4-allylveratrole

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

year 1975

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test:
Insect attractants, both natural and synthetic, play a growing role in integrated insect pest contorl programs. For example, when insects of a target species are lure into a trap baited with an appropriate attractant, the catch indicates the presence and the whereabouts of the population, and this information can be used to restrict the application of conrol measures to those areas that require treatment and to times that will provide maximum benefit. Synthetic materials that were discovered empirically have been used in such monitoring and survey traps for some time, but the number of effective compounds is quite limited. Interest is currently focused on the increacing number of insect sex attractant pheromones that have been identified and made available synthetically during the past 8 years. These substances are produced by one sex to attract the opposite sex for mating; they are generally highly specific and they are effective in survey traps at very low concentrations.
Some of the attractants are effective enough to show promise for the direct control of injurious insect species; they might be used for trapping, or the material could be released into the atmosphere to disorient mate-seeking efforts and thus prevent propagation (Beroza and Knipling, 1972). The amounts of material required in such applications are expected to be relatively minute. Effective traps may contain as little as 10 ug, and it is anticipated that applications in the range of 1-20 g/hectare (0.4-8 g/acre) would be used in the air-permeation method. The specificity of attractants (especially of the pheromones) should be of great value in such applications because only the target species would be affected. However we cannot proceed to large scale field test of attractants and one compound that inhibits the action of a sex attractant) that show promise for use in insect control. Although pesticides under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), as amended by the Federal Environmental Pesticide Control Act of 1972 (FEPCA) and toxicological data are needed for registration of compounds for which such use is contemplated. The substances selected for study have been effective in laboratory and field tests against economically important insect pests (Table 1). Three occur naturally as sex attractant pheromones: disparlure and looplure are emitted by the female gypsy moth and cabbage looper moth, respectively, to attract the male moths. Muscalure, produced by female house fly, is attractive only to the male in laboratory tests but attracted both sexes in field tests (Carlson and Beroza, 1973). Hexalure, though not the natural pheromone of the pink bollworm, resembles a pheromone in its action. (Z)-7-Dodecen-1-ol inhibits the action of the cabbage looper pheromone (Tumlinson et al.,1972). and its disseminitaion may prevent the male from locating the female.
The other four test materials (cue-lure, methyl eugenol, PEP:eugenol (7:3) and tri-medlure) do not appear to be related to any natural pheromones but have been found experimentally to be effective and specific attractants. The reported LD50 of methyl eugenol in rats is 1560 mg/kg (Jenner et al., 1964). Eugenol, a component of the Japanese beetle attractant, is the principal ingridient of oil of cloves: it has uses in perfumes and flavorings, and is used in dentistry as an antiseptic, an analgesic, and a component of dental cement. ITS acute oral LD in rats was as 1930 mg/kg (Sober et al., 1950) and as 2680 mg/kg (Jenner et al., 1964; Hagan et al., 1965). Phenethyl propionate, the other component of the Japanese beetle attractant is also used in perfumes and flavorings.
The test materials are described in Table 1; they are colorless liquids that are soluble in the usual organic solvents. They wre procured from commercial sources according to specifications set up by U.S Department of Agriculture scientists; purity of each material was checked in the Organic Chemicals Synthesis Laboratory, AEQI, in Beltsville by gas chromatography. The limited amounts of material available for testing restricted the number of tests that could be made, particularly at higher dose levels.
Young albino rats of the Sprague Dawley strain (150-240g) were used in the tests of acute oral and inhalation toxicity. Albino rabbits of the New Zealand strain (1.9-3.1 kg) were used for the tests of acute dermal toxicity, eye irritation, and primary skin irritation. Healthy fingerlings (averaage lenght, 37-75 mm) of rainbow trout (Salmo gairdnerii) and bluegill sunfish (Lepomis macrochirus) were used in the tests of toxicity to fish (static fish toxicity studies). The rainbow trout were held at 13 C and the blugills at 18 C.

- Short description of test conditions:
For tests of toxicity to fish, bioassay vessels were lined with disposable polyethylene bags and filled with 12,5 liter reconstituted water (30mg CaSO4, 30mg MgSO4, 48mg NaHCO3 and 2mg KCl added per liter of deionized water). Ten fish were placed in each vessel and allowed 24hr to become acclimated. Claculated amounts of each test material, as 1% or 10% (w/v) solutions in acetone, were then added to the bioassay vessels. Each material was tested at five five concentrations selected on the basis of preliminary screening tests to determine the approximate ranges of toxicity. Control groups of fish in untreated water and in water to which acetone only was added were observed concurrently. The fish were observed for 96 hr and all deaths and/or untoward behavioral reactions were recorded. The concentrations of dissolved oxygen was measured in all solutions in which deaths occured to be sure the test water contained sufficient oxygen; dissolved oxygen concentrations above 4mg/liter (4ppm for the warm-water fish (bluegills) or above 5mg/liter for cold water fish (rainbow trout) were considered adequate. The median lethal concentrations (LC50) of the seven test materials were calculated whenever the data permitted (Licthfiel and Wilcoxon, 1949).
To check on suitability as test subjects, fish from each lot were challenged, under the same experimental conditions with a reference pesticide (toxaphene, at 0.010, 0.018, and 0.056 ppm). The observed 96hr LC50 was usually around 0.02 ppm; with one lot of bluegills there were no deaths at 0.018 ppm and no survivors at 0.056 ppm.
- Parameters analysed / observed:
24, 48, 96 hr LC50

