroy toxin agents. The closest analogue of the present invention is a method for processing of the toxin agent iprite by alkaline hydrolysis followed by biodegradation of generated less toxic substances as disclosed in US Patent No. 6,017,750. The method provides for processing of a mixture of 2,2'-dichlorodiethyl sulfide and bis-(2-(2-chloroethylthio)ethyl) (HT). The process includes - treating HT with a hydrolyzing agent to form a mixture of thiodiglycol (TDG) and bis-(2-(2-hydroxyethylthio)ethyl) ether(s) (T-OH); - neutralizing the TDG and T-OH mixture to a pH sufficient for biodegradation of the mixture; - biodegrading the neutralized TDG and T-OH mixture. The drawbacks of this process are the production in the first stage alkaline hydrolysis rather toxic products of neutralization. This significantly complicates and raises costs for all processes related to further storage, transportation etc, and also raises costs of further biodegradation since special units are required for biodegradation and permanent renewal of microorganisms which die due to high toxicity of alkaline hydrolysis products. 3 WO 2004/066726 PCT/US2004/001175 SUMMARY OF THE INVENTION The object of this invention is the creation of a highly effective method and reagent for the first stage of ecotoxicants (toxin agents, pesticides and their hydrolysates) processing during which the toxicity of initial products is reduced so much so that further biodegradation is possible. Also, the object of this invention is the expansion of the method's operational possibilities, that is expansion of the range of ecoctoxicants, which can be processed by means of the proposed method (all types of solid, liquid gaseous toxin agents, and also pesticides containing functional groups of halogen or pseudohalogen origin, sulfur group (Cl, Br, I, NO 2 , SO 4 , nitrophenyl etc), hydrolysis products of the above-mentioned ecotoxicants). The method can also be applied for sanitation of soils and objects polluted by toxin agents and pesticides. As the result of the first stage of processing, products are produced, which are significantly less toxic as compared to ones in known technologies, including the above-described closest analogue. Besides significantly lower toxicity the product obtained by the present process possesses one more new important property - it has nutrition value for microorganisms. In connection with it, there is the possibility to considerably extend the range of natural microorganisms ("active silt" type) capable to process the product of toxin agents neutralization, generated by using the proposed method. Due to their low toxicity, products produced at the first stage of processing can be transported as common low toxic wastes and exposed to neutralization at pH sufficient for purposes of final biodegradation at conventional waste water biochemical treatment plants. 4 WO 2004/066726 PCT/US2004/001175 A second embodiment of the claimed method is a two stage technological scheme for toxin agents destruction: chemical detoxification of toxin agents and further biodegradation (using microorganisms) of products received at the first stage. In the proposed embodiments of the method, the prior art problems are solved by using as the basic reagent for chemical neutralization of toxin agents and pesticides a reagent containing an amino acid or a mixture of amino acids, or peptide, or derivatives of amino acids and peptides, or their mixture in aqueous or aqueous-organic medium. Hydrolysis products of protein containing factory waste can be applied as a reagent as well. The novelty of the proposed reagent is that, for the first time, an amino acid reagent is proposed as a means for reducing toxicity of toxin agents, pesticides and their hydrolysate where the reagent comprises an amino acid or a mixture of two or more amino acids, or peptide, or a mixture of amino acids and peptides in aqueous organic medium. Products of hydrolysis of protein containing factory waste can also be applied as means for reducing toxin agents toxicity. It is known that amino acids can easily form salts. Besides, amino acids form stable chelated complexes with various ions of metals. At the same time, information on the property of amino acids and peptides to neutralize toxicity of ecotoxicants (toxin agents, pesticides and their hydrolysates) has not been found out. It was found in this instance that amino acid reagents form with the mentioned ecotoxicants low toxic compounds which can not be degraded to common products and wastes of microorganisms' vital activities during biodegradation. More detailed information on the present invention can be found herein below. 