Bactivec
Descripción
Product Description. Bactivec
Active ingredient:
Name of the Active ingredient:
Bacillus thuringiensis var. israelensis serotype H – 14.
Taxonomy:
Kingdom Prokaryotes
Order Eubacteriales
Family Bacillaceae
Genus Bacillus
Origin:
Isolated by Leonard Goldberg and Joel Margalit in 1977 in the Negev desert, Israel.
Characteristics:
Long gram-positive bacillus, forming ellipsoidal spores. Facultative anaerobe, somewhat restricted in the sugar-catabolizing range. During sporulation it forms protein crystals (cuboidal, triangular and diamond-shaped) known as endotoxic crystals, highly specific of mosquito larvae of the genera Culex, Anopheles, Aedes, Mansonia, Psorophora and of Simulids.
Way of action:
Mosquito larvae ingest the spores and the protein crystals. The protein crystals dissolve in an alkaline medium with proteolysis. Toxic fragments are liberated and recognized by specific receptors localized in the epithelial cells of the intestine. This process leads to pore formation in the membranes of the distal portion of the microcapillaries of the cecum mucosa and of the middle part of the intestine. The concomitant changes of the cytoplasm organelles include the disintegration of the endoplasmic reticulum and mitochondrial tumefaction and dilatation of the peri-nuclear space. As a consequence of all these processes, there is hypertrophy, with rupture of the intestinal cells in the middle intestine and in the cecum that leads to ionic unbalance, toxemia and bacteremia that have as a result the death of the larvae.
Composition of the product:
The product commercially known as BACTIVEC contains spores and endotoxic crystals of Bacillus thuringiensis var. israelensis serotype H-14.
Composition (g/L) (%)
Spores and endotoxic crystals of Bacillus thuringiensis var. Israelensis serotype H-14
6
0,6
Inert ingredient
993,9
99,39
Preserver
0,1
0,01
Type of product:
Bioinsecticide.
Class of product:
Slightly toxic, Class IV.
Type of formulation:
Aqueous suspension.
Diseases it controls:
Filariasis, Dengue, Malaria, Yellow Fever, Encephalitis, and Onchocerciasis.
Application:
Application sites:
Mosquito reservoirs with different ecologic characteristics. Domestic water deposits, ponds, dams, drinking troughs, oxidation ponds, ditches, rivers, etc.
Irrigated rice fields:
It is applied by direct aspersion on the surface of the reservoir or aerially, depending on the area of the reservoir.
Application range, dose and volume:
Depending on the area to be treated and on the amount of vegetation present, doses vary between (2 – 5 mL/ m2 ). For black flies, the dose is 25 ppm per minute.
Frequency and application time:
Depending on the ecologic characteristics of the reservoir, on the species present and on their density, the time varies between 15 and 60 days.
Application methods and equipment:
• Terrestrial applications: direct aspersion on the surface of the reservoir using to that effect hand, motor-knapsack and other devices.
• Aerial applications: the recommended dose is to 20 -50 liters of the product per hectare, from an airplane.
AREA TO BE TREATED
(Small scale) AREA TO BE TREATED
(Medium scale) AREA TO BE TREATED
(Big scale >1 ha)
By hand
By motor-knapsack device
By hand-sprayers
By hand
By motor-knapsack device
By hand-sprayers
From the air, using and airplane or helicopter
Protection measures:
This biolarvicide bears no operational risks. Its active ingredient and the concentration of the raw materials it contains have no toxic effects on humans or on other animal species. The product is innocuous and highly specific for mosquito larvae.
Precautions during use and handling:
- Keep the product away from food.
- Avoid direct contact with the eyes.
- Use gloves while applying by hand.
- Wash your hands with soap and water after manipulation.
Precautions for storage.
- Store the product in its original container, tightly closed and labeled, in a well-aired place, not above 40°C and protected from sunlight.
Precautions for final disposal of the biolarvicide and its containers
• Dispose of the containers according to local government regulations.
Host range, susceptible species, effect on species most closely related to the target pests:
Hosts: larvae of the genera Aedes, Culex, Anopheles, Psorophora, Mansonia, Uranotaenia, and Simulids.
Highly pathogen for mosquito larvae.
Infective dose: LC50 = 0.0125 .
Habitat: Larvae, it is not found in soil.
Susceptible species:
Aedes aegypti, Aedes albopictus, Aedes taeniorhynchus, Anopheles albimanus, Anopheles darlingii, Anopheles gambiae, Anopheles Nuñeztovari, Anopheles pseudopunctipennis, Culex quinquefasciatus, Culex nigripalpus, Culex coronator, Culex erraticus, Psorophora spp, Mansonia spp, Uranotaenia sapphirina, Simulium quadrivitatus.
Physiology of Bacillus thuringiensis H - 14:
During its growth, this bacillus has three distinct phases. The vegetative phase (growth and division of the microorganism), the sporulation phase (in which there are changes in the cytoplasm, endonuclear proteolysis and rearrangements within the cell), and the last sporulation phase in which the protein crystals with larvicide activity are synthesized.