GLP compliance:

not specified

Analytical monitoring:

yes

Details on sampling:

not specified

Vehicle:

not specified

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
For tests of toxicity to fish, bioassay vessels were lined with disposable polyethylene bags and filled with 12,5 liter reconstituted water (30mg CaSO4, 30mg MgSO4, 48mg NaHCO3 and 2mg KCl added per liter of deionized water). Ten fish were placed in each vessel and allowed 24hr to become acclimated. Claculated amounts of each test material, as 1% or 10% (w/v) solutions in acetone, were then added to the bioassay vessels.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Source: not specified
- Age at study initiation (mean and range, SD): not specified
- Length at study initiation (length definition, mean, range and SD): 37-75 mm
- Weight at study initiation (mean and range, SD): not specified

ACCLIMATION
- Acclimation period: 24 h
- Acclimation conditions (same as test or not): same as test
- Type and amount of food during acclimation: not specified
- Feeding frequency during acclimation: not specified
- Health during acclimation (any mortality observed): no

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

not specified

Test temperature:

13 C

pH:

not specified

Dissolved oxygen:

not specified

Salinity:

not specified

Conductivity:

not specified

Nominal and measured concentrations:

3.2-10.0 ppm

Details on test conditions:

For tests of toxicity to fish, bioassay vessels were lined with disposable polyethylene bags and filled with 12,5 liter reconstituted water (30mg CaSO4, 30mg MgSO4, 48mg NaHCO3 and 2mg KCl added per liter of deionized water). Ten fish were placed in each vessel and allowed 24hr to become acclimated. Claculated amounts of each test material, as 1% or 10% (w/v) solutions in acetone, were then added to the bioassay vessels. Each material was tested at five five concentrations selected on the basis of preliminary screening tests to determine the approximate ranges of toxicity. Control groups of fish in untreated water and in water to which acetone only was added were observed concurrently. The fish were observed for 96 hr and all deaths and/or untoward behavioral reactions were recorded. The concentrations of dissolved oxygen was measured in all solutions in which deaths occured to be sure the test water contained sufficient oxygen; dissolved oxygen concentrations above 4mg/liter (4ppm for the warm-water fish (bluegills) or above 5mg/liter for cold water fish (rainbow trout) were considered adequate. The median lethal concentrations (LC50) of the seven test materials were calculated whenever the data permitted (Licthfiel and Wilcoxon, 1949).
To check on suitability as test subjects, fish from each lot were challenged, under the same experimental conditions with a reference pesticide (toxaphene, at 0.010, 0.018, and 0.056 ppm). The observed 96hr LC50 was usually around 0.02 ppm; with one lot of bluegills there were no deaths at 0.018 ppm and no survivors at 0.056 ppm.

Reference substance (positive control):

yes

Remarks:

toxaphene

Key result

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 5.6 - < 10 other: ppm

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

6.9 other: ppm

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

6 other: ppm

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Results with reference substance (positive control):

- Results with reference substance valid? yes
- LC50: The observed 96hr LC50 was usually around 0.02 ppm; with one lot of bluegills there were no deaths at 0.018 ppm and no survivors at 0.056 ppm

Sublethal observations / clinical signs:

Chemical	Concentration range (ppm)	LC50 (ppm)
		Rainbow trout			Blugill sunfish
		24 h	48h	96h	24 h	48h	96h
Cue-lure	10-32	ca 21	ca 20	ca 16	18 (16-21)	ca 15	ca 15
Disparlure	0.1-100	>100	>100	>100	>100	>100	>100
Looplure	0.1-100	>100	>100	>100	>100	>100	>100
Looplure inhibitor	1.8-4.4	3.7 (3.4-4.0)	3.7 (3.4-4.0)	3.7 (3.4-4.0)	ca 3.1	2.9 (2.6-3.2)	2.8 (2.5-3.2)
Methyl eugenol	3.2-10.0	>5.6, <10.0	6.9 (5.5-8.6)	6.0 4.9-7.2	ca 8.5	8.1 (7.4-9.0)	8.1 (7.4-9.0)
PEP:eugenol (7:3)	10-32	ca 20	ca 14.5	>10, <13	ca 21	>15, <18	ca 12
Trimedlure	7.8-3.2	11.5 (9.2-14.3)	11.0 (9.0-13.4)	9.6 (8.4-11.0)	14.7 (11.8-18.5)	14.7 (11.8-18.5)	12.1 (10.0-14.3)

Validity criteria fulfilled:

yes

Conclusions:

The measured 96 hour LC50 for rainbow trout was found to be a mean 6.0 ppm with confidence limits ranging 5.5-8.6ppm.

