DESC:FIELD
The present disclosure relates to a biostimulant composition.
DEFINITIONS
As used in the present disclosure, the following terms are generally intended to have the meaning as set forth below, except to the extent that the context in which they are used indicates otherwise.
Biostimulant refers to a substance or microorganism or a combination of both whose primary function when applied to plants, seeds or rhizosphere is to stimulate physiological processes in plants and to enhance its nutrient uptake, growth, yield, nutrition efficiency, crop quality and tolerance to stress, regardless of its nutrient content.
Curd refers to the edible part of cauliflower, which consists of a shoot system with short internodes, branches apices and bracts.
Tiller of rice plant refers to a specialized grain-bearing branch that is formed on the unelongated basal internode and grows independently of the mother stem (culm) by means of its own adventitious roots.
Panicle refers to the terminal component of the rice tiller. Panicle is borne on the uppermost internode of the culm. The panicle bears rice spikelets, which develop into grains.
BACKGROUND
The background information herein below relates to the present disclosure but is not necessarily prior art.
It is well recognized that biotic and abiotic stress prevents essentially all crop systems from achieving their yield potential. Abiotic stresses may be prevented by optimizing plant growth conditions and through provision of water, nutrients and plant growth regulators. In addition to these traditional approaches, biostimulants are increasingly being integrated into production systems with the goal of modifying physiological processes in plants to optimize productivity.
Biostimulants are the substance or microorganism or a combination of both whose primary function when applied to plants, seeds or rhizosphere is to stimulate physiological processes in plants and to enhance its nutrient uptake, growth, yield, nutrition efficiency, crop quality and tolerance to stress, regardless of its nutrient content. This is in contrast to fertilizers, which produce a nutritional response. Many important benefits of biostimulants are based on their ability to influence hormonal activity. Hormones in plants (phytohormones) are chemical messengers regulating normal plant development as well as responses to the environment. Compounds in the biostimulants can alter the hormonal status of the plant and exert large influences over its growth and health.
The sector of the biostimulants in the agricultural field has been growing in recent years. This growth is linked to;
• the constant environmental and societal pressure that farmers are under in relation to the use of synthetic products; and
• to the awareness of the essential role of the relationship between soil micro-organisms and plants.
The continuous and injudicious use of synthetic products (fertilizers) for increasing the crop yield might be hazardous to environment as well as human health, which in turn leads to many fatal diseases.
Therefore, there is, felt a need to provide an alternative biostimulant composition that mitigates the drawbacks mentioned hereinabove or at least provides a useful alternative.
OBJECTS
Some of the objects of the present disclosure, which at least one embodiment herein satisfies, are as follows.
It is an object of the present disclosure to ameliorate one or more problems of the prior art or to at least provide a useful alternative.
An object of the present disclosure is to provide a biostimulant composition.
Another object of the present disclosure is to provide a biostimulant composition that improves the plant/crop health thereby enhancing the crop yield.
Other objects and advantages of the present disclosure will be more apparent from the following description, which is not intended to limit the scope of the present disclosure.
SUMMARY
The present disclosure relates to a biostimulant composition comprises 0.1 mass% to 40 mass% of humic acid, 0.1 mass% to 30 mass% of seaweed extract, 0.1 mass% to 15 mass% of at least one amino acid, 0.02 mass% to 20 mass% of at least one vitamin, 0.01 mass% to 1 mass% of a microbial extract, 5 mass% to 10 mass% of at least one protein hydrolysate, 0.01 mass% to 5 mass% of at least one additive, 0 mass% to 1 mass% of water and 96 mass% to 98 mass% of at least one inert material. The mass% of all the components are with respect to the total mass of the biostimulant composition.
DETAILED DESCRIPTION
The present disclosure relates to a biostimulant composition.
Embodiments are provided so as to thoroughly and fully convey the scope of the present disclosure to the person skilled in the art. Numerous details are set forth, relating to specific components, and methods, to provide a complete understanding of embodiments of the present disclosure. It will be apparent to the person skilled in the art that the details provided in the embodiments should not be construed to limit the scope of the present disclosure. In some embodiments, known processes or well-known apparatus or structures, and well known techniques are not described in detail.
The terminology used, in the present disclosure, is only for the purpose of explaining a particular embodiment and such terminology shall not be considered to limit the scope of the present disclosure. As used in the present disclosure, the forms "a,” "an," and "the" may be intended to include the plural forms as well, unless the context clearly suggests otherwise. The terms "comprises," "comprising," “including,” and “having,” are open ended transitional phrases and therefore specify the presence of stated features, integers, steps, operations, elements, modules, units and/or components, but do not forbid the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. The particular order of steps disclosed in the method and process of the present disclosure are not to be construed as necessarily requiring their performance as described or illustrated. It is also to be understood that additional or alternative steps may be employed.
The terms first, second, third, etc., should not be construed to limit the scope of the present disclosure as the aforementioned terms may be only used to distinguish one element, component, region, layer or section from another component, region, layer or section. Terms such as first, second, third etc., when used herein do not imply a specific sequence or order unless clearly suggested by the present disclosure.
Biostimulants are the substance or microorganism or a combination of both whose primary function when applied to plants, seeds or rhizosphere is to stimulate physiological processes in plants and to enhance its nutrient uptake, growth, yield, nutrition efficiency, crop quality and tolerance to stress, regardless of its nutrient content. This is in contrast to fertilizers, which produce a nutritional response. Many important benefits of biostimulants are based on their ability to influence hormonal activity. Hormones in plants (phytohormones) are chemical messengers regulating normal plant development as well as responses to the environment. Root and shoot growth, as well as other growth responses are regulated by phytohormones. Compounds in the biostimulants alters the hormonal status of the plant and exert large influences over its growth and health.
The sector of the biostimulants in the agricultural field has been growing in recent years. This growth is linked to;
• the constant environmental and societal pressure that farmers are under in relation to the use of synthetic products; and
• to the awareness of the essential role of the relationship between soil micro-organisms and plants.
The continuous and injudicious use of synthetic products (fertilizers) for increasing the crop yield might be hazardous to environment as well as human health, which in turn leads to many fatal diseases.
In an aspect, the present disclosure relates to a biostimulant composition.
The biostimulant composition comprises:
(i) 0.1 mass% to 40 mass% of humic acid;
(ii) 0.1 mass% to 30 mass% of seaweed extract;
(iii) 0.1 mass% to 15 mass% of at least one amino acid;
(iv) 0.02 mass% to 20 mass% of at least one vitamin;
(v) 0.01 mass% to 1 mass% of a microbial extract;
(vi) 5 mass% to 10 mass% of at least one protein hydrolysate;
(vii) 0.01 mass% to 5 mass% of at least one additive;
(viii) 0 mass% to 1 mass% of water; and
(ix) 96 mass% to 98 mass% of at least one inert material,
wherein the mass% of all the components are with respect to the total mass of the biostimulant composition.
In an exemplary embodiment of the present disclosure, the amount of humic acid is 0.96 mass% with respect to the total mass of the biostimulant composition. In another exemplary embodiment of the present disclosure, the amount of humic acid is 38.22 mass% with respect to the total mass of the biostimulant composition.
In an embodiment of the present disclosure, the seaweed extract is Durvillea Potatorum.
In an embodiment of the present disclosure, the seaweed extract comprises alginic acid, micro-nutrients, minerals and organic compounds. Alginic acid is present in maximum proportion in the seaweed extract.
In an exemplary embodiment of the present disclosure, the amount of seaweed extract is 0.7 mass% with respect to the total mass of the biostimulant composition. In another exemplary embodiment of the present disclosure, the amount of seaweed extract is 26.24 mass% with respect to the total mass of the biostimulant composition.
In an embodiment of the present disclosure, a ratio of humic acid to seaweed extract is in the range of 1:1 to 2:1. In an exemplary embodiment of the present disclosure, the ratio of humic acid to seaweed extract is 1.45:1.
In an embodiment of the present disclosure, the amino acids are selected from L-amino acids, D-amino acids and the mixture thereof.
In an embodiment of the present disclosure, the amino acid is at least one selected from the group consisting of proline, alanine, tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, ornithine, arginine, histidine, penicillamine, valine, leucine, isoleucine, and phenylalanine.
In an exemplary embodiment of the present disclosure, the amino acid is selected from proline, alanine, tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, ornithine, arginine, histidine, penicillamine, valine, leucine, isoleucine, phenylalanine and mixture thereof.
In an exemplary embodiment of the present disclosure, the amount of amino acid is 0.24 mass% with respect to the total mass of the biostimulant composition. In another exemplary embodiment of the present disclosure, the amount of amino acid is 9.15 mass% with respect to the total mass of the biostimulant composition.
In an embodiment of the present disclosure, the vitamin is at least one selected from the group consisting of ascorbic acid (vitamin C), thiamin mononitrate (vitamin B1) and tocopherol (vitamin E). In an exemplary embodiment, the vitamin is a combination of ascorbic acid (vitamin C), thiamin mononitrate (vitamin B1) and tocopherol (vitamin E).
In an exemplary embodiment of the present disclosure, the amount of vitamin is 0.465 mass% with respect to the total mass of the biostimulant composition. In another exemplary embodiment of the present disclosure, the amount of vitamin is 16.9 mass% with respect to the total mass of the biostimulant composition.
In an embodiment of the present disclosure, the ratio of at least one amino acid to at least on vitamin is in the range of 0.1:1 to 1:1. In an exemplary embodiment of the present disclosure, the ratio of the amino acid to the vitamin is 0.52:1. In another exemplary embodiment of the present disclosure, the ratio of the amino acid to the vitamin is 0.54:1.
In an embodiment of the present disclosure, the microbial extract is Bacillus extract. In an exemplary embodiment of the present disclosure, the Bacillus extract is Bacillus velezensis.
In an exemplary embodiment of the present disclosure, the amount of microbial extract is 0.05 mass% with respect to the total mass of the biostimulant composition.
In an embodiment of the present disclosure, the protein hydrolysate is a hydrolyzed protein.
In an embodiment of the present disclosure, the hydrolyzed protein is selected from the group consisting of hydrolyzed soybean protein, hydrolyzed black bean protein, hydrolyzed chickpea protein, hydrolyzed green pea protein and hydrolyzed fava bean protein. In an exemplary embodiment of the present disclosure, the protein hydrolysate is hydrolyzed soybean protein (P-Soyatose).
In an exemplary embodiment of the present disclosure, the amount of the protein hydrolysate is 7.48 mass% with respect to the total mass of the biostimulant composition.
In an embodiment of the present disclosure, at least one additive is selected from the group consisting of myo-inositol and calcium gluconate. In an exemplary embodiment of the present disclosure, the additive is myo-inositol. In another exemplary embodiment of the present disclosure, the additive is a combination of myo-inositol and calcium gluconate.
In an exemplary embodiment of the present disclosure, the amount of additive is 0.12 mass% with respect to the total mass of the biostimulant composition. In another exemplary embodiment of the present disclosure, the amount of additive is 2 mass% with respect to the total mass of the biostimulant composition.
In an exemplary embodiment of the present disclosure, optionally the amount of water is 0.4 mass% with respect to the total mass of the biostimulant composition.
In an embodiment of the present disclosure, at least one inert material is selected from the group consisting of bentonite, kaolin, montmorillonite, hectorite, saponite, attapulgite, and sepiolite. In an exemplary embodiment of the present disclosure, the inert material is bentonite.
In an exemplary embodiment of the present disclosure, the amount of the inert material is 97.5 mass% with respect to the total mass of the biostimulant composition.
In an exemplary embodiment of the present disclosure, the biostimulant composition comprises:
(i) 0.96 mass% of humic acid;
(ii) 0.7 mass% of seaweed extract (Durvillea Potatorum);
(iii) 0.24 mass% of at least one amino acid;
(iv) 0.34 mass% of ascorbic Acid (Vitamin C);
(v) 0.074 mass% of thiamin mononitrate (Vitamin B1);
(vi) 0.05 mass% of tocopherol (Vitamin E);
(vii) 0.05 mass% of Bacillus extract (Bacillus velezensis);
(viii) 0.12 mass% of myo-inositol;
(ix) 97.5 mass% of bentonite; and
(x) 0.4 mass% of water;
wherein the mass% of all the components are with respect to the total mass of the biostimulant composition.
In another exemplary embodiment of the present disclosure, the biostimulant composition comprises:
(i) 38.22 mass% of humic acid;
(ii) 26.24 mass% of seaweed extract (Durvillea Potatorum);
(iii) 9.15 mass% of at least one amino acid;
(iv) 12.3 mass% of ascorbic Acid (Vitamin C);
(v) 2.75 mass% of thiamin mononitrate (Vitamin B1);
(vi) 1.83 mass% of tocopherol (Vitamin E);
(vii) 7.48 mass% of hydrolyzed soybean protein (P-soyatose);
(viii) 1.53 mass% of myo-inositol; and
(ix) 0.5 mass% of calcium gluconate;
wherein the mass% of all the components are with respect to the total mass of the biostimulant composition.
In an embodiment of the present disclosure, the biostimulant composition is in a solid form.
In an embodiment of the present disclosure, the solid form is selected from the group consisting of powder, granules, water dispersible granules and wettable powder. In an exemplary embodiment of the present disclosure, the biostimulant composition is powder. In an exemplary embodiment of the present disclosure, the biostimulant composition is in form of granules.
In an embodiment of the present disclosure, the biostimulant composition is in the form of powder having a particle size in the range of 400 micron to 1000 micron. In an exemplary embodiment, the biostimulant composition in powder form has a particle size of 700 micron.
In an embodiment of the present disclosure, the biostimulant composition is used to enhance a yield of crop.
In an embodiment of the present disclosure, one or more of the active ingredients of the biostimulant composition is encapsulated for various purposes, such as to increase the effect/activity, to make it safer, or to obtain a stable and more efficacious formulation.
The biostimulant composition of the present disclosure are useful in improving the crop characteristics such as, increase in tillering, increase in plant height, stronger tillers, greener leaf color, pigment content, photosynthetic activity, less fertilizers needed, more productive tillers, early flowering, early grain maturity, less plant verse (lodging), increased shoot growth, enhanced plant vigor and the like. Thus, the biostimulant composition of the present disclosure ultimately increases the yields of economically/ agriculturally important crops.
In a further aspect, the present disclosure provides a simple process for the preparation of a biostimulant composition.
The process of the present disclosure is described in detail:
In a step, the predetermined amounts of humic acid, seaweed extract, at least one amino acid, at least one vitamin, a microbial extract and at least one additive and a predetermined amount of water are mixed in a blender at a speed in the range of 10 to 20 rpm for a time period in the range of 15 to 30 min under stirring to obtain a liquid mixture followed by spraying the liquid mixture on a predetermined amount of bentonite granules for a time period of 40 min to obtain the biostimulant composition in the form of granules.
In an exemplary embodiment of the present disclosure, the speed is 11 rpm (boot mixture) and the time period is 20 min.
In another embodiment of the present disclosure, a homogenous powder of the biostimulant composition is obtained by adding the predetermined amounts of humic acid, seaweed extract, at least one amino acid, at least one vitamin, at least one protein hydrolysate, and at least one additive in a blender for a time period in the range of 5 to 20 min at a speed in the range of 20 to 30 rpm to obtain a homogenous mixture followed by mixing for 10 min to obtain the biostimulant composition in the form of powder.
In an exemplary embodiment of the present disclosure, the speed is 30 rpm (blender) and the time period is 10 min.
In an exemplary embodiment of the present disclosure, the amount of humic acid is 0.96 mass% with respect to the total mass of the biostimulant composition. In another exemplary embodiment of the present disclosure, the amount of humic acid is 38.22 mass% with respect to the total mass of the biostimulant composition.
In an embodiment of the present disclosure, the seaweed extract is Durvillea Potatorum.
In an exemplary embodiment of the present disclosure, the amount of seaweed extract is 0.7 mass% with respect to the total mass of the biostimulant composition. In another exemplary embodiment of the present disclosure, the amount of seaweed extract is 26.24 mass% with respect to the total mass of the biostimulant composition.
In an embodiment of the present disclosure, a ratio of humic acid to seaweed extract is in the range of 1:1 to 2:1. In an exemplary embodiment of the present disclosure, the ratio of humic acid to seaweed extract is 1.45:1.
In an embodiment of the present disclosure, the amino acids are selected from L - amino acids, D-amino acids and the mixture thereof.
In an embodiment of the present disclosure, the amino acid is at least one selected from the group consisting of proline, alanine, tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, ornithine, arginine, histidine, penicillamine, valine, leucine, isoleucine, and phenylalanine.
In an exemplary embodiment of the present disclosure, the amino acid is proline, alanine, tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, ornithine, arginine, histidine, penicillamine, valine, leucine, isoleucine, phenylalanine and mixture thereof.
In an exemplary embodiment of the present disclosure, the amount of amino acid is 0.24 mass% with respect to the total mass of the biostimulant composition. In another exemplary embodiment of the present disclosure, the amount of amino acid is 9.15 mass% with respect to the total mass of the biostimulant composition.
In an embodiment of the present disclosure, the vitamin is at least one selected from the group consisting of ascorbic acid (vitamin C), thiamin mononitrate (vitamin B1) and tocopherol (vitamin E). In an exemplary embodiment, the vitamin from a combination of ascorbic acid (vitamin C), thiamin mononitrate (vitamin B1) and tocopherol (vitamin E).
In an exemplary embodiment of the present disclosure, the amount of vitamin is 0.464 mass% with respect to the total mass of the biostimulant composition. In another exemplary embodiment of the present disclosure, the amount of vitamin is 16.9 mass% with respect to the total mass of the biostimulant composition.
In an embodiment of the present disclosure, the ratio of at least one amino acid to at least on vitamin is in the range of 0.1:1 to 1:1. In an exemplary embodiment of the present disclosure, the ratio of the amino acid to the vitamin is 0.52:1. In another exemplary embodiment of the present disclosure, the ratio of the amino acid to the vitamin is 0.54:1.
In an embodiment of the present disclosure, the microbial extract is Bacillus extract.
In an exemplary embodiment of the present disclosure, the Bacillus extract is Bacillus velezensis.
In an exemplary embodiment of the present disclosure, the amount of microbial extract is 0.05 mass% with respect to the total mass of the biostimulant composition.
In an embodiment of the present disclosure, the protein hydrolysate is a hydrolyzed protein.
In an embodiment of the present disclosure, the hydrolyzed protein is selected from the group consisting of hydrolyzed soybean protein, hydrolyzed black bean protein, hydrolyzed chickpea protein, hydrolyzed green pea protein and hydrolyzed fava bean protein. In an exemplary embodiment of the present disclosure, the protein hydrolysate is hydrolyzed soybean protein (P-Soyatose).
In an exemplary embodiment of the present disclosure, the amount of the protein hydrolysate is 7.48 mass% with respect to the total mass of the biostimulant composition.
In an embodiment of the present disclosure, at least one additive is selected from the group consisting of myo-inositol and calcium gluconate. In an exemplary embodiment of the present disclosure, the additive is myo-inositol. In another exemplary embodiment of the present disclosure, the additive is a combination of myo-inositol and calcium gluconate.
In an exemplary embodiment of the present disclosure, the amount of additive is 0.12 mass% with respect to the total mass of the biostimulant composition. In another exemplary embodiment of the present disclosure, the amount of additive is 2 mass% with respect to the total mass of the biostimulant composition.
In an exemplary embodiment of the present disclosure, optionally the amount of water is 0.4 mass% with respect to the total mass of the biostimulant composition.
In an embodiment of the present disclosure, at least one inert material is selected from the group consisting of bentonite, kaolin, montmorillonite, hectorite, saponite, attapulgite, and sepiolite. In an exemplary embodiment of the present disclosure, inert material is bentonite.
In an exemplary embodiment of the present disclosure, the amount of inert material is 97.5 mass% with respect to the total mass of the biostimulant composition.
The process of the present disclosure is carried out at ambient temperatures. Thus, the process of the present disclosure is energy efficient.
The process of the present disclosure employs inexpensive and easily available reagents. Thus, the process of the present disclosure is economical.
The foregoing description of the embodiments has been provided for purposes of illustration and not intended to limit the scope of the present disclosure. Individual components of a particular embodiment are generally not limited to that particular embodiment, but, are interchangeable. Such variations are not to be regarded as a departure from the present disclosure, and all such modifications are considered to be within the scope of the present disclosure.
The present disclosure is further illustrated herein below with the help of the following experiments. The experiments used herein are intended merely to facilitate an understanding of the ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the experiments should not be construed as limiting the scope of embodiments herein. These laboratory scale experiments can be scaled up to industrial/commercial scale and the results obtained can be extrapolated to industrial/commercial scale.
EXPERIMENTAL DETAILS:
EXPERIMENT 1: Preparation of a biostimulant composition in accordance with the present disclosure
Two biostimulant compositions (the product A named “Laatu” and the product B named “Danza”) were prepared in accordance with the present disclosure.
Process for preparation of the Product A: (LAATU) in accordance with the present disclosure:
9.56 kg of humic acid powder, 6.62 kg of seaweed powder (Durvillea Potatorum), 2.45 kg of various mixture of amino acids, 3.42 kg of ascorbic acid (vitamin C), 0.74 kg of thiamin mononitrate (vitamin B1), 0.49 kg of tocopherol (vitamin E), 1.23 kg of myo-inositol, 0.49 kg of Bacillus extract (Bacillus velezensis), were mixed in quantity sufficient of water for 20 min at a speed of 11 rpm to obtain a liquid mixture. The so obtained Liquid mixture was sprayed on 975 kg of bentonite granules for 40 min to obtain the biostimulant composition in the form of granules.
Process for preparation of the Product B: (DANZA) in accordance with the present disclosure:
382.19 kg of humic acid powder, 262.38 kg of seaweed powder (Durvillea Potatorum), 91.56 kg of various mixture of amino acids, 122.94 kg of ascorbic acid (vitamin C), 27.5 kg of thiamin mononitrate (vitamin B1), 18.31 kg of tocopherol (vitamin E), 15.25 kg of myo-inositol, 74.75 kg of hydrolyzed soybean protein (P-soytose), and 5.13 kg of calcium gluconate were mixed for 10 min at a speed of 30 rpm to obtain a homogenous mixture. The mixture was mixed for 10 min to obtain the biostimulant composition in the form of a powder.
The so-obtained powder form of the biostimulant composition has a particle size of 700 micron.
The biostimulant compositions of the product A and B obtained above in accordance with the present disclosure were checked for the quality control and packed post quality check.
EXPERIMENT 2: Bio-efficacy study of the product A prepared in Experiment 1 of the present disclosure
1. Effect of the product A on Cauliflower Crop
Technical Details:
Duration of Experiment : 2 years
Season/year : 1st season (Rabi 2012-13);
2nd Season (Rabi 2013-14)
Location of Experiment : Trial done at Varanasi, Uttar Pradesh
Crop and Variety : Cauliflower, Pusa Hybrid - 2
Plot Size : 5.0 m x 3.0 m
Row Spacing : 50 cm X 45 cm