Diagram:
Vegetative phase pre-spore phase spore protein crystals
Genetic characteristics of Bacillus thuringiensis:
The parent strain of Bti, ONR 60A has nine plasmids that have been extracted by the Eckhardt methods (1978). The molecular weights of these plasmids are 3.6 Md, 4.2 Md, 4.8 Md, 9.8 Md, 10.2 Md, 65 Md, 72 Md, 105 Md and 130 Md. In spite of the confusion about which plasmid or which genes codify the protein crystal, it has been demonstrated that the 72 Md-plasmid is the one to which toxicity is associated. This was proved by Clark and Dean (1983), Kamdar et al. (1983), Ward and Ellar (1983), González and Carlton (1984) and the non published results of Clark, Workman and Dean show that all protein crystals are codified by the 72-megadalton plasmid.
Parameters:
• Optimal growth temperature: 28 - 30°C .
• pH: 7.2 - 7.4 .
• Sunlight:
A long exposure to sunlight may lower the stability and effectiveness of the bacillus on account of the presence of ultraviolet radiation.
• Desiccation:
Desiccation inactivates the protein crystals.
• Characteristics:
Serotype H - 14 has all the biochemical and morphologic characteristics that are common to the strains of Bacillus thuringiensis. The differences are found in the flagellar antigens of the vegetative cell, the antigen composition of the delta endotoxin, the type of esterases. There is no beta exotoxin, it is stable to heat and it produces protein crystals of all possible forms, contrary to other varieties, which always produce a diamond-shaped crystal. Hans E. Huber et al. published a paper (Parasporal crystal subunits of Bacillus thuringiensis) in which 10 strains of Bacillus thuringiensis were compared on the basis of their molecular weight in a polyacrylamide gel electrophoresis and by ultracentrifugation. Nine strains being serotypes H - 1 to H - 9 had the same molecular weight, the exception being Bacillus thuringiensis H - 14, with a dimeric structure crystal.
Relationship to known pathogens of plants, vertebrates, invertebrates or other organisms:
There is no relationship to known pathogens of plants, vertebrates, invertebrates or other organisms. This has been demonstrated by numerous studies of toxicity and innocuousness with the strain and with the product. Bacillus thuringiensis has been submitted to the most rigorous tests to ascertain its influence on man and on other animals (Shadduck, 1980). All special tests have proved that it is entirely safe. Only in West African rivers that supply drinking water, more than 240 tons of Bacillus thuringiensis H - 14 were used in 1982 with no undesirable effects (TDR, 1987).
Preservation method. Quality control of the strain:
The paper: "Comparative study by restriction analysis of the strain Bacillus thuringiensis H - 14", used in the production of Bactivec intends to demonstrate the quality of the methods used for the preservation of the strain.
Summary: The identity of the restriction patterns of the chromosomal DNA of the strain actually used in Cuba for the production of the biolarvicide BACTIVEC and the reference strains of the Pasteur Institute of Paris and of the Pushkin Microbiology Institute of Leningrad was demonstrated. There was a quality control of the conservation methodology used. The patterns were obtained as controls for the use in prospective studies. Five strains were used, two of them from the Pasteur Institute, lyophilized, one in paper and one in an ampoule, two from the Pushkin Institute, one in agar slanted culture and the other one lyophilized. The fifth strain was obtained from the commercial product. After purification of the chromosomal DNA of each strain, it was digested with restriction enzymes EcoR I and HindIII. All the strains showed the same restriction patterns, which proves the similarity between the particular strain and the reference strains, its purity and the quality and safety of the conservation methods used at the Pedro Kourí Institute of Tropical Medicine and LABIOFAM.
Manufacturing Methods and Quality Assurance:
Stages of the Production Process:
• Opening of the lyophilized ampoule.
• Purity control and microscopy.
• Adaptation of the liquid medium.
• Purity control and microscopy.
• Multiplication of the solid medium.
• Purity control and microscopy.
• Erlenmeyer sowing.
• Orbital shaker.
• Purity control and microscopy.
• Culture medium preparation.
• Sowing and fermentation process of the industrial inoculum.
• Purity control and microscopy.
• Culture medium preparation.
• Sowing and industrial fermentation process.
• Purity control and microscopy.
• Addition of the preserver.
• Homogenization.
• Fermented draining.
• Bottling.
• Biological test, purity control and microscopy.
• Refrigeration.
Type of Formulation:
Aqueous suspension.
Identity and purpose of the formulation:
Biological insecticide for the struggle against medically important vectors, highly specific against mosquito larvae and black flies. It is innocuous for other species.
Biological Potency and Product Guarantee:
• Methodology of the biological assay.
• Methodology for the determination of the biological potency according to the Manual of the Pasteur Institute for the determination of the biological potency in formulations of unknown activity .
Unintentional Ingredients:
Limits of contaminants:
Less than 103 Colony Forming Units/mL (CFU/mL).
Storage stability test:
Physical and Chemical Properties:
Physical state, color, odor:
The product is a gray liquid suspension containing spores and crystals.
Smell: characteristic.
Solubility: very well soluble.
Density: 1.0 - 1.05.
Corrosiveness: not corrosive.
Inflammability: not inflammable.
Stability: Unstable in alkaline medium.
Compatibility: Not compatible with highly alkaline products.
Adequate diluents: liquids, water.
Human Health and Safety Testing:
This is an innocuous product highly specific for mosquito larvae. This biolarvicide may be used in agricultural and livestock farms for the control of species of medical and veterinary importance breeding massively in permanent and temporary reservoirs (such as cattle troughs, flooded rice fields, ponds for fish and shrimp breeding and other reservoirs). There are domestic and international experiences on the effectiveness of this product on different types of mosquito reservoirs and also on its safety for the accompanying fauna and flora. Some of the results are show in the following tables, some can be found in the literature mentioned and some in technical reports annexed.