Executive summary:

Insect attractants, both natural and synthetic, play a growing role in integrated insect pest contorl programs. For example, when insects of a target species are lure into a trap baited with an appropriate attractant, the catch indicates the presence and the whereabouts of the population, and this information can be used to restrict the application of conrol measures to those areas that require treatment and to times that will provide maximum benefit. Synthetic materials that were discovered empirically have been used in such monitoring and survey traps for some time, but the number of effective compounds is quite limited. Interest is currently focused on the increacing number of insect sex attractant pheromones that have been identified and made available synthetically during the past 8 years. These substances are produced by one sex to attract the opposite sex for mating; they are generally highly specific and they are effective in survey traps at very low concentrations.

Some of the attractants are effective enough to show promise for the direct control of injurious insect species; they might be used for trapping, or the material could be released into the atmosphere to disorient mate-seeking efforts and thus prevent propagation (Beroza and Knipling, 1972). The amounts of material required in such applications are expected to be relatively minute. Effective traps may contain as little as 10 ug, and it is anticipated that applications in the range of 1-20 g/hectare (0.4-8 g/acre) would be used in the air-permeation method. The specificity of attractants (especially of the pheromones) should be of great value in such applications because only the target species would be affected. However we cannot proceed to large scale field test of attractants and one compound that inhibits the action of a sex attractant) that show promise for use in insect control. Although pesticides under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), as amended by the Federal Environmental Pesticide Control Act of 1972 (FEPCA) and toxicological data are needed for registration of compounds for which such use is contemplated. The substances selected for study have been effective in laboratory and field tests against economically important insect pests (Table 1). Three occur naturally as sex attractant pheromones: disparlure and looplure are emitted by the female gypsy moth and cabbage looper moth, respectively, to attract the male moths. Muscalure, produced by female house fly, is attractive only to the male in laboratory tests but attracted both sexes in field tests (Carlson and Beroza, 1973). Hexalure, though not the natural pheromone of the pink bollworm, resembles a pheromone in its action. (Z)-7-Dodecen-1-ol inhibits the action of the cabbage looper pheromone (Tumlinson et al.,1972). and its disseminitaion may prevent the male from locating the female.

The other four test materials (cue-lure, methyl eugenol, PEP:eugenol (7:3) and tri-medlure) do not appear to be related to any natural pheromones but have been found experimentally to be effective and specific attractants. The reported LD50 of methyl eugenol in rats is 1560 mg/kg (Jenner et al., 1964). Eugenol, a component of the Japanese beetle attractant, is the principal ingridient of oil of cloves: it has uses in perfumes and flavorings, and is used in dentistry as an antiseptic, an analgesic, and a component of dental cement. ITS acute oral LD in rats was as 1930 mg/kg (Sober et al., 1950) and as 2680 mg/kg (Jenner et al., 1964; Hagan et al., 1965). Phenethyl propionate, the other component of the Japanese beetle attractant is also used in perfumes and flavorings.

The test materials are described in Table 1; they are colorless liquids that are soluble in the usual organic solvents. They wre procured from commercial sources according to specifications set up by U.S Department of Agriculture scientists; purity of each material was checked in the Organic Chemicals Synthesis Laboratory, AEQI, in Beltsville by gas chromatography. The limited amounts of material available for testing restricted the number of tests that could be made, particularly at higher dose levels.

Young albino rats of the Sprague Dawley strain (150-240g) were used in the tests of acute oral and inhalation toxicity. Albino rabbits of the New Zealand strain (1.9-3.1 kg) were used for the tests of acute dermal toxicity, eye irritation, and primary skin irritation. Healthy fingerlings (averaage lenght, 37-75 mm) of rainbow trout (Salmo gairdnerii) and bluegill sunfish (Lepomis macrochirus) were used in the tests of toxicity to fish (static fish toxicity studies). The rainbow trout were held at 13 C and the blugills at 18 C.

For tests of toxicity to fish, bioassay vessels were lined with disposable polyethylene bags and filled with 12,5 liter reconstituted water (30mg CaSO4, 30mg MgSO4, 48mg NaHCO3 and 2mg KCl added per liter of deionized water). Ten fish were placed in each vessel and allowed 24hr to become acclimated. Claculated amounts of each test material, as 1% or 10% (w/v) solutions in acetone, were then added to the bioassay vessels. Each material was tested at five five concentrations selected on the basis of preliminary screening tests to determine the approximate ranges of toxicity. Control groups of fish in untreated water and in water to which acetone only was added were observed concurrently. The fish were observed for 96 hr and all deaths and/or untoward behavioral reactions were recorded. The concentrations of dissolved oxygen was measured in all solutions in which deaths occured to be sure the test water contained sufficient oxygen; dissolved oxygen concentrations above 4mg/liter (4ppm for the warm-water fish (bluegills) or above 5mg/liter for cold water fish (rainbow trout) were considered adequate. The median lethal concentrations (LC50) of the seven test materials were calculated whenever the data permitted (Licthfiel and Wilcoxon, 1949).