Table 1: Treatment dosage
Sr.No. Treatments Dosage (kg/ha)
Bio-efficacy treatments
T1 the Product A 2.5
T2 the Product A 5
T3 the Product A 10
T4 the Product A 15
T5 control -
T6 the Product A
Phyto-toxicity study 20
RESULTS AND DISCUSSION
Among the different parameters, the significant differences were recorded in average number of leaf per plant, average curd weight, average curd diameter, and yield parameters. In general, the mean number of leaf per plant, curd weight and curd diameter, were recorded more in the Product A treatments as compared to the control.
(i) Average No. of leaf/plant:
Maximum average number of leaf/plant for both the years was recorded in T4 Product A @ 15 kg/ha (22.40) as compared to control (16.60).
(ii) Average Curd diameter (in cm):
For both the years of data, the highest average curd diameter was recorded in Treatments T4 (15.85 cm) followed by T3 (15.80 cm) and T2 (15.60 cm). The lowest average curd diameter was observed in control (13.32 cm).
(iii) Average weight of Curd (in g):
The impact of treatment T4 of the Product A application was found to be significant on average weight of curd. Maximum average weight of curd was found in T4 (1244.6 g) as compared to control (819.76 g).
(iv) Average Yield (q/Acre):
Average of both the years of trial showed that highest yield (88.32 q/ha) was recorded in T4. Lowest yield was recorded in control (66.95 q/ha).
Effect on Phytotoxicity of Cauliflower Crop
No symptoms of any abnormality, toxicity were found either on the leaves, or curds in cauliflower with the application of the Product A up to 20 kg/ha during the entire crop season in both the years of experiment.
Conclusion:
the Product A @10-15 kg/ha has a potential in:
? increasing the number of leaf per plant,
? increasing the curd weight,
? increasing the curd diameter, and
? increasing the yield of cauliflower curds.
There was no phyto-toxicity on cauliflower crop from optimum dose and double the optimum dose of the Product A. Hence, the Product A was found to be safe to cauliflower crop even at much higher dose.
2. Effect of the product A on chilli Crop
Technical Details:
Duration of Experiment : Two years
Season/year : 1st season (Kharif 2012-13);
2nd Season (Kharif 2013-14)
Location of Experiment : Trial done at Varanasi, Uttar Pradesh
Crop and Variety : Chilli, Pusa Jwala
Plot Size : 4.0 m x 5.0 m
Row Spacing : 70 cm X 30 cm