Mortality in Anopheles nuñeztovari larvae exposed to different concentrations of BACTIVEC (Yaneth E. Rojas, Thesis for the Degree of Master of Science, Maracay, June, 1997)
Concentration (ppm) Number of Larvae per replica
Mortality
I II III IV %
0.25 25 3 3 2 2 10
0.5 25 8 10 11 9 38
1.0 25 12 15 17 19 63
2.5 25 21 23 20 25 82
5.0 25 24 23 25 24 96
10.0 25 25 25 25 25 100
Control 25 0 0 0 0 0
Laboratory conditions:
Temperature: 25 - 28°C
pH: 6.9 - 7.2
Larvae: III instar
Determination of the Lethal Time 50 and 95 of BACTIVEC on Anopheles nuñeztovari larvae in laboratory conditions (Yaneth E. Rojas, Thesis for the Degree of Master of Science, Maracay, June, 1997).
Concentration
(10 ppm) Larvae/
Replicate MORTALITY
1 h 2 h 6 h 12 h 18 h 24 h
Exp. No. 1 25 0 1 1 15 23 25
Exp. No. 2 25 0 0 2 14 24 25
Exp. No. 3 25 0 0 1 14 24 25
Exp. No. 4 25 0 1 1 15 23 25
Total 100 0 2 5 58 94 100
Control 25 0 0 0 0 0 0
Lethal Time (LT) Hours
LT50 11.02
LT95 18.95
Standard Deviation: 7.201330 ± 0.6869
Chi2 = 3.202.
TOXICOLOGY:
Both the active ingredient and the product BACTIVEC were tested according to Mammalian safety of microbial agents for vector control: a WHO Memorandum, WHO (1981).
Toxicological studies using different inoculation routes prove the innocuousness of the product for mice, rats, guinea pigs and rabbits.
• Subchronic toxicity:
The tests using Balb/c white rats and guinea pigs extended over 90 days. The animals received the product orally with the feed in doses of 20 grams per animal.
There were no clinical symptoms in treated animals. The progeny of pregnant females consuming the product was followed to the second generation.
• Chronic toxicity:
Reproduction Study with at least two generations in a rodent species (rat)
The tests using Balb/c white rats extended over 90 days. The animals were administered the product orally with feed in doses of 20 grams per animal. There were no clinical symptoms in the treated animals. The offspring of pregnant females consuming the product was followed to the second generation.
Safety studies with the Active Ingredient (Montero et al., Compilation of toxicological studies, 1990 – 1992).
Animals Inoculation Dose (PPM) Inoculation Route Total Animals Effect
Oral SC IP
Albino rats (Balb/c) 5 X X X 10 There were no external symptoms of damage
25 X X X 10
100 X X X 10
Control - - - 10
White Winstar Mice 5 X X X 10 There were no external symptoms of damage
25 X X X 10
100 X X X 10
Control - - - 10
Guinea Pigs 5 X X X 10 There were no external symptoms of damage
25 X X X 10
100 X X X 10
Control - - 10
SC: subcutaneous
IP: intraperitoneal
Safety studies of BACTIVEC:
Safety studies of BACTIVEC (Montero et al., Compilation of toxicological studies, 1990 - 1992)
Animals Inoculation Dose (PPM) Inoculation Route Total Animals Effect
Oral SC IP
Albino rats (Balb/c) 5 X X X 10 There were no external symptoms of damage
25 X X X 10
100 X X X 10
Control - - - 10
White Winstar Mice 5 X X X 10 There were no external symptoms of damage
25 X X X 10
100 X X X 10
Control - - - 10
Guinea Pigs 5 X X X 10 There were no external symptoms of damage
25 X X X 10
100 X X X 10
Control - - - 10
Fishes of the Poecilia, Limia, Gambusia and Rivulus genera 5
25
100 X There were no external symptoms of damage
Results of the inoculation of organs and of Histology for the Active Ingredient and BACIVEC. (Montero et al., Compilation of Toxicological Studies, 1990 - 1992).
Animals Inoculated organs Culture Media Histology 28 days after exposure
Albino rats
White mice
Guinea pigs
Fish
The inoculated organs were Liver, Spleen, and Heart The culture media used were NYPC, Sabouraud, Brower thioglycollate, and Nutrient Agar. None of the studied groups showed an increase of the size of parenchymatous organs or of the heart. There were no pneumatic foci in the lungs.
None of the culture media used in the test showed signs of growth.
Toxicity Studies on target fauna.
TAXONOMY
INVERTEBRATES
Operational doses
Overdoses
Overdoses
AI
EFFECT
Diptera
Tipulidae
Tipula spp
Psychodidae
Psychoda spp
Culicidae
A. aegypti
A.. albopictus
A .taeniorhynchus
An. albimanus
An. darlingii
An. gambiae
An. nuñeztovari
An. pseudopunctipennis.
C. quinquefasciatus
C. nigripalpus
C. coronator
C. erraticus
Psorophora spp
Mansonia spp
Uranotaenia sapphirina
Simulidae
Simulium quadrivitatus
Chironomidae
Chironomus spp.
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N.S.
N.S.
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
AI: Active Ingredient
Toxicity studies in non-target fauna. (Montero et al., Compilation of Studies, 1992, 1994, 1995).