To check on suitability as test subjects, fish from each lot were challenged, under the same experimental conditions with a reference pesticide (toxaphene, at 0.010, 0.018, and 0.056 ppm). The observed 96hr LC50 was usually around 0.02 ppm; with one lot of bluegills there were no deaths at 0.018 ppm and no survivors at 0.056 ppm.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test:
Insect attractants, both natural and synthetic, play a growing role in integrated insect pest contorl programs. For example, when insects of a target species are lure into a trap baited with an appropriate attractant, the catch indicates the presence and the whereabouts of the population, and this information can be used to restrict the application of conrol measures to those areas that require treatment and to times that will provide maximum benefit. Synthetic materials that were discovered empirically have been used in such monitoring and survey traps for some time, but the number of effective compounds is quite limited. Interest is currently focused on the increacing number of insect sex attractant pheromones that have been identified and made available synthetically during the past 8 years. These substances are produced by one sex to attract the opposite sex for mating; they are generally highly specific and they are effective in survey traps at very low concentrations.
Some of the attractants are effective enough to show promise for the direct control of injurious insect species; they might be used for trapping, or the material could be released into the atmosphere to disorient mate-seeking efforts and thus prevent propagation (Beroza and Knipling, 1972). The amounts of material required in such applications are expected to be relatively minute. Effective traps may contain as little as 10 ug, and it is anticipated that applications in the range of 1-20 g/hectare (0.4-8 g/acre) would be used in the air-permeation method. The specificity of attractants (especially of the pheromones) should be of great value in such applications because only the target species would be affected. However we cannot proceed to large scale field test of attractants and one compound that inhibits the action of a sex attractant) that show promise for use in insect control. Although pesticides under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), as amended by the Federal Environmental Pesticide Control Act of 1972 (FEPCA) and toxicological data are needed for registration of compounds for which such use is contemplated. The substances selected for study have been effective in laboratory and field tests against economically important insect pests (Table 1). Three occur naturally as sex attractant pheromones: disparlure and looplure are emitted by the female gypsy moth and cabbage looper moth, respectively, to attract the male moths. Muscalure, produced by female house fly, is attractive only to the male in laboratory tests but attracted both sexes in field tests (Carlson and Beroza, 1973). Hexalure, though not the natural pheromone of the pink bollworm, resembles a pheromone in its action. (Z)-7-Dodecen-1-ol inhibits the action of the cabbage looper pheromone (Tumlinson et al.,1972). and its disseminitaion may prevent the male from locating the female.
The other four test materials (cue-lure, methyl eugenol, PEP:eugenol (7:3) and tri-medlure) do not appear to be related to any natural pheromones but have been found experimentally to be effective and specific attractants. The reported LD50 of methyl eugenol in rats is 1560 mg/kg (Jenner et al., 1964). Eugenol, a component of the Japanese beetle attractant, is the principal ingridient of oil of cloves: it has uses in perfumes and flavorings, and is used in dentistry as an antiseptic, an analgesic, and a component of dental cement. ITS acute oral LD in rats was as 1930 mg/kg (Sober et al., 1950) and as 2680 mg/kg (Jenner et al., 1964; Hagan et al., 1965). Phenethyl propionate, the other component of the Japanese beetle attractant is also used in perfumes and flavorings.
The test materials are described in Table 1; they are colorless liquids that are soluble in the usual organic solvents. They wre procured from commercial sources according to specifications set up by U.S Department of Agriculture scientists; purity of each material was checked in the Organic Chemicals Synthesis Laboratory, AEQI, in Beltsville by gas chromatography. The limited amounts of material available for testing restricted the number of tests that could be made, particularly at higher dose levels.
Young albino rats of the Sprague Dawley strain (150-240g) were used in the tests of acute oral and inhalation toxicity. Albino rabbits of the New Zealand strain (1.9-3.1 kg) were used for the tests of acute dermal toxicity, eye irritation, and primary skin irritation. Healthy fingerlings (averaage lenght, 37-75 mm) of rainbow trout (Salmo gairdnerii) and bluegill sunfish (Lepomis macrochirus) were used in the tests of toxicity to fish (static fish toxicity studies). The rainbow trout were held at 13 C and the blugills at 18 C.