Table 2: Treatment dosage
Sr.No. Treatments Dosage (kg/ha)
Bio-efficacy treatments
T1 the Product A 2.5
T2 the Product A 5
T3 the Product A 10
T4 the Product A 15
T5 Control -
T6 the Product A
Phyto-toxicity study 20
RESULTS AND DISCUSSION
Among the different parameters, the significant differences were recorded in average height of plant, average number of flower per plant, average number of fruit per plant, average fruit weight, average fruit length, and average yield of chillies. In general, the mean height of plant, number of flower per plant, number of fruit per plant, fruit weight, fruit length, fruit diameter were recorded more in the Product A treatments as compared to the control. The treatments of the Product A (T3, T4 and T6) were effective in increasing yield of chillies. The color of fruits were bright green with uniform size in case of the Product A treated plants.
(i) Plant height (cm):
Maximum plant height for both the years was recorded in T4 Product A @ 15 kg/ha (54.76 cm) as compared to control (42.66 cm).
(ii) Number of flower/plant:
For both the years of data, the highest number of flowers per plant were recorded in Treatments T4 (106.31) followed by T3 (104.27) and T2 (103.97 cm). The lowest number of flowers per plant was observed in control (82.34).
(iii) Number of fruits/plant:
For both the years of data, the maximum number of fruits per plant was recorded in Treatments T4 (68.06). The minimum number of fruits per plant was observed in control (43.53).
(iv) Fruit length (cm):
For both the years of data, the maximum fruit length was recorded in Treatments T4 (8.76 cm). The minimum fruit length was observed in control (7.58 cm).
(v) Fruit diameter (cm):
Maximum fruit diameter for both the years was recorded in T4 Product A @ 15 kg/ha (0.95 cm) as compared to control (0.74 cm).
(vi) Weight of 10 fruits (g):
Highest weight of 10 fruits for both the years was recorded in T4 Product A @ 15 kg/ha (5.41 g) as compared to control (4.49 g).
(vii) Yield of green chilies (q/Acre):
Average of both the years of trial showed that highest yield (64.94 q/ha) was
recorded in T4. Lowest yield was recorded in control (40.66 q/ha).
(viii) Effect on Phytotoxicity of Chilli Crop
No symptoms of any abnormality, toxicity were found either on the leaves or fruits in chillies with the application of the Product A up to 20kg/ha.
Conclusion:
the Product A @ 10-15 kg/ha has a potential in:
? increasing the height of the plant,
? increasing the number of flower/plant,
? increasing the number of fruit/ plant,
? increasing the fruit length,
? increasing the fruit diameter,
? increasing the weight of fruit, and
? increasing the yield of chillies
There was no phyto - toxicity on chilli crop from optimum dose and double the optimum dose of the Product A. Hence, it can be concluded that the Product A is safe to Chilli crop even at much higher doses (even at double the optimum dose).
3. Effect of the product A on Okra Crop
Technical Details:
Duration of Experiment : Two years
Season/year : 1st season (Kharif 2012-13);
2nd Season (Kharif 2013-14)
Location of Experiment : Trial done at Varanasi, Uttar Pradesh
Crop and Variety : Okra, Parbhani Kranti
Plot Size : 4.0 m x 3.0 m
Row Spacing : 60 cm X 4305 cm

Table 3: Treatment dosage
Sr.No. Treatments Dosage (kg/ha)
Bio-efficacy treatments
T1 the Product A 2.5
T2 the Product A 5
T3 the Product A 10
T4 the Product A 15
T5 Control -
T6 the Product A
Phyto-toxicity study 20
RESULTS AND DISCUSSION
Among the different parameters, the significant differences were recorded in average height of plant, average number of flower per plant, average number of fruit per plant, average fruit weight, average fruit length, average fruit diameter and average yield of okra. In general, the mean height of plant, number of flower per plant, number of fruit per plant, fruit weight, fruit length, fruit diameter were recorded more in the Product A treatments as compared to the control. The treatments of the Product A (T3, T4 and T6) were effective in increasing yield of okra. The color of fruits was bright green with uniform size in case of the Product A treated plants.
(i) Plant height (cm):
Maximum plant height for both the years was recorded in T4 Product A @ 15 kg/ha (135.67 cm) as compared to control (113.00 cm).
(ii) Number of flower/plant:
For both the years of data, the highest number of flowers per plant were recorded in Treatments T4 (28.67) followed by T3 (26.66) and T2 (25.00). The lowest number of flowers per plant was observed in control (17.00).
(iii) Number of fruits/plant:
For both the years of data, the maximum number of fruits per plant was recorded in Treatments T4 (23.33). The minimum number of fruits per plant was observed in control (17.00).
(iv) Fruit length (cm):
For both the years of data, the maximum fruit length was recorded in Treatments T4 (16.96 cm). The minimum fruit length was observed in control (12.33 cm).
(v) Fruit diameter (cm):
Maximum fruit diameter for both the years was recorded in T4 Product A @ 15 kg/ha (2.63 cm) as compared to control (1.36 cm).
(vi) Weight of 10 fruits (g):
Highest weight of 10 fruits for both the years was recorded in T4 Product A @ 15 kg/ha (93.66 g) as compared to control (81.00 g).
(vii) Yield of okra (q/Acre):
Average of both the years of trial showed that highest yield (49.98 q/ha) was recorded in T4. Lowest yield was recorded in control (42.60 q/ha).
Effect on Phytotoxicity of Okra Crop:
No symptoms of any abnormality, toxicity were found either on the leaves or fruits in okra with the application of the Product A up to 20kg/ha.
Conclusion:
the Product A @ 10 - 15 kg/ha has a potential in:
? increasing the height of the plant,
? increasing the number of flower/plant,
? increasing the number of fruit/ plant,
? increasing the fruit length,
? increasing the fruit diameter,
? increasing the weight of fruit, and
? increasing the yield of okra (16.8-17.3%).
There was no phyto-toxicity on okra crop from optimum dose and double the optimum dose of the Product A. Hence, it can be concluded that the Product A was safe to Okra crop even at double the optimum dose.
4. Effect of the product A on Paddy (Rice) Crop
Technical Details:
Duration of Experiment : Two years
Season/year : 1st season (Kharif 2014);
2nd Season (Kharif 2015)
Location of Experiment : Trial done at West Bengal
Crop and Variety : Rice, MTU 7029
Plot Size : 4.0 m x 5.0 m
Row Spacing : 20 cm X 15 cm

Table 4: Treatment dosage
Sr.No. Treatments Dosage (kg/ha)
Bio-efficacy treatments
T1 the Product A 5
T2 the Product A 10
T3 the Product A 15
T4 Control -
T5 the Product A
Phyto-toxicity study 20
RESULTS AND DISCUSSION
(i) Plant height:

Plant height was recorded at 30, 60 and 90 days after transplanting (DAT). At 30 days after transplanting, the plant height was not affected by the different treatments. The plant height at this stage could not show the effect of the application of the Product A in both the seasons. Data revealed that at 60 days after transplanting stage, the crop started showing the effect of application of the Product A and a significant increase in the plant height was recorded in all the treatments in comparison to control. Treatments T3 (First year - 94.5 cm, Second year – 94.9 cm) and T2 (First year - 85.4 cm, Second year – 94.1 cm) recorded maximum plant height. At 90 days similar result were obtained from second year of study. Crop clearly showed the effect of different levels of the Product A and resulted in significant increase in plant height from control.
(ii) Crop growth rate (g/m2/day) (CGR):

The data of average Crop Growth Rate as recorded on 20-40 DAT. 40-60 DAT and 60-80 DAT. The CGR value during 20-40 DAT was not significantly affected. In first year, during 40-60 DAT, 60-80 DAT the highest value of Crop Growth Rate was recorded in Treatments T3 (9.53 g/m2 and 10.13 g/m2) respectively, followed by T2 (9.19 g/m2 and 9.93 g/m2) and Tl (8.00 g/m2 and 9.87 g/m2). The lowest CGR was observed in control (7.10 g/m2 and 8.28 g/m2). The same trend was observed during second year of trial.
(iii) Number of tillers per m2:

The impact of the Product A application was found to be non-significant at 30 DAT, The highest number of tiller was observed during 60 DAT thereafter overall reduction in number of tillers was observed at 90 days after transplanting. Data during both 60 and 90 DAT revealed that the number of tillers was significantly affected by the application of the Product A in all the treatments. It was observed that in first year of experimentation, during 60 DAT, maximum number of tillers was recorded in case or T3 (615) followed by T2 (610) and in second year of trial highest number of tiller was recorded in T2 (625.7). At 90 DAT the maximum number of tillers was recorded in case of T2 (582.6) followed by T3 (541) in first year and in second year T2 (549.4) recorded maximum tillers followed by T1 (530.8).
(iv) Yield attributing characters:

During both the years of trial, the number of panicles/m2 was significantly improved over control, by the application of the Product A. The highest number of panicles/m2 was recorded in T2 (514) followed by T3 (509) and T1 (493). The data on number of grains per panicle also showed similar trend as in case of number of panicles/m2. Highest number of grains per panicle (208) was found in treatments T2, followed by T3 (204). All other treatments also showed a significantly higher number of grains per panicle over control.
(v) Effect on Grain characteristics:

During both the years of trial, the grain size in terms of grain length and grain breadth was significantly improved over control, by the application of the Product A. In first year, the highest grain length and grain breadth was observed in T3 (6.57 cm and 2.51 cm respectively) followed by T2 (6.55 cm and 2.51 cm respectively) and T1 (6.39 cm and 2.37 cm respectively). Similar trend was recorded in second year trial. Test weight was highest in T2 (22.93 and 22.89) followed by T3 (22.90 and 22.82) during first year and second year.
Effect on Phytotoxicity of Paddy (Rice) Crop:
Data revealed that there was no phytotoxicity symptoms viz., yellowing, necrosis, epinasty/ hyponasty, vein clearing, leaf injury and stunting etc. after the application of the Product A at 10 kg/ha and 20 kg/ha during the entire crop season in both the years of experiment.
Conclusion:
a. Application of the Product A at the dose of 10 kg/ha was found to be optimum in Kharif rice for overall growth and development of plants. These treatments also produced higher CGR (crop growth rate), yield components and grain yield of rice due to effective increase in the yield contributing characters like no. of panicle and grains per panicle.
b. There was no phyto-toxicity on rice crop from optimum dose and double the optimum dose of the product A applied either at tillering or at Panicle emergence. Hence, it can be concluded that the Product A was safe to Rice Crop even at double the optimum dose.
5. Effect of the product A on Tomato Crop
Technical Details:
Duration of Experiment : Two years
Season/year : 1st season (Rabi 2012-13);
2nd Season (Rabi 2013-14)
Location of Experiment : Trial done at Varanasi, Uttar Pradesh
Crop and Variety : Tomato, CO- 3
Plot Size : 4.0 m x 5.0 m
Row Spacing : 70 cm X 60 cm

Table 5: Treatment dosage
Sr.No. Treatments Dosage (kg/ha)
Bio-efficacy treatments
T1 the product A 2.5
T2 the product A 5
T3 the product A 10
T4 the product A 15
T5 Control -
T6 the product A
Phyto-toxicity study 20
RESULTS AND DISCUSSION
Among the different parameters, the significant differences were recorded in average height of plant, number of flower per plant, number of fruit per plant, fruit weight, fruit diameter and yield parameters. In general, the mean number of fruit per plant, fruit weight and fruit diameter were recorded more in the Product A treatments as compared to the control. The treatments of the Product A (T3, T4 and T6) increased yield of fruits. The color of fruits was bright red with firm lustrous skin in case of the Product A treated plants.
(i) Plant height (cm):
Maximum plant height for both the years was recorded in T4 Product A @ 15 kg/ha (65.36 cm) as compared to control (55.20 cm).
(ii) Number of flower/plant:
For both the years of data, the highest number of flowers per plant were recorded in Treatments T4 (34.21) followed by T3 (32.48) and T2 (32.34 cm). The lowest number of flowers per plant was observed in control (21.94).
(iii) Number of fruits/plant:
For both the years of data, the maximum number of fruits per plant was recorded in Treatment T4 (24.41) which was followed by T3 (23.08) and T2 (22.41). The minimum number of fruits per plant was observed in control (14.61).
(iv) Fruit diameter (cm):
Maximum fruit diameter for both the years was recorded in T4 Product A @ 15 kg/ha (5.50 cm) as compared to control (4.28 cm).
(v) Average weight of 10 fruits (g):
Highest weight of 10 fruits for both the years was recorded in T4 Product A @ 15 kg/ha (742 g) as compared to control (524 g).
(vi) Yield of tomato (q/Acre):
Average of both the years of trial showed that highest yield (134.60 q/ha) was recorded in T4. Lowest yield was recorded in control (88.61 q/ha).
Effect on Phytotoxicity of Tomato Crop:
No symptoms of any abnormality, toxicity were found either on the leaves or fruits in tomato with the application of the Product A up to 20kg/ha.
Conclusion:
The Product A @ 10 - 15 kg/ha has a potential in:
? increasing the height of the plant,
? increasing the number of flower/plant,
? increasing the number of fruit/ plant,
? increasing the fruit diameter,
? increasing the weight of fruit, and
? increasing the yield of tomato.
There was no phyto-toxicity on tomato crop from optimum dose and double the optimum dose of the Product A. Hence, it can be concluded that the Product A was safe to Tomato Crop even at double the optimum dose.
EXPERIMENT 3: Bio-efficacy study of the product B prepared in Experiment 1
1. Effect of the product A on Paddy (Rice) Crop
Technical Details:
Duration of Experiment : Two years
Season/year : 1st season (Kharif 2014);
2nd Season (Kharif 2015)
Location of Experiment : Trial done at West Bengal
Crop and Variety : Rice, MTU 7029
Plot Size : 4.0 m x 5.0 m
Row Spacing : 20 cm X 15 cm

Table 6: Treatment dosage
Sr.No. Treatments Dosage (kg/ha)
Bio-efficacy treatments
T1 The Product B 5
T2 The Product B 10
T3 The Product B 15
T4 Control -
T5 The Product B
Phyto-toxicity study 20
RESULTS AND DISCUSSION
(i) Plant height:
Plant height was recorded at 30, 60 and 90 days after transplanting (DAT). At 30 days after transplanting the plant, height was not affected by the different treatments. The plant height at this stage could not show the effect as the application of the Product B in both the seasons.
Data revealed that at 60 days after transplanting, stage the crop started showing the effect of application of the Product B and a significant increase in the plant height was recorded in all the treatments in comparison to control. Treatments T3 (First year - 94.5 cm, Second year – 94.9 cm) and T2 (First year - 85.4 cm, Second year – 94.1 cm) recorded maximum plant height. At 90 days similar result were obtained from second year of study. Crop clearly showed the effect of different levels of the Product B and resulted in significant increase in plant height from control.
(ii) Crop growth rate (g/m2/day) (CGR):
The data of average Crop Growth Rate as recorded on 20-40 DAT. 40-60 DAT and 60-80 DAT. The CGR value during 20-40 DAT was not significantly affected. In first year, during 40-60 DAT, 60-80 DAT the highest value of Crop Growth Rate was recorded in Treatments T3 (9.53 g/m2 and 10.13 g/m2) respectively, followed by T2 (9.19 g/m2 and 9.93 g/m2) and Tl (8.00 g/m2 and 9.87 g/m2). The lowest CGR was observed in control (7.10 g/m2 and 8.28 g/m2). The same trend was observed during second year of trial.
(iii) Number of tillers per m2:
The impact of the Product B application was found to be non-significant at 30 DAT, The highest number of tiller was observed during 60 DAT thereafter overall reduction in number of tillers was observed at 90 days after transplanting. Data during both 60 and 90 DAT revealed that the number of tillers was significantly affected by the application of the Product B in all the treatments. It was observed that in first year of experimentation, during 60 DAT, maximum number of tillers was recorded in case or T3 (615) followed by T2 (610) and in second year of trial highest number of tiller was recorded in T2 (625.7). At 90 DAT the maximum number of tillers was recorded in case of T2 (582.6) followed by T3 (541) in the first year and in the second year T2 (549.4) recorded maximum tillers followed by T1 (530.8).
(iv) Yield attributing characters:
During both the years of trial, the number of panicles/m2 was significantly improved over control, by the application of the Product B. The highest number of panicles/m2 was recorded in T2 (514) followed by T3 (509) and T1 (493). The data on number of grains per panicle also showed similar trend as in case of number of panicles/m2. Highest number of grains per panicle (208) was found in treatments T2, followed by T3 (204). All other treatments also showed a significantly higher number of grains per panicle over control.
(v) Effect on Grain characteristics:
During both the years of trial, the grain size in terms of grain length and grain breadth was significantly improved over control, by the application of the Product B. In first year, the highest grain length and grain breadth was observed in T3 (6.57 cm and 2.51 cm respectively) followed by T2 (6.55 cm and 2.51 cm respectively) and T1 (6.39 cm and 2.37 cm respectively). Similar trend was recorded in second year trial. Test weight was highest in T2 (22.93 and 22.89) followed by T3 (22.90 and 22.82) during first year and second year.
Effect on Phytotoxicity of Paddy (Rice) Crop:
Data revealed that there was no phytotoxicity symptoms viz., yellowing, necrosis, epinasty/ hyponasty, vein clearing, leaf injury and stunting etc. after the application of the Product B at 10 kg/ha and 20 kg/ha during the entire crop season in both the years of experiment.
Conclusion:
a. Application of the Product B at the dose of 10 kg/ha is found to be optimum in Kharif rice for overall growth and development of plants. These treatments also produced higher CGR, yield components and grain yield of rice due to effective increase in the yield contributing characters like no. of panicle and grains per panicle.
b. There was no phyto - toxicity on rice crop from optimum dose and double the optimum dose of the Product B applied either at tillering or at Panicle emergence. Hence, it can be concluded that the Product B was safe to Rice Crop even at double the optimum dose.
2. Effect of the product A on Tomato Crop
Technical Details:
Duration of Experiment : Two years
Season/year : 1st season (Rabi 2012-13);
2nd Season (Rabi 2013-14)
Location of Experiment : Trial done at Varanasi, Uttar Pradesh
Crop and Variety : Tomato, CO- 3
Plot Size : 4.0 m x 5.0 m
Row Spacing : 70 cm X 60 cm