TAXONOMY
INVERTEBRATES
Product Overdose
(100 ppm) Product
operational dose EFFECT
Coelenterata
Hydrozoa
Hydra spp
Turbellaria
Planaridae
Dugesia spp
Mollusca
Tarebia granifera
Pomacea paludosa
Fossaria cubensis
Physa cubensis
Pseudosuccinea columela
Biomphalaria peregrina
Pyrguphorus coronatus
Drepanotrema anatinum
Helisoma duryi
Crustacea
Cladocera
Daphnia magna
Ostracoda
Cypridae spp.
Copepoda
Cyclops vernalis
Mesocyclops spp.
Insecta
Ephemeroptera
Baetidae
Ephemerillidae
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N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
Odonata
Anisoptera
Ashnidae
Anax junius
Orthemis ferruginea
Tramea abdominalis
Libellulidae
Eritrodiplax umbrata
Zygoptera
Coenagrionidae
Ishnura ramburii
Hemiptera
Notonectidae
Notonecta indica
Buenoa platycnemis
Nepidae
Ranatra fabrioli
Belostomatidae
Belostoma minor
Nauconidae
Pelocoris pauji
Coleoptera
Hidrophilidae
Trapisternum proximus
Trapisternum lateralis
Dysticidae
Dineutus longimanus
Hidrophilidae
Diptera
Musca domestica
Lepidoptera
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N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
Study of the toxic irritant effect on the eye mucosa and on the skin of BACTIVEC (Summary of toxicological tests).
OPHTHALMIC IRRITABILITY.
Animals T Doses Cornea Iris Conjunctiva
Observation Period
Conclusions
New Zealand albino rabbits
Males
Weight: 1.8 - 2.0 Kg
22ºC
0.1 mL
x
x
x
1, 24, 48 and 72 hours There was no erythema or abnormal secretions.
Primary irritation index: 0 – 10
There were no toxic lesions.
DERMAL IRRITABILITY
Animals Application area Doses Permanency period upon the skin Observation Period Conclusions
New Zealand albino rabbits
Males
Weight 1.8 – 2.0 Kg
Dorsal area of the rump
0.5 mL
4 hours
1, 24, 48 and 72 hours There were no signs of skin irritation.
Primary irritation index: 0 – 0.4
Legend:
T: Temperature
Reference
Food and feed residue studies.
BACTIVEC is a biolarvicide having a proven permanency of 7 - 60 days. The spores contained in the product cannot germinate and recycle in the environment. The permanency period depends on the ecological characteristics in the reservoirs in which it is applied and on the species present.
Residual effect of BACTIVEC in reservoirs polluted by Culex quinquefasciatus. (Elsa Ramírez, Thesis for the Degree of Master of Science, 1992).
Titer at the beginning
Titer at the end % M. 24h % M.
48 h % M.
4 W % M.
8 W
% M.
12 W % M
16 W
5 X 106
3 X 104
90
100
65
50
49
40
Legend:
M: Mortality
W: Week
Persistency of BACTIVEC in semi-field experiments with larvae of Culex quinquefasciatus. (Victoria Elisa Soto de Sosa, Thesis for the Degree of Master of Science, 1993).
Container No. % Mortality
48 hours 1 W 2 W 3 W 4 W 5 W 6 W
1-2
3-4
5-6
7-8
9-10
Control
100
100
100
100
100
0
100
100
100
100
100
0
100
100
100
100
100
0
100
100
100
100
100
0
100
100
100
100
100
0
100
100
100
100
100
0
84
84
84
84
83
0
Legend:
W: Week
Study of the stability of BACTIVEC in storage conditions. (Elsa Ramírez, Thesis for the Degree of Master of Science, 1992).
Lot No. Storage time (years) T (°C) Color Smell pH Titer
(sp/mL)
X 109 Efficacy
(% in 5 ppm)
1 3 30 There was no change Ch. 4.6 0.6 97.3
2 4 2 Ch. 4.9 2.6 100
3 4 8 Ch. 7.0 7.1 60
4 3 30 Ch. 5.0 1.0 71
5 3 2 Ch. 4.9 4.5 61
6 3 8 Ch. 5.4 2.9 97.3
7 1 2 Ch. 4.8 3.2 88.0
8 1 30 Ch. 4.7 3.7 99.0
T: storage temperature.
No: number.
Ch.: characteristic.
Efficacy and stability of different lots of Bactivec at different concentrations in larvae of Culex quinquefasciatus. (Elsa Ramírez, Thesis for the Degree of Master of Science, 1992.)
No t(years) pH T (°C) Titer
(sp/mL)109 Mortality (%)
103 ppm 102ppm 5 ppm 1 ppm
1 4 4.6 30 0.6 100 100 97.3 22
2 1 4.7 30 3.7 100 100 99.0 22
3 2 4.8 4 3.2 100 100 88.0 30
4 3 4.9 4 1.0 100 100 97.3 23
5 4 4.9 4 2.6 100 100 100 22
6 3 5.0 30 4.5 100 100 71.0 23
7 3 5.4 14 2.9 100 100 61.0 19
8 4 7.0 14 7.1 100 100 60.0 16
Legend:
t: time since elaboration.
There were no significant differences in the storage time or in the different concentrations of BACTIVEC with a were no significant differences in the storage time or in the different concentrations of BACTIVEC with a degree of signification of p
Active ingredient:
Name of the Active ingredient:
Bacillus thuringiensis var. israelensis serotype H – 14.