- Short description of test conditions:
For tests of toxicity to fish, bioassay vessels were lined with disposable polyethylene bags and filled with 12,5 liter reconstituted water (30mg CaSO4, 30mg MgSO4, 48mg NaHCO3 and 2mg KCl added per liter of deionized water). Ten fish were placed in each vessel and allowed 24hr to become acclimated. Claculated amounts of each test material, as 1% or 10% (w/v) solutions in acetone, were then added to the bioassay vessels. Each material was tested at five five concentrations selected on the basis of preliminary screening tests to determine the approximate ranges of toxicity. Control groups of fish in untreated water and in water to which acetone only was added were observed concurrently. The fish were observed for 96 hr and all deaths and/or untoward behavioral reactions were recorded. The concentrations of dissolved oxygen was measured in all solutions in which deaths occured to be sure the test water contained sufficient oxygen; dissolved oxygen concentrations above 4mg/liter (4ppm for the warm-water fish (bluegills) or above 5mg/liter for cold water fish (rainbow trout) were considered adequate. The median lethal concentrations (LC50) of the seven test materials were calculated whenever the data permitted (Licthfiel and Wilcoxon, 1949).
To check on suitability as test subjects, fish from each lot were challenged, under the same experimental conditions with a reference pesticide (toxaphene, at 0.010, 0.018, and 0.056 ppm). The observed 96hr LC50 was usually around 0.02 ppm; with one lot of bluegills there were no deaths at 0.018 ppm and no survivors at 0.056 ppm.
- Parameters analysed / observed:
24, 48, 96 hr LC50

GLP compliance:

not specified

Analytical monitoring:

yes

Details on sampling:

not specified

Vehicle:

not specified

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
For tests of toxicity to fish, bioassay vessels were lined with disposable polyethylene bags and filled with 12,5 liter reconstituted water (30mg CaSO4, 30mg MgSO4, 48mg NaHCO3 and 2mg KCl added per liter of deionized water). Ten fish were placed in each vessel and allowed 24hr to become acclimated. Claculated amounts of each test material, as 1% or 10% (w/v) solutions in acetone, were then added to the bioassay vessels.

Test organisms (species):

Lepomis macrochirus

Details on test organisms:

TEST ORGANISM
- Common name:
bluegill sunfish
- Source: not specified
- Age at study initiation (mean and range, SD):
not specified
- Length at study initiation (length definition, mean, range and SD):
not specified
- Weight at study initiation (mean and range, SD): not specified



ACCLIMATION
- Acclimation period: 24 h
- Acclimation conditions (same as test or not): same as test
- Type and amount of food during acclimation: not specified
- Feeding frequency during acclimation: not specified
- Health during acclimation (any mortality observed): no

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

not specified

Test temperature:

18 C

pH:

not specified

Dissolved oxygen:

not specifed

Salinity:

not specified

Conductivity:

not specified

Nominal and measured concentrations:

3.2-10.0 ppm

Details on test conditions:

For tests of toxicity to fish, bioassay vessels were lined with disposable polyethylene bags and filled with 12,5 liter reconstituted water (30mg CaSO4, 30mg MgSO4, 48mg NaHCO3 and 2mg KCl added per liter of deionized water). Ten fish were placed in each vessel and allowed 24hr to become acclimated. Claculated amounts of each test material, as 1% or 10% (w/v) solutions in acetone, were then added to the bioassay vessels. Each material was tested at five five concentrations selected on the basis of preliminary screening tests to determine the approximate ranges of toxicity. Control groups of fish in untreated water and in water to which acetone only was added were observed concurrently. The fish were observed for 96 hr and all deaths and/or untoward behavioral reactions were recorded. The concentrations of dissolved oxygen was measured in all solutions in which deaths occured to be sure the test water contained sufficient oxygen; dissolved oxygen concentrations above 4mg/liter (4ppm for the warm-water fish (bluegills) or above 5mg/liter for cold water fish (rainbow trout) were considered adequate. The median lethal concentrations (LC50) of the seven test materials were calculated whenever the data permitted (Licthfiel and Wilcoxon, 1949).
To check on suitability as test subjects, fish from each lot were challenged, under the same experimental conditions with a reference pesticide (toxaphene, at 0.010, 0.018, and 0.056 ppm). The observed 96hr LC50 was usually around 0.02 ppm; with one lot of bluegills there were no deaths at 0.018 ppm and no survivors at 0.056 ppm.

Reference substance (positive control):

yes

Remarks:

toxaphene

Key result

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

ca. 8.5 other: ppm

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

8.1 other: ppm

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

8.1 other: ppm

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Results with reference substance (positive control):

- Results with reference substance valid? yes
- LC50: The observed 96hr LC50 was usually around 0.02 ppm; with one lot of bluegills there were no deaths at 0.018 ppm and no survivors at 0.056 ppm

Sublethal observations / clinical signs:

Chemical	Concentration range (ppm)	LC50 (ppm)
		Rainbow trout			Blugill sunfish
		24 h	48h	96h	24 h	48h	96h
Cue-lure	10-32	ca 21	ca 20	ca 16	18 (16-21)	ca 15	ca 15
Disparlure	0.1-100	>100	>100	>100	>100	>100	>100
Looplure	0.1-100	>100	>100	>100	>100	>100	>100
Looplure inhibitor	1.8-4.4	3.7 (3.4-4.0)	3.7 (3.4-4.0)	3.7 (3.4-4.0)	ca 3.1	2.9 (2.6-3.2)	2.8 (2.5-3.2)
Methyl eugenol	3.2-10.0	>5.6, <10.0	6.9 (5.5-8.6)	6.0 4.9-7.2	ca 8.5	8.1 (7.4-9.0)	8.1 (7.4-9.0)
PEP:eugenol (7:3)	10-32	ca 20	ca 14.5	>10, <13	ca 21	>15, <18	ca 12
Trimedlure	7.8-3.2	11.5 (9.2-14.3)	11.0 (9.0-13.4)	9.6 (8.4-11.0)	14.7 (11.8-18.5)	14.7 (11.8-18.5)	12.1 (10.0-14.3)

Validity criteria fulfilled:

yes

Conclusions:

The measured 96 hour LC50 for rainbow trout was found to be a mean 8.1ppm with confidence limits ranging 7.4-9.0ppm.