Table 7: Treatment dosage
Sr.No. Treatments Dosage (kg/ha)
Bio-efficacy treatments
T1 the Product B 2.5
T2 the Product B 5
T3 the Product B 10
T4 the Product B 15
T5 Control -
T6 the Product B
Phyto-toxicity study 20
RESULTS AND DISCUSSION
Among the different parameters the significant differences were recorded in average height of plant, number of flower per plant, number of fruit per plant, fruit weight, fruit diameter and yield parameters. In general, the mean number of fruit per plant, fruit weight and fruit diameter were recorded more in the Product B root application as compared to control. The root application of the Product B (T3, T4 and T6) increased yield of fruits. The color of fruits was bright red with firm lustrous skin in case of the Product B treated plants.
(i) Plant height (cm):
Maximum plant height for both the years was recorded in T4 Product B @ 15 kg/ha (65.36 cm) as compared to control (55.20 cm).
(ii) Number of flower/plant:
For both the years of data, the highest number of flowers per plant were recorded in Treatments T4 (34.21) followed by T3 (32.48) and T2 (32.34 cm). The lowest number of flowers per plant was observed in control (21.94).
(iii) Number of fruits/plant:
For both the years of data, the maximum number of fruits per plant was recorded in Treatment T4 (24.41). The minimum number of fruits per plant was observed in control (14.61).
(iv) Fruit diameter (cm):
Maximum fruit diameter for both the years was recorded in T4 Product B @ 15 kg/ha (5.50 cm) as compared to control (4.28 cm).
(v) Average weight of 10 fruits (g):
Highest weight of 10 fruits for both the years was recorded in T4 Product B @ 15 kg/ha (742 g) as compared to control (524 g).
(vi) Yield of tomato (q/Acre):
Average of both the years of trial showed that highest yield (134.60 q/ha) was recorded in T4. Lowest yield was recorded in control (88.61 q/ha).
Effect on Phytotoxicity of Tomato Crop:
No symptoms of any abnormality, toxicity were found either on the leaves or fruits in tomato with root application of the Product B up to 20 kg/ha.
Conclusion:
From the above finding it can be concluded that the Product B used as root application @ 10-15 kg/ha has a potential in:
? increasing the height of the plant,
? increasing the number of flower/plant,
? increasing the number of fruit/plant,
? increasing the fruit diameter,
? increasing the weight of fruit, and
? increasing the yield of tomato.
There was no phyto-toxicity on tomato crop from optimum dose and double the optimum dose of the Product B.
EXPERIMENT NO: 4 Toxicological studies of the Product A prepared in accordance with the present disclosure
Testing laboratory: International Institute of Biotechnology and Toxicology (IIBAT), Padappai, Kancheepuram District, Tamil Nadu.
Test Guideline : All the test were conducted as per OECD test guidelines. In accordance with the OECD Principles of Good Laboratory Practices (GLP) along with the Integrated Management System (IMS) as per ISO 9001:2015; ISO 14001:2015 and ISO 45001:2018. OECD Guidelines for Testing of Chemicals:
• Acute oral toxicity – acute toxic class method (no. 423);
• Acute dermal toxicity – fixed dose method (no. 402);
• Acute inhalation toxicity testing (no. 403);
• Acute dermal irritation/corrosion study (no. 404);
• In vivo eye irritation (no. 405);
• Fish acute toxicity testing (no. 203);
• Acute oral toxicity test (no. 213);
• Acute contact toxicity testing (no. 214); and
• Earthworm acute toxicity test (no. 207).
Status of study: Complete [under Good Laboratory Practices (GLP) accreditation]
1. Acute oral toxicity study in wistar rats:
An acute oral toxicity study of the Product A, was carried out in healthy, young adult female Wistar rats. The animals were acclimatized for 5 days prior to dosing. Animals were housed in groups of 3 per cage. The study comprised of two steps involving 3 animals per step. The test item was prepared shortly prior to administration. Required quantity of test item was weighed, mixed with distilled water. The test item was administered to overnight fasted animals in a single dose by gavage using intubation needle fitted with a graduated syringe. The dose volume was maintained as 10 mL/kg body weight for all the animals.
In Step 1 of the experiment, 3 female rats were dosed with the test item at the dose level of 2000 mg/kg b.w and observed for mortality and clinical signs. Since no mortalities and clinical signs were observed in these animals, a Step 2 experiment with another set of 3 female rats was performed with the same dose level (i.e. 2000 mg/kg b.w.). All the animals of Step-1 and Step-2 were observed at 30 min, 1h, 2h, 3h and 4h post-dosing and at once daily thereafter for 14 days for morbidity, mortality and clinical signs of toxicity. The body weight of the animals was recorded prior to dosing (Day 0) and at weekly intervals thereafter. All the animals were euthanized by CO2 asphyxiation at the end of the observation period and subjected to gross pathological examination.
Inference: No mortality and clinical signs was observed in any of the animals of Step 1 and Step 2 experiments. Body weight gain was noticed in all animals during the 14 days observation period. The gross pathology examination of the animals did not reveal any gross lesions. The acute oral LD50 of the Product A in female rats was >2000 mg/kg b.w. with a LD50 cut-off value of 5000 mg/kg b.w. The test item, the Product A thus classified as Category 5 or Unclassified according to the Globally Harmonized System (GHS) of classification.
2. Acute dermal toxicity in wistar rats:
An acute dermal toxicity study of the Product A, was carried out in Wistar rats. A range finding study was carried out using 1 female rat in order to establish the dose level for the main study. In the range finding study, the test item was pulverized using mortar and pestle, and a known weight of the powdered test item at the dose level of 2000 mg/kg b.w. was moistened with minimum volume of distilled water (250 µL) so as to prepare a paste and thereafter applied uniformly over the exposed area of dorsal/flank skin clipped area. After the application, the test item was held in contact with the skin with a porous gauze dressing and bandaged with non-irritating adhesive tape for 24 hour exposure period. Restrainer (neck collar) was used to prevent the ingestion of the test item from the application site. After the exposure period, the dressing was removed and the residual test item was wiped gently from the skin using cotton soaked in potable well water of IIBAT (International Institute of Biotechnology and Toxicology) processed by reverse osmosis system. Following test item application, the animal was observed during first 30 min, and at 1, 2, 4 and 6 hours for clinical signs of toxicity after the beginning of the exposure and thereafter once daily for 14 days. Mortality or clinical signs of toxicity was not found in the range finding study. Therefore, the main study was performed with 2 female animals with same dose level (2000 mg/kg b.w.) to confirm the classification outcome.
In the Main Study, the test item was evenly applied to the fur clipped area of the rats following the same procedure used in the range finding study. Following test item application, the animals were observed first 30 min, and at, 1, 2, 4 and 6 hours for mortality or clinical signs of toxicity and thereafter once daily for 14 days. The body weights of individual animals were recorded prior to the test item application, weekly and at the termination of the study. All the animals were subjected to gross pathological examination at the end of 14 - day observation period.
Inference: Mortality: No mortality during the study
Signs and symptoms: Animals treated with the test item did not exhibit any mortality or clinical signs of toxicity for the entire obsevation period. No skin reactions at the site of appplication were observed following the removal of the test items. Rats treated with the test item showed normal body weight during the study. Gross pathology examination of the animals did not reveal any macroscopic lesions.
Result: LD50: >2000 mg/kg b.wt. Therefore, the test item the Product A falls under the Category 5/Unclassified as per the Globally Harmonized System of classification and labeling of chemicals (GHS).
3. Acute inhalation toxicity in wistar rats
An acute inhalation toxicity study with the Product A, was conducted in Wistar rats. The study was performed using inhalation equipment (Nose-only) fabricated by CH.Technologies, Inc., USA. Restraint tubes (along with animal) were attached to the ports of the chamber. One of the ports was assigned for collection of test atmosphere (aerosols) concentration and for reading the dynamic measurements of the chamber on a regular basis during the exposure. A limit study was performed with 6 Wistar rats (3/sex) at the maximum attainable concentration i.e., 1.10 mg/L of the Product A. Concentration above 1.10 mg/L could not be achieved in the limit test due to the physicochemical nature of the test item. The animals were exposed continuously for 4 hours through nose-only inhalation.
Inference:
Mortality: No mortality was observed in any of the exposed animals for 14 days.
Signs and symptoms: Change in body coat colour, lethargy, mild respiratory distress durinh 1st and 2nd hour of post exposure. All the animals were found to be apparently normal from day 1 till end.
Result: LC50: >1.10 mg/l
4. Acute dermal irritation/corrosion study in new Zealand white rabbits
An acute dermal irritation/corrosion study of the Product A was conducted in New Zealand White rabbits. An initial test was performed on 1 animal (male rabbit) using a single patch. A weight of 0.5001 g of finely powdered test item using pestle and mortar was moistened with distilled water (300 µl) and evenly applied to the fur clipped area (6 cm2) of left side skin of the rabbit and covered with a gauze patch, which was held in place with non-irritating tape the right side remained as untreated. The patch was loosely held in contact with the skin by means of a suitable semi-occlusive dressing for duration of 4 hours. Restrainers (neck collars) were used to prevent the ingestion of the test item from the application site. At the end of the exposure period, the patches were removed and the residual test item was gently wiped off from the application site, using cotton soaked with distilled water. Following test item application, the animal was observed once daily for morbidity and mortality. Skin reactions at the application site were scored immediately (3 min) after the removal of patch (for initial test alone), 60 min 24, 48 and 72 hour after patch removal following the Draize method. Untreated control site was also scored for comparative purpose. Body weight gain of individual animals were recorded prior to the test item application and at the termination of the experiment. As the results, the initial test did not reveal any irritation, a confirmatory test using 2 additional animals (male rabbits) was carried out following the same procedure to confirm the non-irritant nature of the test item.
Inference: No mortality occurred. All animals were found to be apparently normal from the day of application to the day of termination of the experiment. Test item related skin reactions were not noticed at the application site in any of the treated rabbits. The treated site of all the three animals was comparable to the untreated control site. Mean scores (24, 48 and 72 h) of all three animals were 0 for erythema, eschar and oedema. No treatment-related effects were noticed in body weight. No irritation potential of the test item was noticed and the mean irritation scores of the animals were not falling under any of the categories of the GHS classification and labeling of chemicals. Therefore, the test item Product A was classified to be a non- irritant to the skin of New Zealand White rabbits.
5. In vivo eye irritation in new Zealand white rabbits
Acute In vivo eye irritation/corrosion study with the Product A was conducted in New Zealand White rabbits. In the initial test, 100 mg of the finely powdered test item was instilled into the conjunctival sac of the left eye of 1 male rabbit. The right eye of the rabbit served as the control. The ocular irritation potential was evaluated and graded at 1, 24, 48 and 72 h after the test item instillation. The animal did not exhibit any ocular lesion following instillation of the test item. To confirm the findings of the initial test, the confirmatory test was conducted with 2 additional animals (male) using the same procedure and ocular lesions were evaluated for 3 days. The animals were also observed for clinical signs of toxicity, morbidity and mortality. The individual body weights of the animals were recorded prior to treatment and at the termination of the experiment.
Inference: No mortality occurred. All animals were found to be apparently normal from the day of test item instillation to the last day of observation. No treatment related changes were noticed in the conjunctivae, cornea and iris of the rabbits. The mean scores (24, 48 and 72 h) of all three animals were 0. No treatment-related effects were noticed in body weight. No test item related ocular lesions were observed and the mean irritation scores of the animals did not fall under any of the categories of the GHS classification and labeling of chemicals. Therefore, the test item Product A was considered to be a non-irritant to the eye of New Zealand White Rabbits.
6. Acute oral toxicity study in chicken
Acute Oral Toxicity of the Product A was tested in Chicken, as per OECD test guidelines as given above. Known weight of the test item was mixed with distilled water, thereafter administered orally to 5 Chicken (5 treated chicken), at the dose level of 2000 mg/kg body weight. 5 control Chicken, were similarly treated with distilled water alone. All birds were observed individually for toxicity signs and mortality thrice on day 0 and thereafter daily for 14 days. Body weight of each bird was recorded just prior to administration of dose (0 day) and on days 3, 7 and 14 after dosing. Food consumption and water consumption was measured daily. At the end of the test, all surviving birds were humanely euthanized by carbon dioxide asphyxiation and gross pathology was carried out. Gross pathology conducted on birds dosed with 2000 mg/kg b.w. and control did not revealed any treatment related lesions.
Inference: No mortality and clinical signs of toxicity were observed in birds treated with 2000 mg/kg b.w. of the Product A. Similarly, no mortality and clinical signs of toxicity were observed in control birds for the entire experimental period. Compared to control birds, there was no significant change observed in the body weight of birds treated with 2000 mg/kg b.w. on day 3, 7 and day 14.There was no significant change observed in the consumption of feed of the birds treated with the Product A from day 1 to day 14 when compared with control birds. There was no significant change observed in the consumption of water in birds treated with the Product A from day 1 to day 14 when compared with control birds. Based upon the above Limit Dose Test, the LD50 of the Product A for Chicken, was considered as greater than 2000 mg/kg b.wt.
7. Acute toxicity study in freshwater fish (poecilia reticulata)
A study was conducted to evaluate the acute toxicity of the Product A to fresh water fish, Poecilia reticulata. The semi-static bioassay procedure was followed as per OECD test guidelines as mentioned above.
In a range finding experiment, groups of 7 fish each in 2 replicates were exposed to control and test item concentrations of 0.5, 1, 10, 50 and 100 mg/L the Product A, respectively for 96 hours. The fish were observed for mortality and abnormal behaviour twice on the day (2 ± 0.5 hour and 5 ± 1 hour) of exposure, thereafter, at the end of 24, 30, 48, 54, 72, 78 and 96 hours throughout the experimental period.
Inference: The results showed that no mortality and toxicity signs (abnormal behaviour) were occurred in control and all test item concentrations at the end of 96 hours.
Further, a limit test was conducted with control and test item concentration of 100 mg/L in 2 replicates at 7 fish/ replicate for 96 hours. The fish were observed for mortality and abnormal behaviour twice on the day of exposure, 2 ± 0.5 hour and 5 ± 1 hour thereafter, at the end of 24, 30, 48, 54, 72, 78 and 96 hours throughout the experimental period.
Inference: No mortality and toxicity sign (abnormal behaviour) were observed in the control and 100 mg/L concentration, throughout the observation period of 96 hours. During the experiment (Limit test), pH ranged from 7.46 to 7.96. Dissolved oxygen ranged from 87.6 to 99.8% and temperature ranged from 22.7 to 23.1°C in control and 100 mg/L. Based on the above findings, the 24, 48, 72 and 96 hours LC50 of the Product A to freshwater fish, Poecilia reticulata was greater than 100 mg/L.
8. Acute oral toxicity to honeybees (apis cerana indica)
For range finding experiment, dose viz., 10, 25, 50, 75 and 100 µg/bee was administered to bees orally. Consumption was observed to be 9.50, 23.13, 45.38, 66.36 and 94.44 µg/bee. As no mortality was observed in any of the doses in the range finding experiment, a limit test was conducted with a single dose of 100 µg/bee to demonstrate that LD50 of the Product Awas >100 µg/bee (nominal dose). In parallel to the test item, in the limit test, dimethoate technical (reference item) was also evaluated at the doses viz., 0.045, 0.068, 0.101 and 0.152 µg a.i./bee along with a control (acetone + 50% w/v sucrose solution). Consumption was observed to be 0.042, 0.060, 0.082 and 0.126 µg a.i./bee Bees in the test item treatments were offered the test solutions prepared in 50% w/v sucrose solution. Bees in the reference item treatments were offered the test solutions prepared in acetone + 50% w/v sucrose solution. The test solutions and controls were offered to the bees for 6 hours with dosing volume of 200 µL/Group. Assessments of mortality and abnormal behavioural effects were carried out at 4, 24 and 48 hours of dosing.