Taxonomy:
Kingdom Prokaryotes
Order Eubacteriales
Family Bacillaceae
Genus Bacillus
Origin:
Isolated by Leonard Goldberg and Joel Margalit in 1977 in the Negev desert, Israel.
Characteristics:
Long gram-positive bacillus, forming ellipsoidal spores. Facultative anaerobe, somewhat restricted in the sugar-catabolizing range. During sporulation it forms protein crystals (cuboidal, triangular and diamond-shaped) known as endotoxic crystals, highly specific of mosquito larvae of the genera Culex, Anopheles, Aedes, Mansonia, Psorophora and of Simulids.
Way of action:
Mosquito larvae ingest the spores and the protein crystals. The protein crystals dissolve in an alkaline medium with proteolysis. Toxic fragments are liberated and recognized by specific receptors localized in the epithelial cells of the intestine. This process leads to pore formation in the membranes of the distal portion of the microcapillaries of the cecum mucosa and of the middle part of the intestine. The concomitant changes of the cytoplasm organelles include the disintegration of the endoplasmic reticulum and mitochondrial tumefaction and dilatation of the peri-nuclear space. As a consequence of all these processes, there is hypertrophy, with rupture of the intestinal cells in the middle intestine and in the cecum that leads to ionic unbalance, toxemia and bacteremia that have as a result the death of the larvae.
Composition of the product:
The product commercially known as BACTIVEC contains spores and endotoxic crystals of Bacillus thuringiensis var. israelensis serotype H-14.
Composition (g/L) (%)
Spores and endotoxic crystals of Bacillus thuringiensis var. Israelensis serotype H-14
6
0,6
Inert ingredient
993,9
99,39
Preserver
0,1
0,01
Type of product:
Bioinsecticide.
Class of product:
Slightly toxic, Class IV.
Type of formulation:
Aqueous suspension.
Diseases it controls:
Filariasis, Dengue, Malaria, Yellow Fever, Encephalitis, and Onchocerciasis.
Application:
Application sites:
Mosquito reservoirs with different ecologic characteristics. Domestic water deposits, ponds, dams, drinking troughs, oxidation ponds, ditches, rivers, etc.
Irrigated rice fields:
It is applied by direct aspersion on the surface of the reservoir or aerially, depending on the area of the reservoir.
Application range, dose and volume:
Depending on the area to be treated and on the amount of vegetation present, doses vary between (2 – 5 mL/ m2 ). For black flies, the dose is 25 ppm per minute.
Frequency and application time:
Depending on the ecologic characteristics of the reservoir, on the species present and on their density, the time varies between 15 and 60 days.
Application methods and equipment:
• Terrestrial applications: direct aspersion on the surface of the reservoir using to that effect hand, motor-knapsack and other devices.
• Aerial applications: the recommended dose is to 20 -50 liters of the product per hectare, from an airplane.
AREA TO BE TREATED
(Small scale) AREA TO BE TREATED
(Medium scale) AREA TO BE TREATED
(Big scale >1 ha)
By hand
By motor-knapsack device
By hand-sprayers
By hand
By motor-knapsack device
By hand-sprayers
From the air, using and airplane or helicopter
Protection measures:
This biolarvicide bears no operational risks. Its active ingredient and the concentration of the raw materials it contains have no toxic effects on humans or on other animal species. The product is innocuous and highly specific for mosquito larvae.
Precautions during use and handling:
- Keep the product away from food.
- Avoid direct contact with the eyes.
- Use gloves while applying by hand.
- Wash your hands with soap and water after manipulation.
Precautions for storage.
- Store the product in its original container, tightly closed and labeled, in a well-aired place, not above 40°C and protected from sunlight.
Precautions for final disposal of the biolarvicide and its containers
• Dispose of the containers according to local government regulations.
Host range, susceptible species, effect on species most closely related to the target pests:
Hosts: larvae of the genera Aedes, Culex, Anopheles, Psorophora, Mansonia, Uranotaenia, and Simulids.
Highly pathogen for mosquito larvae.
Infective dose: LC50 = 0.0125 .
Habitat: Larvae, it is not found in soil.
Susceptible species:
Aedes aegypti, Aedes albopictus, Aedes taeniorhynchus, Anopheles albimanus, Anopheles darlingii, Anopheles gambiae, Anopheles Nuñeztovari, Anopheles pseudopunctipennis, Culex quinquefasciatus, Culex nigripalpus, Culex coronator, Culex erraticus, Psorophora spp, Mansonia spp, Uranotaenia sapphirina, Simulium quadrivitatus.
Physiology of Bacillus thuringiensis H - 14:
During its growth, this bacillus has three distinct phases. The vegetative phase (growth and division of the microorganism), the sporulation phase (in which there are changes in the cytoplasm, endonuclear proteolysis and rearrangements within the cell), and the last sporulation phase in which the protein crystals with larvicide activity are synthesized.