Executive summary:

Insect attractants, both natural and synthetic, play a growing role in integrated insect pest contorl programs. For example, when insects of a target species are lure into a trap baited with an appropriate attractant, the catch indicates the presence and the whereabouts of the population, and this information can be used to restrict the application of conrol measures to those areas that require treatment and to times that will provide maximum benefit. Synthetic materials that were discovered empirically have been used in such monitoring and survey traps for some time, but the number of effective compounds is quite limited. Interest is currently focused on the increacing number of insect sex attractant pheromones that have been identified and made available synthetically during the past 8 years. These substances are produced by one sex to attract the opposite sex for mating; they are generally highly specific and they are effective in survey traps at very low concentrations.

Some of the attractants are effective enough to show promise for the direct control of injurious insect species; they might be used for trapping, or the material could be released into the atmosphere to disorient mate-seeking efforts and thus prevent propagation (Beroza and Knipling, 1972). The amounts of material required in such applications are expected to be relatively minute. Effective traps may contain as little as 10 ug, and it is anticipated that applications in the range of 1-20 g/hectare (0.4-8 g/acre) would be used in the air-permeation method. The specificity of attractants (especially of the pheromones) should be of great value in such applications because only the target species would be affected. However we cannot proceed to large scale field test of attractants and one compound that inhibits the action of a sex attractant) that show promise for use in insect control. Although pesticides under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), as amended by the Federal Environmental Pesticide Control Act of 1972 (FEPCA) and toxicological data are needed for registration of compounds for which such use is contemplated. The substances selected for study have been effective in laboratory and field tests against economically important insect pests (Table 1). Three occur naturally as sex attractant pheromones: disparlure and looplure are emitted by the female gypsy moth and cabbage looper moth, respectively, to attract the male moths. Muscalure, produced by female house fly, is attractive only to the male in laboratory tests but attracted both sexes in field tests (Carlson and Beroza, 1973). Hexalure, though not the natural pheromone of the pink bollworm, resembles a pheromone in its action. (Z)-7-Dodecen-1-ol inhibits the action of the cabbage looper pheromone (Tumlinson et al.,1972). and its disseminitaion may prevent the male from locating the female.

The other four test materials (cue-lure, methyl eugenol, PEP:eugenol (7:3) and tri-medlure) do not appear to be related to any natural pheromones but have been found experimentally to be effective and specific attractants. The reported LD50 of methyl eugenol in rats is 1560 mg/kg (Jenner et al., 1964). Eugenol, a component of the Japanese beetle attractant, is the principal ingridient of oil of cloves: it has uses in perfumes and flavorings, and is used in dentistry as an antiseptic, an analgesic, and a component of dental cement. ITS acute oral LD in rats was as 1930 mg/kg (Sober et al., 1950) and as 2680 mg/kg (Jenner et al., 1964; Hagan et al., 1965). Phenethyl propionate, the other component of the Japanese beetle attractant is also used in perfumes and flavorings.

The test materials are described in Table 1; they are colorless liquids that are soluble in the usual organic solvents. They wre procured from commercial sources according to specifications set up by U.S Department of Agriculture scientists; purity of each material was checked in the Organic Chemicals Synthesis Laboratory, AEQI, in Beltsville by gas chromatography. The limited amounts of material available for testing restricted the number of tests that could be made, particularly at higher dose levels.

Young albino rats of the Sprague Dawley strain (150-240g) were used in the tests of acute oral and inhalation toxicity. Albino rabbits of the New Zealand strain (1.9-3.1 kg) were used for the tests of acute dermal toxicity, eye irritation, and primary skin irritation. Healthy fingerlings (averaage lenght, 37-75 mm) of rainbow trout (Salmo gairdnerii) and bluegill sunfish (Lepomis macrochirus) were used in the tests of toxicity to fish (static fish toxicity studies). The rainbow trout were held at 13 C and the blugills at 18 C.