5 WO 2004/066726 PCT/US2004/001175 The instant method does not have the drawbacks of the prior art methods mentioned above and possesses a number of advantages in comparison with them. Technical results obtained when using the proposed method of toxin agents treatment are as follows: - Providing destruction of toxin agents, pesticides and their hydrolysates with further bio-utilization of their degradation products, that is the possibility to use the product, received as the result of treatment by microorganisms directly as soil improving additives for soils of general purposes including agricultural ones. - Significant extension of the ecotoxicants range - all types of toxin agents and pesticides, containing functional groups of haloid or pseudohaloid nature, sulfur groups (Cl, Br, I, NO 2 , So 4 2 -, nitrophenyl etc.), products of interaction of which with the amino acid reagent can be exposed to effective biodegradation. This indicates rather wide universality of the method - Possibility to utilize this method for sanitation of soils and objects contaminated with toxin agents and pesticides - Use of highly effective and available reagent for detoxification of toxin agents and pesticides - Significantly less toxicity of neutralization products compared to known technologies in which alkaline hydrolysis is applied; toxin agents neutralization products can be utilized by microorganisms during their vital activities, thus the range of microorganisms capable to treat neutralized by the proposed method toxin agents (pesticides) is expanded 6 WO 2004/066726 PCT/US2004/001175 - Possibility to transport neutralization products generated at the first stage as low toxic waste and possibility of their biodegradation at conventional waste water biochemical treatment plants. DETAILED DESCRIPTION OF THE INVENTION IMPLEMENTATION The instant method for neutralization of toxin agents, pesticides or their hydrolysates comprises a compound of toxin agent, pesticide or their hydrolysate with an amino acid reagent. The amino acid reagent comprises at least one of the following amino acids: alanine, arginine, aspartic, asparagine, valine, histidine, glycine, glutamic, glutamine, isoleucine, leucine, lysine, methionine, praline, serine, tyrosine, threonine, tryptophane, phenylalanine, cysteine, cystine or a mixture of these amino acids in various combinations, or peptides, or derivatives of amino acids and peptides or a mixture of amino acids and peptides in aqueous or aqueous organic medium. In the given below examples (laboratory experiments) the amino acid reagent contained one amino acid or a mixture of two or more amino acids provided pH maintained at the given range. Also, as an amino acid reagent for industrial purposes an alkaline hydrolysis product of protein containing factory waste containing mixture of amino acid salts and peptides can be used. For further biodegradation the low toxic product obtained as the result of interaction of amino acid reagent with toxin agent, pesticide or hydrolysate of toxin agent or pesticide is neutralized to pH of about 7 to about 8 sufficient to provide the possibility for its further biodegradation. Biodegradation is carried out in a conventional way with the use of microorganisms, such as "active silt" to provide a neutralized biomass. 7 WO 2004/066726 PCT/US2004/001175 Example 1. Detoxification and biodegradation of lewisite under the action of an amino acid reagent and microorganisms. An clewisite (2-chlorovinyldichloroarsine) sample was prepared via interacting arsenic trichloride with acetylene in the presence of aluminum chloride and it was isolated via double distillation. Characteristics - boiling point 75-790 mm Hg, d 20 =1 .875, u 2 0 D = 1.06092 - corresponded to the literature data. Detoxification of lewisite was carried out via the following method. In a 50 ml Ehrlenmeir flask equipped with a magnetic stirrer there was placed a solution of 124 mg of lewisite in 30 ml of 0.1 M of amino acid reagent (pH 7-12), it was stirred at room temperature for 5 hours and kept overnight. In a day pH of the solution was reduced to 7.0 and it was passed to evaluate acute toxicity in per os tests, conducted on rats. LD 5 0 , per os, of the detoxification products exceeded 5000 mg/kg. The same solution (the reactionary mass after detoxification of lewisite by 0.1 M solution of the amino acid reagent) was evaluated as a substrate for vital functions of bacteria Streptococcus thermophylis and an activated sludge. The use was made of the starting solution as well as its dilutions of the 10-'+ 10 concentration. In each test tube there were placed 10 ml of the solution with the 106 titre, and there followed incubation at 370 C for a day. In a day there was recorded the growth of bacteria in all the test tubes. The samples of the starting concentration of the reactive mass were passed to evaluate acute toxicity in per os tests conducted on rats. It was established that LD 5 O, per os, of the solutions