Diagram:
Vegetative phase pre-spore phase spore protein crystals
Genetic characteristics of Bacillus thuringiensis:
The parent strain of Bti, ONR 60A has nine plasmids that have been extracted by the Eckhardt methods (1978). The molecular weights of these plasmids are 3.6 Md, 4.2 Md, 4.8 Md, 9.8 Md, 10.2 Md, 65 Md, 72 Md, 105 Md and 130 Md. In spite of the confusion about which plasmid or which genes codify the protein crystal, it has been demonstrated that the 72 Md-plasmid is the one to which toxicity is associated. This was proved by Clark and Dean (1983), Kamdar et al. (1983), Ward and Ellar (1983), González and Carlton (1984) and the non published results of Clark, Workman and Dean show that all protein crystals are codified by the 72-megadalton plasmid.
Parameters:
• Optimal growth temperature: 28 - 30°C .
• pH: 7.2 - 7.4 .
• Sunlight:
A long exposure to sunlight may lower the stability and effectiveness of the bacillus on account of the presence of ultraviolet radiation.
• Desiccation:
Desiccation inactivates the protein crystals.
• Characteristics:
Serotype H - 14 has all the biochemical and morphologic characteristics that are common to the strains of Bacillus thuringiensis. The differences are found in the flagellar antigens of the vegetative cell, the antigen composition of the delta endotoxin, the type of esterases. There is no beta exotoxin, it is stable to heat and it produces protein crystals of all possible forms, contrary to other varieties, which always produce a diamond-shaped crystal. Hans E. Huber et al. published a paper (Parasporal crystal subunits of Bacillus thuringiensis) in which 10 strains of Bacillus thuringiensis were compared on the basis of their molecular weight in a polyacrylamide gel electrophoresis and by ultracentrifugation. Nine strains being serotypes H - 1 to H - 9 had the same molecular weight, the exception being Bacillus thuringiensis H - 14, with a dimeric structure crystal.
Relationship to known pathogens of plants, vertebrates, invertebrates or other organisms:
There is no relationship to known pathogens of plants, vertebrates, invertebrates or other organisms. This has been demonstrated by numerous studies of toxicity and innocuousness with the strain and with the product. Bacillus thuringiensis has been submitted to the most rigorous tests to ascertain its influence on man and on other animals (Shadduck, 1980). All special tests have proved that it is entirely safe. Only in West African rivers that supply drinking water, more than 240 tons of Bacillus thuringiensis H - 14 were used in 1982 with no undesirable effects (TDR, 1987).
Preservation method. Quality control of the strain:
The paper: "Comparative study by restriction analysis of the strain Bacillus thuringiensis H - 14", used in the production of Bactivec intends to demonstrate the quality of the methods used for the preservation of the strain.
Summary: The identity of the restriction patterns of the chromosomal DNA of the strain actually used in Cuba for the production of the biolarvicide BACTIVEC and the reference strains of the Pasteur Institute of Paris and of the Pushkin Microbiology Institute of Leningrad was demonstrated. There was a quality control of the conservation methodology used. The patterns were obtained as controls for the use in prospective studies. Five strains were used, two of them from the Pasteur Institute, lyophilized, one in paper and one in an ampoule, two from the Pushkin Institute, one in agar slanted culture and the other one lyophilized. The fifth strain was obtained from the commercial product. After purification of the chromosomal DNA of each strain, it was digested with restriction enzymes EcoR I and HindIII. All the strains showed the same restriction patterns, which proves the similarity between the particular strain and the reference strains, its purity and the quality and safety of the conservation methods used at the Pedro Kourí Institute of Tropical Medicine and LABIOFAM.
Manufacturing Methods and Quality Assurance:
Stages of the Production Process:
• Opening of the lyophilized ampoule.
• Purity control and microscopy.
• Adaptation of the liquid medium.
• Purity control and microscopy.
• Multiplication of the solid medium.
• Purity control and microscopy.
• Erlenmeyer sowing.
• Orbital shaker.
• Purity control and microscopy.
• Culture medium preparation.
• Sowing and fermentation process of the industrial inoculum.
• Purity control and microscopy.
• Culture medium preparation.
• Sowing and industrial fermentation process.
• Purity control and microscopy.
• Addition of the preserver.
• Homogenization.
• Fermented draining.
• Bottling.
• Biological test, purity control and microscopy.
• Refrigeration.
Type of Formulation:
Aqueous suspension.
Identity and purpose of the formulation:
Biological insecticide for the struggle against medically important vectors, highly specific against mosquito larvae and black flies. It is innocuous for other species.
Biological Potency and Product Guarantee:
• Methodology of the biological assay.
• Methodology for the determination of the biological potency according to the Manual of the Pasteur Institute for the determination of the biological potency in formulations of unknown activity .
Unintentional Ingredients:
Limits of contaminants:
Less than 103 Colony Forming Units/mL (CFU/mL).
Storage stability test:
Physical and Chemical Properties:
Physical state, color, odor:
The product is a gray liquid suspension containing spores and crystals.
Smell: characteristic.
Solubility: very well soluble.
Density: 1.0 - 1.05.
Corrosiveness: not corrosive.
Inflammability: not inflammable.
Stability: Unstable in alkaline medium.
Compatibility: Not compatible with highly alkaline products.
Adequate diluents: liquids, water.
Human Health and Safety Testing:
This is an innocuous product highly specific for mosquito larvae. This biolarvicide may be used in agricultural and livestock farms for the control of species of medical and veterinary importance breeding massively in permanent and temporary reservoirs (such as cattle troughs, flooded rice fields, ponds for fish and shrimp breeding and other reservoirs). There are domestic and international experiences on the effectiveness of this product on different types of mosquito reservoirs and also on its safety for the accompanying fauna and flora. Some of the results are show in the following tables, some can be found in the literature mentioned and some in technical reports annexed.