For tests of toxicity to fish, bioassay vessels were lined with disposable polyethylene bags and filled with 12,5 liter reconstituted water (30mg CaSO4, 30mg MgSO4, 48mg NaHCO3 and 2mg KCl added per liter of deionized water). Ten fish were placed in each vessel and allowed 24hr to become acclimated. Claculated amounts of each test material, as 1% or 10% (w/v) solutions in acetone, were then added to the bioassay vessels. Each material was tested at five five concentrations selected on the basis of preliminary screening tests to determine the approximate ranges of toxicity. Control groups of fish in untreated water and in water to which acetone only was added were observed concurrently. The fish were observed for 96 hr and all deaths and/or untoward behavioral reactions were recorded. The concentrations of dissolved oxygen was measured in all solutions in which deaths occured to be sure the test water contained sufficient oxygen; dissolved oxygen concentrations above 4mg/liter (4ppm for the warm-water fish (bluegills) or above 5mg/liter for cold water fish (rainbow trout) were considered adequate. The median lethal concentrations (LC50) of the seven test materials were calculated whenever the data permitted (Licthfiel and Wilcoxon, 1949).

To check on suitability as test subjects, fish from each lot were challenged, under the same experimental conditions with a reference pesticide (toxaphene, at 0.010, 0.018, and 0.056 ppm). The observed 96hr LC50 was usually around 0.02 ppm; with one lot of bluegills there were no deaths at 0.018 ppm and no survivors at 0.056 ppm.

- Parameters analysed / observed:

24, 48, 96 hr LC50

Reference 3

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

yes

Specific details on test material used for the study:

The test item information as per Test Item Data Sheet and Certificate of Analysis is presented below:
Name of Test Item : Methyl Eugenol
Chemical Name (IUPAC) : 4-allyl-1,2-dimethoxybenzene
CAS No. : 93-15-2
Physical Appearance (with colour) : Pale yellow liquid
Batch No. : 40003010419
Purity (As per Certificate of Analysis) : 99.24%
Batch Produced by (Name and Address) : YASHO INDUSTRIES LIMITED
Plot No. 2514/2515, Phase IV, G. I. D. C., Vapi - 396195, Gujarat, India
Date of Manufacture : Feb 2019
Date of Expiry : Jan 2021
Storage conditions : Ambient (21 to 29°C)
Test Item Code by Test Facility : D819-001

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

The solubility of Methyl Eugenol in the test medium was found to be low in reverse osmosis water (tap water) according to an internal method.
Therefore, individual WAFs was determined by adding the appropriate amount of test item to dilution water reverse osmosis water (tap water) at loading rates of (e.g.1.0, 10.0, 50.0, 75 and 100.0 mg/L). Each mixing vessel was filled to achieve minimal headspace given the constraints of the vessels and closed with foil-covered stoppers. The mixture was stirred using a magnetic stirrer at minimum of 100 RPM (the duration of mixing was 6 hours, selected based on WAF equilibration trail). Same method was employed during every renewal period.
The mixtures were allowed to settle and equilibrate to test temperature (before removing the test solution). The aqueous portions of WAFs were collected using steri- pipette and transferred to test vessels. Same method was employed for control group using reverse osmosis water (tap water).
The preparation of test media on day 0 during range finding study and main study are presented as Appendix 2 and Appendix 5 respectively.
During Range finding study and main study TOC was analyzed on day 0 (fresh) and on day 1 (spent) sample from all the group.

Test organisms (species):

Cyprinus carpio

Details on test organisms:

Fish Species : Cyprinus carpio

(Cyprinus carpio)
Justification for Selection of Species : Cyprinus carpio (common carp) is readily available species and has been historically shown to be a suitable model for acute toxicity studies. Moreover, it is recommended by OECD Test Guideline No. 203, and by other regulatory test guidelines.
Source of Supply : In-house maintained Cyprinus carpio (common carp) procured from Fisheries Research and Information Center (Inland), Hebbal, Bangalore, Karnataka, India.


Total Length of Fish : The length was measured a day prior to the exposure. The total length of fish used in the range finding study ranged from 2.9 to 3.8 cm and ranged from 3.0 to 3.8 cm during main study respectively.
Refer Appendix 1 & 4.
No. of Groups and
N No. of Fish per
Group : Six groups [1 control and 5 treatment groups] consisting of 7 fish in each group were used during the range finding study.
Seven groups [1 control and 6 treatment groups] consisting of 7 fish in each group were used during the main study.


6.5 Acclimatization
Forty two and forty nine healthy fish were collected with an additional of 10 fish for acclimatization during the range finding study and main study respectively. Healthy fish were acclimatized for 7 days prior to test initiation both during range finding study and main study respectively. No mortality was observed during acclimatization period.
Fish were fed ad libitum daily with commercial feed pellets (Kijaro Grow). Feeding was stopped approximately 24 hour prior to commencement of the exposure to test item.
Water temperature during acclimatization was between 22.1 to 22.9oC during range finding study and 22.8 to 22.7°C during main study. Hardness of the water measured once during the period of acclimatization (at start) was 220 and 208 mg CaCO3/L during range finding study and main study respectively. Prior to acclimatization the glass aquaria was labeled for identification (study no., study code, no. of fish acclimatized, acclimatization start and acclimatization end date).
6.7 Test Vessels
Thoroughly cleaned rectangular glass aquaria having the water holding capacity of 50 liters were used as test vessels. The test vessels were labeled for identification (study no., test item code, study code, group no. and nominal loading rate, exposure start and exposure end dates) prior to experiment initiation.
6.8 Feeding
Fish were fed ad libitum daily with commercial feed pellets (Kijaro Grow) during acclimatization. Feeding was stopped approximately 24 hours prior to commencement of the exposure to test item.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