containing Streptococcus thermophylis strains and activated sludge exceeded 5000 mg/kg. On investigating all levels of doses, no clinical symptoms of intoxication were recognized. Morphological studies of internal conducted in 14 days after introduction of detoxification products revealed complete absence of symptoms of 8 WO 2004/066726 PCT/US2004/001175 toxic action. No mutagenic and carcinogenic properties of the detoxification products were revealed. Example 2. Detoxification of lewisite under the action of lysine. In a 30 ml Ehrlenmeir flask equipped with a magnetic stirrer there were placed 62 mg of a-lewisite, which was identical to lewisite used in the example 1, and 15 ml of 0.1 M solution of sodium lysinate (pH 8-12). The mixture was stirred at room temperature for 5 hours, the solution was kept overnight. Next day pH of the solution was reduced to 7,0 and the solution was passed to evaluate acute toxicity in per os tests conducted on rats. LD 50 , per os, of the detoxification products exceeded 5000 mg/kg. Morphological studies of internal, conducted in 14 days after introduction of the detoxification products, revealed a complete absence of any symptoms of toxic action. The detoxification products revealed no mutagenic and carcinogenic properties (Eims test with metabolic activation - salmonella/microsomes, strains TA-98 and TA-100). Example 3. Detoxification of lewisite under the action of cysteine. In a 30 ml Ehrlenmeir flask equipped with a magnetic stirrer there were placed 62 mg of ca-lewisite, which was identical to that used in the example 1, and 15 ml of 0.1 M solution of the cysteine sodium salt (pH 8-12). The mixture was stirred at room temperature for 5 hours, the solution was kept overnight. In a day pH of the solution was reduced to 7.0 and it was passed to evaluate acute toxicity in per os tests conducted on rats. LD 50 , per os of the detoxification products exceeded 5000 mg/kg. Morphological studies of internal, conducted in 14 days after introduction of the detoxification products revealed a complete absence of any symptoms of toxic action. The detoxification products revealed no mutagenic and carcinogenic 9 WO 2004/066726 PCT/US2004/001175 properties (Eims test with metabolic activation - salmonella/microsomes, strains TA-98 and TA-100). Example 4. Detoxification of yperite under the action of the amino acid reagent. An yperite sample (2,2'-dichlorodiethyl sulfide) was prepared via the Meyer method from 2,2'-dioxyethylsulfide and concentrated hydrochloric acid and was isolated by double distillation. The characteristics - boiling point 95-97o/10 MM Hg, d 20 ou=1,275, u 20 D = 1,528 - corresponded to the literature data. Detoxification of yperite was conducted via the following method. Into a 50 ml Ehrlenmeyer flask equipped with a magnetic stirrer there were placed 80 mg of yprite in 25 ml of 0.1M of the amino acid reagent and they were stirred at room temperature for 5 hours. The solution was kept overnight, in a day pH was reduced to pH=7.0 and it was passed to evaluate acute toxicity in per os tests conducted on rats. LD 5 0 , per os, of the detoxification products exceeded 5000 mg/kg. Morphological studies of internal, conducted in 14 days after introduction of the detoxification products, revealed a complete absence of any symptoms of toxic action. The detoxification products revealed no mutagenic and carcinogenic properties (Eims test with metabolic activation - salmonella/microsomes, strains TA-98 and TA-100). Example 5. Detoxification and biodegradation of the hydrolysate of the compound V, under the action of the amino acid reagent and microorganisms. A sample of the hydrolysate of the compound V, was obtained in the result of the hydrolysis of O-isobutyl-S-2-diethylaminoethyl-methylthiophosphonate with water in the ratio 7:100 (volume) conducted at room temperature over 3 months. Evaluation of acute toxicity in per os tests, conducted on rats, revealed 10 WO 2004/066726 PCT/US2004/001175 that LD 50 , per os, of the hydrolysate of the compound V, is 750 mg/kg. Clinical picture of the intoxication was a curare-like one of the depolarizing type. Detoxification of the hydrolysate of the compound V, was conducted in the following way. In a 100 ml Ehrlemneyer flask equipped with a magnetic stirrer there were placed 5 ml of the hydrolysate of the compound V,,, 10 ml of 0,1 M of amino acid reagent (pH 8-12) and 35 ml of water, they were stirred at room temperature for 2.hours and kept overnight. Evaluation of acute toxicity in per os tests conducted on rats revealed that LDSa, per os, of the obtained reactive mass exceeded 5000 mg/kg. Hydrolysate Vx (LD 50 , per os, 750 mg/kg) was evaluated as a possible substrate for vital activity of a series of microorganisms (Streptococcus thermophylis, Bacillus subtilis, Escherichia coli, Streptococcus