Mortality in Anopheles nuñeztovari larvae exposed to different concentrations of BACTIVEC (Yaneth E. Rojas, Thesis for the Degree of Master of Science, Maracay, June, 1997)
Concentration (ppm) Number of Larvae per replica
Mortality
I II III IV %
0.25 25 3 3 2 2 10
0.5 25 8 10 11 9 38
1.0 25 12 15 17 19 63
2.5 25 21 23 20 25 82
5.0 25 24 23 25 24 96
10.0 25 25 25 25 25 100
Control 25 0 0 0 0 0
Laboratory conditions:
Temperature: 25 - 28°C
pH: 6.9 - 7.2
Larvae: III instar
Determination of the Lethal Time 50 and 95 of BACTIVEC on Anopheles nuñeztovari larvae in laboratory conditions (Yaneth E. Rojas, Thesis for the Degree of Master of Science, Maracay, June, 1997).
Concentration
(10 ppm) Larvae/
Replicate MORTALITY
1 h 2 h 6 h 12 h 18 h 24 h
Exp. No. 1 25 0 1 1 15 23 25
Exp. No. 2 25 0 0 2 14 24 25
Exp. No. 3 25 0 0 1 14 24 25
Exp. No. 4 25 0 1 1 15 23 25
Total 100 0 2 5 58 94 100
Control 25 0 0 0 0 0 0
Lethal Time (LT) Hours
LT50 11.02
LT95 18.95
Standard Deviation: 7.201330 ± 0.6869
Chi2 = 3.202.
TOXICOLOGY:
Both the active ingredient and the product BACTIVEC were tested according to Mammalian safety of microbial agents for vector control: a WHO Memorandum, WHO (1981).
Toxicological studies using different inoculation routes prove the innocuousness of the product for mice, rats, guinea pigs and rabbits.
• Subchronic toxicity:
The tests using Balb/c white rats and guinea pigs extended over 90 days. The animals received the product orally with the feed in doses of 20 grams per animal.
There were no clinical symptoms in treated animals. The progeny of pregnant females consuming the product was followed to the second generation.
• Chronic toxicity:
Reproduction Study with at least two generations in a rodent species (rat)
The tests using Balb/c white rats extended over 90 days. The animals were administered the product orally with feed in doses of 20 grams per animal. There were no clinical symptoms in the treated animals. The offspring of pregnant females consuming the product was followed to the second generation.
Safety studies with the Active Ingredient (Montero et al., Compilation of toxicological studies, 1990 – 1992).
Animals Inoculation Dose (PPM) Inoculation Route Total Animals Effect
Oral SC IP
Albino rats (Balb/c) 5 X X X 10 There were no external symptoms of damage
25 X X X 10
100 X X X 10
Control - - - 10
White Winstar Mice 5 X X X 10 There were no external symptoms of damage
25 X X X 10
100 X X X 10
Control - - - 10
Guinea Pigs 5 X X X 10 There were no external symptoms of damage
25 X X X 10
100 X X X 10
Control - - 10
SC: subcutaneous
IP: intraperitoneal
Safety studies of BACTIVEC:
Safety studies of BACTIVEC (Montero et al., Compilation of toxicological studies, 1990 - 1992)
Animals Inoculation Dose (PPM) Inoculation Route Total Animals Effect
Oral SC IP
Albino rats (Balb/c) 5 X X X 10 There were no external symptoms of damage
25 X X X 10
100 X X X 10
Control - - - 10
White Winstar Mice 5 X X X 10 There were no external symptoms of damage
25 X X X 10
100 X X X 10
Control - - - 10
Guinea Pigs 5 X X X 10 There were no external symptoms of damage
25 X X X 10
100 X X X 10
Control - - - 10
Fishes of the Poecilia, Limia, Gambusia and Rivulus genera 5
25
100 X There were no external symptoms of damage
Results of the inoculation of organs and of Histology for the Active Ingredient and BACIVEC. (Montero et al., Compilation of Toxicological Studies, 1990 - 1992).
Animals Inoculated organs Culture Media Histology 28 days after exposure
Albino rats
White mice
Guinea pigs
Fish
The inoculated organs were Liver, Spleen, and Heart The culture media used were NYPC, Sabouraud, Brower thioglycollate, and Nutrient Agar. None of the studied groups showed an increase of the size of parenchymatous organs or of the heart. There were no pneumatic foci in the lungs.
None of the culture media used in the test showed signs of growth.
Toxicity Studies on target fauna.
TAXONOMY
INVERTEBRATES
Operational doses
Overdoses
Overdoses
AI
EFFECT
Diptera
Tipulidae
Tipula spp
Psychodidae
Psychoda spp
Culicidae
A. aegypti
A.. albopictus
A .taeniorhynchus
An. albimanus
An. darlingii
An. gambiae
An. nuñeztovari
An. pseudopunctipennis.
C. quinquefasciatus
C. nigripalpus
C. coronator
C. erraticus
Psorophora spp
Mansonia spp
Uranotaenia sapphirina
Simulidae
Simulium quadrivitatus
Chironomidae
Chironomus spp.
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N.S.
N.S.
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
AI: Active Ingredient
Toxicity studies in non-target fauna. (Montero et al., Compilation of Studies, 1992, 1994, 1995).