Results: The test item, the Product A recorded 0% mortality at the dose of 100 µg/bee (92.93 µg/bee based on the actual consumption) at 48 hours of dosing. There was no mortality in the control groups (50 % w/v sucrose solution and acetone + 50 % w/v sucrose solution) at the assessment made 48 hours of dosing. No sub lethal (abnormal behavioural) effects were observed at any point of observation after dosing for test item and reference item. The 48-hour Oral LD50 value for the Product A was determined to be > 92.93 µg/bee, based on actual consumption. The LD50 of the reference item was 0.102 (0.083 - 0.149) µg dimethoate/bee at 24 hours of dosing. The test met validity criteria, as mortality in the controls did not exceed 10%.
Conclusion
The effects of the Product A were assessed in oral honey bee toxicity test conducted in the laboratory. The 48h Oral LD50 value for the Product A was determined to be > 92.93 µg/bee, based on actual consumption.
9. Acute contact toxicty to honeybees (apis cerana indica)
Range finding experiment was conducted with the doses viz., 10, 25, 50, 75 and 100 µg/bee against bees were selected. As no mortality was observed in any of the doses in the range finding experiment, a limit test was conducted with a single dose of 100 µg/bee to demonstrate that LD50 of the Product Awas > 100 µg/bee. In parallel to the test item, in the limit test, dimethoate technical (reference item) was also evaluated at the doses viz., 0.045, 0.068, 0.101 and 0.152 µg a.i./bee along with a control (acetone). Further, bees were treated with test item the Product A mixed with deionized water dosed by topical application on the dorsal side of thorax of each bee with dosing volume of 1 µL. For reference item, bees were treated with dimethoate technical dissolved in acetone and dosed by topical application on the dorsal side of thorax of each bee. Assessments of mortality and abnormal behavioural effects were carried out at 4, 24 and 48 hours of dosing.
Results:
The test item, the Product A recorded 0.0% mortality at the dose of 100 µg/bee at 48 hours of dosing. There was no mortality in any of the control groups (deionized water, deionized water + Triton X-100 and acetone) during the assessment made upto 48 hours of dosing. No sub lethal (abnormal behavioural) effects were observed at any point of observation after dosing for test item and reference item. The 48-hour Contact LD50 value for the Product A was determined to be > 100 µg/bee. The LD50 of the reference item was 0.124 (0.114 – 0.139) µg dimethoate/bee at 24 hours of dosing. The test met validity criteria, as mortality in the controls did not exceed 10%.

Conclusion
The effects of the Product A were assessed in contact honeybee toxicity test conducted in the laboratory. The 48h contact LD50 value for the Product A was determined to be > 100 µg/bee.

10. Acute toxicity to earthworm (eisenia fetida)
The acute toxicity of the Product A to the earthworm, Eisenia fetida (Savigny 1826), was estimated in a 14-day soil exposure laboratory. A non-replicated range finding test was conducted prior to definitive test with viz., 10, 33, 100, 333, 667 and 1000 mg of the Product A/kg dry soil. No mortality was observed in any of the tested concentrations in the range finding experiment. Based on the results of range finding test, a limit test with four replicates of 10 clitellated adult earthworms were exposed to nominal concentrations of 1000 mg of the Product A/kg dry soil. An untreated control (Deionised water) was also replicated four times, with ten earthworms in each replicate.
Earthworms were assessed for mortality after 7 and 14 days of exposure, behavioral abnormalities on 0, 7 and 14 days of exposure and Earthworm body weights were assessed on day 0 and day 14.

Table 8: Summary of Results
Treatment Group Control
(Deionised water) 1000 mg/kg of the Product A
Mortality after
14 days [%] 0.00 0.00
Biomass change after
14 days [%]1 -13.70 -14.94
14 day LC50 Greater than 1000 mg/kg dry soil
NOEC related to mortality 1000 mg/kg dry soil
NOEC related to biomass 1000 mg/kg dry soil
1Student t-test (p = 0.05);
-Minus symbol indicates reduction in body weight
Conclusion
According to the results of this study, 14-day LC50 of the Product A was determined to be greater than 1000 mg/kg dry soil. The 14-day No-Observed-Effect Concentration (NOEC) with respect to biomass and mortality was determined to be 1000 mg of the Product A/kg dry soil wt. The 14-day LC50 of the reference compound (2-Chloroacetamide) in the recent experiment (June - July, 2021) was 31.82 m
Table 9: Toxicological Profile of the Product A as per experiment no.4 of the present disclosure
Sr. no. Study Title Toxicity Results
Acute Toxicity
1 Acute Oral Rat LD50 LD50 >2000 mg/kg b.wt
2 Acute Dermal Rat LD50 LD50 >2000 mg/kg b.wt
3 Acute Inhalation LC50 4 hr. LC50 >1.10 mg/L
4 Primary Skin Irritation Rabbit Non- irritant to skin
5 Mucous Membrane Irritation Rabbit Non-irritant to eyes
Ecotoxicity
6 Acute toxicity to Bird LD50 >2000 mg/kg
7 Acute toxicity to fresh water fish , carp LC50 96 hours LC50>100 mg/l
8 Acute contact / oral toxicity to honeybee, Apis cerana indica LD50 (Oral) (48h) >92.93 µg a.i/bee
LD50 (Contact) >100 µg a.i/bee
9 Acute toxicity to Earthworms LC 50 LC50> 1000 mg/kg

EXPERIMENT NO: 5 Toxicological studies of the Product B prepared in accordance with the present disclosure
1. Acute oral toxicity study in wistar rats
An acute oral toxicity study of the Product B, was carried out in female Wistar Rats and acclimatized for 5 days prior to dosing. Animals were housed in stepwise of 3 per cage. The study comprised of four steps involving 3 animals per step. The test item was prepared shortly prior to administration. The required quantity of test item was weighed, mixed with distilled water and administered in a single dose by gavage using intubation needle fitted with a graduated syringe. The animals were fasted overnight prior to dosing and the feed was withheld for 3h post dosing. The Step -1 of the study was conducted at the dose level of 300 mg/kg b.w. with three female rats. Animals did not exhibit any clinical signs of toxicity, morbidity or mortality. To confirm the observations, the Step-2 experiments was conducted at the same dose level of 300 mg/kg b.w. These animals also did not exhibit any clinical signs of toxicity, morbidity or mortality. Since there were no mortality in both Step -1 and Step -2 animals, the Step -3 experiments was conducted at the dose level of 2000 mg/kg b.w. using 3 female rats. None of these animals exhibited any clinical signs of toxicity, morbidity or mortality. Therefore, Step -4 was conducted, to confirm these finding using the same dose level of 2000 mg/kg b.w. with three more female animals. Following the dosing, all Step-1, 2, 3, and Step-4 animals were observed once daily for clinical signs of toxicity, morbidity and mortality for a period of 14 days. Additionally, on Day 0, all animals were observed for clinical signs of toxicity at 30 min, 1h, 2h, 3h and 4h post dosing. Body weights of the animals were recorded prior to dosing and at weekly intervals thereafter. All the animals were sacrificed on Day 14 by CO2 asphyxiation and subjected to gross pathology examination.
Inference: None of the animals treated with the test item at 300 and 2000 mg/kg.b.w. showed any clinical signs of toxicity or mortality. Body weight gain was observed during the study period. The gross pathology examination did not reveal any lesions in any of the animals. Thus, it is concluded that the acute oral LD50 of the Product B in female rats is > 2000-5000 mg/kg b.w. with a LD50 cut-off value of 5000 mg/kg b.w. The test item was classified Category 5 or Unclassified according to the Globally Harmonized System (GHS) of classification
2. Acute dermal toxicity in wistar rats
An acute dermal toxicity study of the Product B, was carried out in Wistar rats. A range finding study involving 3 steps was carried out in order to established the dose level for the main study. In step -1 of the range finding study, 1 female rat was treated with 200 mg/kg.b.w. of the test item. The required quantity of the test item was moistened with approximately 200-250µl of distilled water so as to prepare a paste and then evenly applied to the fur clipped skin area of the rats. Test item requirement per animal was calculated based on the bodyweight of each animal on the day of test item application. After the application, the test item was held in contact with the skin with a porous gauze dressing and bandaged with non-irritating adhesive tape for 24-hour exposure period. After the exposure period, the dressing was removed and the residual test item was wiped gently from the skin using cotton soaked in potable well water of IIBAT processed by reverse osmosis system. No signs of toxicity, morbidity or mortality was observed in this animal. Therefore, the step -2 of the range finding study was performed with another female rat at the dose level of 1000 mg/kg b.w. Since there was no mortality/morbidity at 1000 mg/kg dose, the step-3 experiment of the range finding study was performed at the dose level of 2000 mg/kg b.w. The animals of the range finding study were observed for clinical signs of toxicity during the first 30 min and at 1, 2, 4 and 6 hours after the beginning of the exposure and once daily thereafter, for 14 days. Since there was no mortality or morbidity and clinical signs at the 200, 1000 and 2000 mg/kg dose level, the main study was performed with two female animals with the dose level of 2000 mg/kg b.w. to confirm the classification outcome.
In the Main Study, the test item was evenly applied to the fur clipped area of the rats following the same procedure used in the range finding study. The animals were observed for clinical signs of toxicity during the first 30 min and at 1, 2, 4 and 6 hours after the beginning of the exposure and once daily thereafter, for 14 days. The body weights of individual animals were recorded prior to the test item application, weekly and at the termination of the study. All the animals were subjected to gross pathological examination at the end of 14 - day observation period.
Inference: Animals treated with the test item did not exhibit any mortality or clinical signs of toxicity for the entire observation period. No adverse skin reactions at the site of application were observed following the removal of the test item. Rats treated with the test item showed body weight gain during the study. Gross pathology examination of the animals did not reveal any macroscopic lesions. Thus, the acute Dermal LD50 of the Product B in Wistar rat is >2000 mg/kg b.w. Therefore, the test item the Product B falls under the Category 5/Unclassified as per the Globally Harmonized System of classification and labeling of chemicals (GHS).
3. Acute inhalation toxicity in wistar rats
An acute inhalation toxicity study with the Product B, was conducted in Wistar rats. The study was performed using inhalation equipment (Nose-only) fabricated by
CH. Technologies, Inc., USA. Restraint tubes (along with animal) were attached to the ports of the chamber. One of the ports was assigned for collection of test atmosphere (aerosols) concentration and for reading the dynamic measurements of the chamber on a regular basis during the exposure. A limit test was performed with 6 Wistar rats (3/sex) at the maximum attainable concentration (1.34 mg/L) of the Product B. The animals were exposed to 1.34 mg/L of the Product B (mean actual chamber concentration) continuously for 4 hours through nose-only inhalation. Animals were observed for clinical signs of toxicity, injury, morbidity and mortality for 14 days. Body weights of individual animals were recorded on day 0, 1, 3, 7 and 14 of the experimental period. At the end of the 14-day observation period, the animals were sacrificed by intraperitoneal injection of Thiopentone I.P. (ThiosolTM sodium) and subjected to gross pathology examination. Exposure atmosphere characterization and mortality data of the Limit test are summarized below in table 10:
Table 10:
Group Exposure Concentration
(mg/L) MMAD
(µm) GSD No of Animals Mortality %
Mortality (M + F)
Male Female Male Female
G1 1.34 3.37 2.75 3 3 0 0 0