212-224 mg/L CaCO3

Test temperature:

21.9-22.7 C

pH:

6.78-7.15

Dissolved oxygen:

78.2-89.5%

Salinity:

-

Conductivity:

-

Nominal and measured concentrations:

2.0, 3.8, 7.3, 13.9, 26.3 and 50.0 mg/L

Reference substance (positive control):

not specified

Key result

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LOELR

Effect conc.:

3.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

8.72 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Validity criteria fulfilled:

yes

Conclusions:

The 96 hours No Observed Effect Loading rate (NOEL) and Lowest Observed Effect Loading rate (LOEL) is 2.0 and 3.8 mg/L and acute median lethal loading rate (LL50) value of Methyl Eugenol on fresh water fish Cyprinus carpio is 8.72 mg/L.

Executive summary:

The test itemMethyl Eugenol,obtained from Yasho Industries Limited., was tested for acute toxicity on fish as per the OECD Guidelines for Testing of Chemicals (Section 2), Effects on Biotic Systems, Guideline No. 203, “Fish Acute Toxicity Test” adopted on 17 July 1992 and Guidance Document on Toxicology for registration of pesticides in India, 2017

The freshwater fishCyprinus carpiowas exposedover 96 hourstoMethyl Eugenolto determine the 96 hours median lethal loading rate (LL₅₀).

Range Finding Study

Range finding study was conducted with5 nominal loading rates of 1.0, 10.0, 50.0, 75.0 and 100.0 mg/L of Methyl Eugenolalong with control group.

No clinical sign of toxicity was observed in the control group and at the nominal loading rate of 1.0 mg/L during the 96 hour exposure period.

Clinical signs of easy catching, lethargy, and loss of equilibrium were observed at the nominal loading rate of 10.0 and 50.0 mg/L during the 96 hour exposure period.

Clinical signs of lethargy and easy catching were observed at the nominal loading rate of 75.0 and 100.0 mg/L during the 96 hour exposure period.

Mortalities of 71.4, 100.0, 100.0 and 100.0% were observed at the tested nominal loading rates of10.0, 50.0, 75.0 and 100.0 mg/L. No mortalities were observed in the control group and at the nominal loading rate of 1.0 mg/L.

Main study

Based on the results of dose range finding study, main was conducted at the nominal loading rates of 2.0, 3.8, 7.3, 13.9, 26.3 and 50.0 mg/L ofMethyl Eugenolalong with control group.

No clinical sign of toxicity was observed in the control group and at the nominal loading rate of 2.0 mg/L during the 96 hour exposure period.

Clinical sign of Lethargy was observed at the nominal loading rate of 3.8 mg/L during the 96 hour exposure period.

Clinical signs of Lethargy, easy catching and loss of equilibrium were observed at the nominal loading rate of 7.3 and 13.9 mg/L during the 96 hour exposure period.

Clinical signs of Lethargy and easy catching was observed at the nominal loading rate of 26.3 and 50.0 mg/L during the 96 hour exposure period.

Mortalities 14.3, 42.9, 71.4, 100.0 and 100.0% were observed at the tested nominal loading rates of 3.8, 7.3, 13.9, 26.3 and 50.0 mg/L. No mortalities were observed in the control group and at the nominal loading rate of 6.7 mg/L.

TOC Analysis

The TOC analysis of the nominal loading rates during the main study is 2.150, 3.646, 5.910, 9.434, 17.540 and 35.840 Day 0 (Fresh) and 2.098, 3.958, 5.757, 9.725, 16.66 and 33.56 mg at the nominal loading rates of 2.0, 3.8, 7.3, 13.9, 26.3 and                          50.0 mg/L on Day 1 (Aged).

Description of key information

The fish were observed for 96 hr and all deaths and/or untoward behavioral reactions were recorded. The concentrations of dissolved oxygen was measured in all solutions in which deaths occured to be sure the test water contained sufficient oxygen; dissolved oxygen concentrations above 4mg/liter (4ppm for the warm-water fish (bluegills) or above 5mg/liter for cold water fish (rainbow trout) were considered adequate. The median lethal concentrations (LC50) of the seven test materials were calculated whenever the data permitted (Licthfiel and Wilcoxon, 1949). To check on suitability as test subjects, fish from each lot were challenged, under the same experimental conditions with a reference pesticide (toxaphene, at 0.010, 0.018, and 0.056 ppm). The measured 96 hour LC50 for rainbow trout was found to be a mean 6.0ppm with confidence limits ranging 5.5-8.6ppm and for bluegill sunfish the mean 8.1ppm with confidence limits ranging 7.4-9.0ppm.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

8.72 mg/L