TAXONOMY
INVERTEBRATES
Product Overdose
(100 ppm) Product
operational dose EFFECT
Coelenterata
Hydrozoa
Hydra spp
Turbellaria
Planaridae
Dugesia spp
Mollusca
Tarebia granifera
Pomacea paludosa
Fossaria cubensis
Physa cubensis
Pseudosuccinea columela
Biomphalaria peregrina
Pyrguphorus coronatus
Drepanotrema anatinum
Helisoma duryi
Crustacea
Cladocera
Daphnia magna
Ostracoda
Cypridae spp.
Copepoda
Cyclops vernalis
Mesocyclops spp.
Insecta
Ephemeroptera
Baetidae
Ephemerillidae
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N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
Odonata
Anisoptera
Ashnidae
Anax junius
Orthemis ferruginea
Tramea abdominalis
Libellulidae
Eritrodiplax umbrata
Zygoptera
Coenagrionidae
Ishnura ramburii
Hemiptera
Notonectidae
Notonecta indica
Buenoa platycnemis
Nepidae
Ranatra fabrioli
Belostomatidae
Belostoma minor
Nauconidae
Pelocoris pauji
Coleoptera
Hidrophilidae
Trapisternum proximus
Trapisternum lateralis
Dysticidae
Dineutus longimanus
Hidrophilidae
Diptera
Musca domestica
Lepidoptera
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+
+
+
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+
+
+
+
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N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
Study of the toxic irritant effect on the eye mucosa and on the skin of BACTIVEC (Summary of toxicological tests).
OPHTHALMIC IRRITABILITY.
Animals T Doses Cornea Iris Conjunctiva
Observation Period
Conclusions
New Zealand albino rabbits
Males
Weight: 1.8 - 2.0 Kg
22ºC
0.1 mL
x
x
x
1, 24, 48 and 72 hours There was no erythema or abnormal secretions.
Primary irritation index: 0 – 10
There were no toxic lesions.
DERMAL IRRITABILITY
Animals Application area Doses Permanency period upon the skin Observation Period Conclusions
New Zealand albino rabbits
Males
Weight 1.8 – 2.0 Kg
Dorsal area of the rump
0.5 mL
4 hours
1, 24, 48 and 72 hours There were no signs of skin irritation.
Primary irritation index: 0 – 0.4
Legend:
T: Temperature
Reference
Food and feed residue studies.
BACTIVEC is a biolarvicide having a proven permanency of 7 - 60 days. The spores contained in the product cannot germinate and recycle in the environment. The permanency period depends on the ecological characteristics in the reservoirs in which it is applied and on the species present.
Residual effect of BACTIVEC in reservoirs polluted by Culex quinquefasciatus. (Elsa Ramírez, Thesis for the Degree of Master of Science, 1992).
Titer at the beginning
Titer at the end % M. 24h % M.
48 h % M.
4 W % M.
8 W
% M.
12 W % M
16 W
5 X 106
3 X 104
90
100
65
50
49
40
Legend:
M: Mortality
W: Week
Persistency of BACTIVEC in semi-field experiments with larvae of Culex quinquefasciatus. (Victoria Elisa Soto de Sosa, Thesis for the Degree of Master of Science, 1993).
Container No. % Mortality
48 hours 1 W 2 W 3 W 4 W 5 W 6 W
1-2
3-4
5-6
7-8
9-10
Control
100
100
100
100
100
0
100
100
100
100
100
0
100
100
100
100
100
0
100
100
100
100
100
0
100
100
100
100
100
0
100
100
100
100
100
0
84
84
84
84
83
0
Legend:
W: Week
Study of the stability of BACTIVEC in storage conditions. (Elsa Ramírez, Thesis for the Degree of Master of Science, 1992).
Lot No. Storage time (years) T (°C) Color Smell pH Titer
(sp/mL)
X 109 Efficacy
(% in 5 ppm)
1 3 30 There was no change Ch. 4.6 0.6 97.3
2 4 2 Ch. 4.9 2.6 100
3 4 8 Ch. 7.0 7.1 60
4 3 30 Ch. 5.0 1.0 71
5 3 2 Ch. 4.9 4.5 61
6 3 8 Ch. 5.4 2.9 97.3
7 1 2 Ch. 4.8 3.2 88.0
8 1 30 Ch. 4.7 3.7 99.0
T: storage temperature.
No: number.
Ch.: characteristic.
Efficacy and stability of different lots of Bactivec at different concentrations in larvae of Culex quinquefasciatus. (Elsa Ramírez, Thesis for the Degree of Master of Science, 1992.)
No t(years) pH T (°C) Titer
(sp/mL)109 Mortality (%)
103 ppm 102ppm 5 ppm 1 ppm
1 4 4.6 30 0.6 100 100 97.3 22
2 1 4.7 30 3.7 100 100 99.0 22
3 2 4.8 4 3.2 100 100 88.0 30
4 3 4.9 4 1.0 100 100 97.3 23
5 4 4.9 4 2.6 100 100 100 22
6 3 5.0 30 4.5 100 100 71.0 23
7 3 5.4 14 2.9 100 100 61.0 19
8 4 7.0 14 7.1 100 100 60.0 16
Legend:
t: time since elaboration.
There were no significant differences in the storage time or in the different concentrations of BACTIVEC with a were no significant differences in the storage time or in the different concentrations of BACTIVEC with a degree of signification of p
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