Inference: No mortality was observed. Clinical signs of lethargy was observed in all animals on day 0 post exposure (one hour). Additionally, during exposure, the fur color of animals (around the head) changed to pale black due to the colour of test item and not due to toxicity of the test item. The fur color of all the animals changed to normal on day 1. All the animals recovered from clinical signs of toxicity and were found to be apparently normal from day 1 till day 14 of the observation period. When compared with day 0, slight body weight reduction was observed in all animals on day 1. A gradual body weight increase was observed in all animals on day 3 onwards. Gross pathological examination did not reveal any macroscopic lesions in both the sexes. Thus, the acute inhalation LC50 of the Product B was considered to be >1.34 mg/L in Wistar rats. The Product B is thus classified as Category 4 as per the Globally Harmonized System of classification of chemicals
4. Acute dermal irritation/corrosion study in new Zealand white rabbits
An acute dermal irritation /corrosion study of the Product B, was conducted in New Zealand White rabbits. An initial test was performed in 1 animal using a single dermal patch. A weight of 0.5 g of the powdered test item was moistened with distilled water (250µl) and evenly applied to the fur clipped area (6 cm2) of left side skin of the rabbit. The clipped right side of the animal remained untreated and served as control site. The treated and untreated sites were covered with porous gauze dressing material, which was held in place with non-irritating tape for 4h. Thereafter the dressing was removed and the application site was cleaned with potable well water (IIBAT) processed by reverse osmosis system. Following test item application, the animal was observed once daily for morbidity and mortality. Skin reactions at the application site were scored immediately after the removal of patch (for initial test alone), at 60 min and then at 24, 48 and 72 h after patch removal following the Draize method. Untreated control site was also scored for comparative purpose. Body weights of animal was recorded prior to the test item application and at the termination of the study. As the results of the initial test did not reveal any irritation, a confirmatory test using 2 additional animals was carried out following the same procedure to confirm the non-irritant nature of the test item.
Inference: No morbidity/ mortality observed in the animals. All animals were found to be apparently normal from the day of application to the day of termination of the experiment. Test item related skin reactions were not noticed at the application site in any of the treated rabbits. The treated site of all the 3 animals was comparable to the untreated control site. Mean irritation scores (24, 48 and 72 h) of all 3 animals were 0 for erythema, eschar and oedema. No treatment-related adverse effects were noticed in body weight. No irritation potential of the test item was noticed and the mean irritation scores of the animals were not falling under any of the categories of the GHS classification and labeling of chemicals. Therefore, the test item the Product B was found ‘Non- irritant’ to the skin of New Zealand White rabbits and ‘Not classified’ under the GHS classification.
5. In vivo eye irritation in new Zealand white rabbits
An acute eye irritation / corrosion study with the Product B was conducted in New Zealand White rabbits. In the initial test, 66.7 mg (equivalent of 0.1 mL) of the finely powdered test item was applied into the conjunctival sac of the left eye of 1 rabbit. The right eye of the rabbit served as the control. The ocular irritation potential was evaluated and graded at 1, 24, 48 and 72 hours after the application. The animal did not exhibit any ocular lesion following application of the test item. To confirm the findings of the initial test, the confirmatory test was conducted with 2 additional animals using the same procedure and ocular lesions were evaluated and graded up to 72 hours. The animals were also observed for clinical signs of toxicity, morbidity and mortality. The individual body weights of the animals were recorded prior to treatment and at the termination of the experiment.
Inference: No mortality occurred in this study. All animals were found apparently normal from the day of test item application to the last day of observation. No treatment related changes were noticed in the cornea, iris or conjunctiva of the rabbits. The mean scores (24, 48 and 72 h) of all 3 animals was ‘zero’. A normal gain in the body weight was observed in initial and confirmatory test animals. No test item related ocular lesions were observed at 1, 24, 48 and 72 hours and the mean irritation scores of the animals did not fall under any of the categories of the GHS classification and labeling of chemicals. Therefore, the test item the Product B was found ‘Non-irritant’ to the eye of New Zealand White Rabbits and ‘Not classified’ under the GHS classification.
6. Acute oral toxicity study in chicken
Acute oral toxicity of the Product B was tested in Chicken. Known weight of the test item was mixed with distilled water, thereafter administered orally to 5 chicken, at the dose level of 2000 mg/kg body weight. 5 control chicken, were similarly treated but with distilled water alone. All birds were observed individually for toxicity signs and mortality thrice on day 0 and thereafter once daily for 14 days. Body weight of each bird was recorded just prior to administration of dose (0 day) and on days 3, 7 and 14 after dosing. Feed consumption and water consumption were measured daily. At the end of the test, all birds were humanely euthanized by carbon dioxide asphyxiation and gross pathology was carried out. Gross pathology conducted on birds dosed with 2000 mg/kg b.w. did not reveal any treatment related lesions compared with control birds.
Inference: No mortality and clinical signs of toxicity were observed in birds treated with 2000 mg/kg b.w. of the Product B. Similarly, no mortality and clinical signs of toxicity were observed in control birds for the entire experimental period.
Compared to control birds, there was no significant change observed in the body weight of birds treated with 2000 mg/kg b.w. on day 3, 7 and day 14. There was no significant change observed in the consumption of feed of the birds treated with the Product B from day 1 to day 14 when compared with control birds. There was no significant change observed in the consumption of water in birds treated with the Product B from day 1 to day 3, day 5 to day 10 and day 12 to day 14 when compared with control birds. On day 4 and day 11, there was a significant change observed in the consumption of water in birds treated with the Product B when compared with control birds. Based upon the above Limit Dose Test, the LD50 of the Product B for chicken, was considered as greater than 2000 mg/kg b.w.
7. Acute toxicity study in freshwater fish (poecilia reticulata)
A study was conducted to evaluate the acute toxicity of the Product B to fresh water fish, Poecilia reticulata. The semi-static bioassay procedure was followed as per as per OECD test guidelines to determine 24, 48, 72 and 96 hours LC50. In a range finding experiment, groups of 7 fish each in 2 replicates were exposed to control and test item concentrations of 0.5, 1, 10, 50 and 100 mg/L of the Product B, respectively for 96 hours. The fish were observed for mortality and abnormal behaviour twice on the day (2 ± 0.5 hour and 5 ± 1 hour) of exposure, thereafter, at the end of 24, 30, 48, 54, 72, 78 and 96 hours throughout the experimental period. The results showed that no mortality and toxicity signs (abnormal behaviour) were occurred in control and all test item concentrations at the end of 96 hours.
Based on the results of range finding experiment, a limit test was conducted with control and test item concentration of 100 mg/L in 2 replicates at 7 fish/ replicate for 96 hours. The fish were observed for mortality and abnormal behaviour twice on the day of exposure, 2 ± 0.5 hour and 5 ± 1 hour thereafter, at the end of 24, 30, 48, 54, 72, 78 and 96 hours throughout the experimental period.
Inference: No mortality and toxicity sign (abnormal behaviour) were observed in the control and 100 mg/L concentration, throughout the observation period of 96 hours. During the experiment (Limit test), pH ranged from 7.46 to 7.96. Dissolved oxygen ranged from 87.6 to 99.8% and temperature ranged from 22.7 to 23.1°C in control and 100 mg/L. Based on the above findings, the 24, 48, 72 and 96 hours LC50 of the Product B to freshwater fish, Poecilia reticulata was greater than 100 mg/L.
8. Acute oral toxicity to honeybees (apis cerana indica)
For range finding experiment, nominal doses viz., 10, 25, 50, 75 and 100 µg/bee was administered to bees orally. Actual doses were observed to be 8.72, 22.68, 43.56, 66.73 and 84.66 µg/bee based on actual consumption. Based on the results of the range finding experiment, nominal doses viz., 7.5, 15, 30, 60 and 120 µg/bee with geometric factor of 2.0 was administered to bees orally for the definitive experiment. The actual dose was observed to 6.60, 13.65, 26.47, 54.95 and 102.42 µg/bee based on actual consumption. In parallel to the test item, in the definitive experiment, dimethoate technical (reference item) was also evaluated at the doses viz., 0.045, 0.068, 0.101 and 0.152 µg a.i./bee along with a control (acetone + 50% w/v sucrose solution mixture). Bees in the test item treatments were offered the test solutions prepared in 50% w/v sucrose solution. Bees in the reference item treatments were offered the test solutions prepared in acetone + 50% w/v sucrose solution. The test solutions and controls were offered to the bees for 6 hours with dosing volume of 200 µL/Group. Assessments of mortality and abnormal behavioural effects were carried out at 4, 24 and 48 hours of dosing.
Results:
The test item, the Product B recorded 0% mortality in the dose of 7.5 µg/bee (6.60 µg/bee based on the actual consumption) and 16.67% mortality at maximum dose of 120 µg/bee (102.42 µg/bee based on the actual consumption) at 48 hours of dosing. Control group (50% w/v sucrose solution) recorded 0% mortality at the assessment at 48 hours of dosing. No sub lethal (abnormal behavioural) effects were observed at any point of observation after dosing for test item.
Reference item recorded 80.0% mortality at the highest tested nominal doses of 0.152 µg a.i./bee (0.131 µg a.i./bee based on actual consumption) in the reference tem treatment group at 48 hours of dosing. The lowest dose 0.045 µg a.i./bee (0.041 µg a.i./bee based on actual consumption) recorded 6.67% mortality at 48 hours after dosing. The control group (50% w/v sucrose solution + acetone mixture) recorded 0% mortality upto 48 hours after dosing. Observations of sublethal (abnormal behavioural) effects included, moribund at 4 hours after dosing in the reference item. No sub lethal effects were observed at 24 and 48 hours after dosing in the reference item. The 48-hour Oral LD50 value for the Product B was determined to be > 120 µg /bee (equivalent to 102.42 µg/bee), based on actual consumption. The LD50 of the reference item was 0.107 (95% confidence limits: 0.095 - 0.129) µg dimethoate/bee (based on actual consumption) at 24 hours of dosing. The test met the validity criteria, as mortality in the controls did not exceed 10% (No mortality recorded in the control groups upto 48 hours after dosing).
Conclusion
The effects of the Product B were assessed in oral honey bee toxicity test conducted in the laboratory. The 48h Oral LD50 value for the Product B was determined to be 120 µg /bee (equivalent to 102.42 µg/bee), based on actual consumption.
9. Acute contact toxicty to honeybees (apis cerana indica)
Range finding experiment was conducted with the doses viz., 10, 25, 50, 75 and 100 µg/bee. Based on the results of the range finding experiment, doses viz., 6.25, 12.5, 25, 50 and 100 µg/bee with geometric factor of 2.0 was assessed against honeybees for definitive experiment. In parallel to the test item, in the definitive experiment, dimethoate technical (reference item) was also evaluated at the doses viz., 0.045, 0.068, 0.101 and 0.152 µg a.i./bee along with a control (acetone). In the present study, bees were treated with test item the Product B mixed with deionised water dosed by topical application on the dorsal side of thorax of each bee with dosing volume of 1 µL. For reference item, bees were treated with dimethoate technical dissolved in acetone and dosed by topical application on the dorsal side of thorax of each bee. Assessments of mortality and abnormal behavioural effects were carried out at 4, 24 and 48 hours of dosing.
Results:
The test item, the Product B recorded 0% mortality upto the dose of 12.5 µg/bee and 13.33% mortality at maximum dose of 100 µg/bee at 48 hours of dosing. Control groups (deionised water, deionised water + Triton X-100 and acetone) recorded 0% mortality at the assessment at 48 hours of dosing. No sub lethal (abnormal behavioural) effects were observed at any point of observation after dosing for test item. Reference item recorded the mortality of 83.33% at the highest tested nominal doses of 0.152 µg a.i./bee and the lowest dose 0.045 µg a.i./bee recorded 6.67% mortality at 48 hours after dosing. Observations of sublethal (abnormal behavioural) effects included, moribund (0.152 µg a.i./bee) at 4 hours after dosing in the reference item. No sub lethal effects were observed at 24 and 48 hours after dosing in the reference item. The 48-hour Contact LD50 value for the Product B was determined to be > 100 µg/bee. The LD50 of the reference item was 0.127 (95% Confidence limits: 0.112 - 0.152) µg dimethoate/bee at 24 hours of dosing. The test met the validity criteria, as mortality in the controls did not exceed 10% (All the controls recorded 0% mortality upto 48 hours after dosing).