lactis). The test tubes, each one containing 10 ml of the hydrolysate V<, were sown with the 4 strains. After a day of incubation at 37 0 C all the sowings were killed. The reactive mass obtained after detoxification of the hydrolysate of the compound V. with the amino acid reagent was evaluated as a substrate for vital activity of bacteria and an activated sludge. The samples of the reactive mass of the starting concentration were passed to evaluate acute and chronic toxicity in per os tests conducted on rats. It was established that LD 5 0 , per os, of the reactive mass with the strains of streptococcus termophylis as well as with activated sludge exceeds 5000 mg/kg. No clinical picture of intoxication was observed. In chronic toxicity evaluation tests, each of the 5 days rats got 5000 mg/kg. Thus, LD 50 , per os, exceeded 25000 mg/kg. Morphological studies of internal, conducted in 14 days after introduction of the detoxification products revealed no symptoms of toxic action. The detoxification products revealed no mutagenic and carcinogenic properties (Eims test with metabolic activation salmonella/microsomes, strains TA-98 and TA-100). Control investigation of 11 WO 2004/066726 PCT/US2004/001175 toxicity of the amino acid reagent (pH=7.0) in the chronic test revealed that LDso per os exceeded 25000 ing/kg (5000 mg/kg, daily, 5 days). Example 6. Detoxification and biodegradation of methylparathion (methaphos) under the action of lysine and microorganisms. A sample of methylparathion (0,0-dimethyl-0-p nitrophenylthiophosphate) was obtained by the Schrader method interacting 0,0 dimethylchlorothiophosphate with sodium salt of p-nitrophenol and was isolated by crystallization. The 1H and 31 P NMR spectra data and melting point 34-36 0 C corresponded to the literature data. Detoxification of methylparathion was conducted by the following method. In a 100 ml Ehrlnmeyer flask equipped with a magnetic stirrer there were placed 5 g of parathion and 57 ml of 0.1 M of sodium lysinate (pH 8-12). The mixture was stirred at temperature of 50 0 C for 3.5 hours and kept overnight. In a day pH of the solution was reduced to 7.0 and the solution was passed to evaluate acute toxicity in per os tests conducted on rats. LD 50 per os of the detoxification products of methylparathion exceeded 5000 mg/kg. Morphological studies of internal, conducted in 14 days after introduction of the detoxification products revealed complete absence of symptoms of toxic action. The detoxification products revealed no mutagenic and carcinogenic properties (Eims test with metabolic activation - salmonella/microsomes, strains TA-98 and TA-100). The reactive mass obtained on the detoxification of methylparathion with sodium lysinate was evaluated as a substrate for vital functions of bacteria, activated sludge and Rodococcus. In each test tube there were placed 10 ml of the reactive mass solution and there were added 2 ml of bacteria cultures with the titre of 106, then the tubes were incubated for a day at 37 0 C. In a day there was marked a growth of bacteria in all the investigated samples. The study of acute toxicity of 12 WO 2004/066726 PCT/US2004/001175 samples in per os test conducted on rats revealed that LD 50 per os of the samples with the strains of activated sludge as well as with the strains Rodococcus exceeded 5000 mg/kg. Although the subject of the invention gives a detailed description of the realization of the invention, it should be understood that this is only illustrative and the inventions are not limited to the abovementioned actual examples. The temperature conditions and time of carrying out of the reaction given in the claims are interrelated. Specific conditions should be selected depending on the required reaction rate and production capabilities. The upper limit of the temperature range (about 95 degree. C.) can be used in industrial conditions, particularly, to neutralize pesticides or to increase productivity (reducing time of reaction carrying out). However, this will require additional expenses to heat up the mixture. Carrying out the neutralization of toxicity of the instant method in situ is possible at the temperature of about 10 degree. C. At lower temperatures the process rate is, however, slowed down. Minimal time to carry out reaction is defined by time of ecotoxicant toxicity neutralization. Regardless of the temperature at which reaction is carried out, increase of incubation time does not affect the result, i.e. toxicity neutralization. For the experts in this field of technology, it should be clear that within the framework of the claimed method, the conditions of the method can be varied within the limits given in the claim. Such variations can be introduced without deviation from the essence of the invention, which is given in the claims. 13