Conclusion
The effects of the Product B were assessed in contact honeybee toxicity test conducted in the laboratory. The 48h contact LD50 value for the Product B was determined to be > 100 µg/bee.

10. Acute toxicity to earthworm (eisenia fetida):
The acute toxicity of the Product B to the earthworm, Eisenia fetida (Savigny 1826), was estimated in a 14-day soil exposure laboratory study conducted as per OECD test guideline 207 as mentioned above. A non-replicated range finding test was conducted prior to definitive test with viz., 10, 33, 100, 333, 667 and 1000 mg of the Product B/kg dry soil. No mortality was observed in any of the tested concentrations in the range finding experiment. Based on the results of range finding test, 4 replicates of 10 clitellated adult earthworms were exposed to nominal concentrations of 1000 mg of the Product B/kg dry soil. An untreated control (deionised water) was also replicated 4 times, with 10 earthworms in each replicate (Limit Test). Earthworms were assessed for mortality after 7 and 14 days of exposure, behavioral abnormalities on 0, 7 and 14 days of exposure and Earthworm body weights were assessed on day 0 and day 14.
Table 11 Summary of Results
Treatment Group Control
(Deionised water) 1000 mg/kg of the Product B
Mortality after
14 days [%] 0.00 0.00
Biomass change after
14 days [%]1# - 8.93 - 7.80n.s
14 day LC50 Greater than 1000 mg/kg dry soil
NOEC related to mortality 1000 mg/kg dry soil
NOEC related to biomass 1000 mg/kg dry soil
n.s = Not significantly different compared to the control; 1Student’s T-test (p = 0.05); # Minus symbol indicates reduction in body weight
Conclusion
It was concluded that 14-day LC50 of the Product B was determined to be greater than 1000 mg/kg dry soil. The 14-day No-Observed-Effect Concentration (NOEC) with respect to mortality and biomass was determined to be 1000 mg Product B/kg dry soil. The 14-day LC50 of the reference item (2-Chloroacetamide) in the recent experiment (June, 2021) was 31.82 mg/kg dry soil.
Table 12: Toxicological Profile of the Product B as per experiment no.5 of the present disclosure
Sr. no. Study Title Toxicity Results
Acute Toxicity
1 Acute Oral Rat LD50 LD50 >2000 – 5000 mg/kg b.wt
2 Acute Dermal Rat LD50 LD50 >2000 mg/kg bw
3 Acute Inhalation LC50 4 hr LC50>1.34 mg/L air
4 Primary Skin Irritation Rabbit Non-irritant to skin
5 Mucous Membrane Irritation Rabbit Non-irritant to eyes
Ecotoxicity
6 Acute toxicity to Bird LD50 >2000 mg/kg
7 Acute toxicity to fresh water fish, carp LC50 96 hrs. LC50>100 mg/l
8 Acute contact / oral toxicity to honeybee, Apis cerana indica LD50 (Oral) (48h) >120 µg a.i/bee
LD50 (Contact) >100 µg a.i/bee
9 Acute toxicity to Earthworms LC 50 LC50> 1000 mg/kg
The above-mentioned results of field evaluations of the biostimulant composition as well as their toxicological profiles provided the following inferences and prove that the biostimulant composition of the present disclosure are highly effective to be used either as liquid or as solid formulations on wide range of agricultural crops:
1. The biostimulant composition provides better crop yield (average yield increase by 10 % to 25 %) of various crops under natural field conditions as compared to the control.
2. The quality of fruits and vegetables (size and weight of fruits/vegetables), cereal grains (grain characteristics, number of tillers, plant height, crop growth rate, and the like) and other plant commodities was observed to be enhanced in various treatments of the biostimulant composition.
3. Biostimulant composition gives multiple nutrients to plants by way of different types of biostimulants (active ingredients) being used in composition, thereby, enhancing overall plant health.
4. The various ingredients of the Biostimulant composition have been used in a particular ratio, which proves that the Biostimulant composition of the present disclosure is better in all respects, catering different needs of crops at different time intervals during crop cycle.
5. Soil application as well as foliar application of the biostimulant composition are the two unique types of methods, which are very much practical, providing convenience to farmers as per their crop needs.
6. The biostimulant composition have been found to be safe to mammals, environment, non-target organisms, aquatic organisms, and the like, (if ingested, contacted to skin or inhaled) as evident from available toxicological data.
TECHNICAL ADVANCEMENTS
The present disclosure described herein above has several technical advantages including, but not limited to, the realization of
(a) a biostimulant composition that;
• provides better crop yield (average yield increase by 10 % to 25 %) of various crops under natural field conditions as compared to the control;
• enhances the quality of fruits and vegetables (increases size and weight of fruits/vegetables), cereal grains (grain characteristics, number of tillers, plant height, crop growth rate, and the like) and other plant commodities; and
• is safe for mammals, environment, non-target organisms, aquatic organisms and the like (if ingested, contacted to skin or inhaled); and
(b) a process for the preparation of a biostimulant composition that;
• is simple, economical and environment-friendly.
Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired object or results.
Any discussion of documents, acts, materials, devices, articles or the like that has been included in this specification is solely for the purpose of providing a context for the disclosure. It is not to be taken as an admission that any or all of these matters form a part of the prior art base or were common general knowledge in the field relevant to the disclosure as it existed anywhere before the priority date of this application.
The numerical values mentioned for the various physical parameters, dimensions or quantities are only approximations and it is envisaged that the values ten percent higher/lower than the numerical values assigned to the parameters, dimensions or quantities fall within the scope of the disclosure, unless there is a statement in the specification specific to the contrary.
While considerable emphasis has been placed herein on the components and component parts of the preferred embodiments, it will be appreciated that many embodiments can be made and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. These and other changes in the preferred embodiment as well as other embodiments of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation. ,CLAIMS:WE CLAIM:
1. A biostimulant composition comprising:
(i) 0.1 mass% to 40 mass% of humic acid;
(ii) 0.1 mass% to 30 mass% of seaweed extract;
(iii) 0.1 mass% to 15 mass% of at least one amino acid;
(iv) 0.02 mass% to 20 mass% of at least one vitamin;
(v) 0.01 mass% to 1 mass% of a microbial extract;
(vi) 5 mass% to 10 mass% of at least one protein hydrolysate;
(vii) 0.01 mass% to 5 mass% of at least one additive;
(viii) 0 mass% to 1 mass% of water; and
(ix) 96 mass% to 98 mass% of at least one inert material,
wherein the mass% of all the components are with respect to the total mass of the biostimulant composition.
2. The biostimulant composition as claimed in claim 1, wherein a ratio of the humic acid to the seaweed extract is in the range of 1:1 to 2:1.
3. The biostimulant composition as claimed in claim 1, wherein a ratio of at least one amino acid to at least one vitamin is in the range of 0.1:1 to 1:1.
4. The biostimulant composition as claimed in claim 1, wherein the seaweed extract is Durvillea Potatorum.
5. The biostimulant composition as claimed in claim 4, wherein the seaweed extract comprises of an alginic acid, micro-nutrients, minerals and complex organic compounds.
6. The biostimulant composition as claimed in claim 1, wherein said amino acid is at least one selected from the group consisting of proline, alanine, tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, ornithine, arginine, histidine, penicillamine, valine, leucine, isoleucine, and phenylalanine.

7. The biostimulant composition as claimed in claim 1, wherein said vitamin is at least one selected from the group consisting of ascorbic acid, thiamin mononitrate and tocopherol.
8. The biostimulant composition as claimed in claim 1, wherein said microbial extract is Bacillus extract.
9. The biostimulant composition as claimed in claim 1, wherein said Bacillus extract is Bacillus velezensis.
10. The biostimulant composition as claimed in claim 1, wherein said protein hydrolysate is a hydrolyzed protein.
11. The biostimulant composition as claimed in claim 10, wherein said hydrolyzed protein is selected from the group consisting of hydrolyzed soybean protein, hydrolyzed black bean protein, hydrolyzed chickpea protein, hydrolyzed green pea protein and hydrolyzed fava bean protein.
12. The biostimulant composition as claimed in claim 1, wherein said additive is at least one selected from the group consisting of myo-inositol and calcium gluconate.
13. The biostimulant composition as claimed in claim 1, wherein said inert material is at least one selected from the group consisting of bentonite, kaolin, montmorillonite, hectorite, saponite, attapulgite, and sepiolite.
14. The biostimulant composition as claimed in claim 1, wherein said composition is in solid form.
15. The biostimulant composition as claimed in claim 14, wherein said solid form is selected from the group consisting of powder, granules, water dispersible granules and wettable powder.
16. The biostimulant composition as claimed in claim 15, wherein said powder has a particle size in the range of 400 micron to 1000 micron.
17. The biostimulant composition as claimed in claim 1 is used to enhance a yield of crop.
Dated this 25th day of April, 2023

______________________________
MOHAN RAJKUMAR DEWAN, IN/PA – 25
of R.K.DEWAN & CO.
Authorized Agent of Applicant