Claims (12)

1. A method for neutralization of ecotoxicants comprising mixing the ecotoxicant with a neutralizing component comprising an amino acid reagent and neutralizing the exotoxicants.

2. The method of claim 1 further comprising stirring and reacting of the mixture of ecotoxicant and amino acid reagent at a temperature of about 10 degree C to about 95 degree C for a period of time sufficient to neutralize toxicity.

3. The method of claims 1 or 2 further comprising stirring of the ecotoxicant and amino acid reagent mixture at a temperature of about 95 degree C for a period of time sufficient to neutralize toxicity, but not less than for half an hour.

4. The method of any claims 1 to 3 wherein the amino acid reagent comprise an amino acid or a mixture of amino acids or a peptide or derivative of amino acids and peptides or mixture of amino acids and peptides in aqueous or organic-aqueous medium.

5. The method of any claims 1 to 3 wherein said amino acid reagent comprise at least one of the following amino acids: alanine, arginine, aspartic, asparagine, valine, histidine, glycine, glutamic, glutamine, isoleucine, leucine, lysine, methionine, praline, serine, tyrosine, threonine, tryptophane, phenylalanine, cysteine, cystine.

6. The method of any claims 1 to 3 wherein said amino acid reagent comprises hydrolysis product of protein containing factory waste.

7. A method of ecotoxicants neutralization comprising the following steps: 14 WO 2004/066726 PCT/US2004/001175 " Mixing an ecotoxicant with a neutralizing reagent * Neutralizing the mixture of ecotoxicant and neutralizing to a pH appropriate further biodegradation " Biodegrading the mixture to form a neutralized biomass and waste water wherein the neutralization reagent is an amino acid reagent.

8. The method claim 7 further comprising stirring and reacting at a temperature of about 10 degree C to about 95 degree C for a period of time sufficient for toxicity neutralization.

9. The method of claims 7 or 8 further comprising stirring and reacting at a temperature of about 10 degree C to about 95 degree C for a period of time sufficient for toxicity neutralization, but not less than for half an hour.

10. The method of any of claims 7 to 9 wherein the amino acid reagent comprise an amino acid or a mixture of amino acids or a peptide or derivative of amino acids and peptides or mixture of amino acids and peptides in aqueous or organic-aqueous medium.

11. The method of any of claims from 7 to 9 wherein said amino acid comprises at least one of the following amino acids: alanine, arginine, aspartic, asparagine, valine, histidine, glycine, glutamic, glutamine, isoleucine, leucine, lysine, methionine, praline, serine, tyrosine, threonine, tryptophane, phenylalanine, cysteine, cystine.

12. The method of any of claims 7 to 9 wherein the amino acid reagent comprises the hydrolysis product of industrial protein containing wastes. 15