4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-5-oxo-4,5-dihydro-1H-tetrazole-1-carboxamide

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:

15 Mar - 19 Sep 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: Japanese guidelines of November 25, 1965

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Principles of method if other than guideline:

The study follows an outdated guideline and does not meet nowadays guideline requirements. However, the study was basically conducted similar to OECD guideline 203.

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

yes

Remarks:

DMSO

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 1262 mg (active ingredient 1250 mg) were weighed in 50 mL of special grade dimethyl sulfoxide (DMSO) to reach a 2.5% (w/v) stock solution. This stock solution was added to the test water at the concentrations of 1.0, 1.3, 1.7, 2.2, 3.0, 4.0 and 5.0 mg/L. Only 10 mL of DMSO was added to the control water. Positive control: 50 mg of DMSO added to 50 mL pure water to reach a 0.1% (w/w) solution. This stock solution was added to the test water at the concentrations of 0. 06, 0.08, 0.10, 0.13, and 0.17 mg/L. The control was only with the test water.
- Controls: vehicle control, positive control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): special DMSO
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0.2 mL DMSO/L for test concentration 5 mg/L; 0.16 mL DMSO/L for test concentration 4 mg/L; 0.11 mL DMSO/L for test concentration 3 mg/L; 0.08 mL DMSO/L for test concentration 2.2 mg/L; 0.06 mL DMSO/L for test concentration 1.7 mg/L; 0.05 mL DMSO/L for test concentration 1.3 mg/L; 0.04 mL DMSO/L for test concentration 1.0 mg/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout (reported as Salmo gairdneri irideus)
- Source: obtained from Fuji rainbow trout farm cooperative society (Shizuoka, Japan)
- Length at study termination (length definition, mean, range and SD): 6.3 ± 0.3 cm
- Weight at study termination (mean and range, SD): 3.8 ± 0.6 g

ACCLIMATION
- Acclimation period: 20 days
- Acclimation conditions (same as test or not): same
- Type and amount of food during acclimation: dry food for fish (Oriental Yeast Industrial Co., Ltd., Japan);
- Feeding frequency during acclimation: twice a day

FEEDING DURING TEST : no (not fed 24 hours before the beginning and duration of the test)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

13 - 17 °C

pH:

7.0 - 7.3 (for details see Table 1 under "Any other information on materials and methods incl. tables" below)

Dissolved oxygen:

5.8 - 8.0 mg/L (for details see Table 2 under "Any other information on materials and methods incl. tables" below)

Nominal and measured concentrations:

solvent control, 1.0, 1.3, 1.7, 2.2, 3.0, 4.0, and 5.0 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel:
Material, size, headspace, fill volume: All-glass test chambers (600 mm width x 300 mm depth x 360 mm height); fill volume: 50 L
- Aeration: yes
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per vehicle control (replicates): 1
- Biomass loading rate: 0.8 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Well activated carbon-filtered tap water was used as the test water.
- Intervals of water quality measurement: The dissolved oxygen and pH levels were measured before the beginning of the test and at 24, 48, 72, and 96 hours, thereafter.

OTHER TEST CONDITIONS
- Photoperiod: 13 hrs light (5 a.m. to 6 p.m.), 11 hrs dark
- Light: Fluorescent lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The number of dead or affected fishes in each test chamber was counted at 1, 3, 6, 24, 48, 72, and 96 hours after the beginning of the test.

Reference substance (positive control):

yes

Remarks:

Pentachlorphenol-sodium (PCP-Na)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities:
Control group:
Neither dead nor affected fish were observed throughout the test period.

1.0 and 1.3 mg/L groups:
Neither dead nor affected fish were observed in these groups throughout the test period.

1.7 mg/L group:
After 24 hours from the beginning of the test, loss of equilibrium, body color turn dark and lying on one side were apparent in some fishes. There was one dead fish at 48 hours. Then, after 72 hours to 96 hours, the survival fishes recovered from loss of equilibrium and body color turning dark.

2.2 mg/L group:
After 3 hours from the beginning of the test, loss of equilibrium, depression of spontaneous motor activity and body color turning dark were apparent in some fishes. At 24 hours, lying on one side was apparent in some fishes. There was one dead fish at 24 hours in this group.

3.0 mg/L group:
After 3 hours from the beginning of the test, loss of equilibrium, depression of spontaneous motor activity and body color turning dark were apparent in some fishes. At 24 hours, lying on one side was apparent in some fishes. There was one dead fish at 24 hours, 2 dead fishes at 48 hours and one dead fish at 72 hours in this group.

4.0 mg/L group:
After 3 hours from the beginning of the test, loss of equilibrium, depression of spontaneous motor activity, body color turning dark and lying on one side were apparent in some fishes. There were 7 dead fishes at 24 hours and one dead fish at 72 hours in this group.

5.0 mg/L group:
After one hour from the beginning of the test, loss of equilibrium, depression of spontaneous motor activity and body color turning dark were apparent in some fishes. At 3 hours, lying on one side was apparent in some fishes. There were 9 dead fishes at 24 hours and one dead fish at 48 hours in this group.

At 24 hours, the dissolved oxygen concentrations of the test water were decreased in all test groups. So each test water was aerated gently.

Results with reference substance (positive control):

LC50 (96 h) = 0.074 mg/L

Reported statistics and error estimates:

The 50% lethal concentrations (LC50) were calculated on the basis of mortality at 24, 48, 72, and 96 hours after the beginning of the test by the Doudroff method.

Sublethal observations / clinical signs:

Table 1: Acute toxicity to rainbow trout

Group	Concentration (mg/L)	No. of fish	No. of dead fish (after x hrs)							Mortality (%) after x hrs				LC50 (mg/L) after x hrs
			1	3	6	24	48	72	96	24	48	72	96	24	48	72	96
1	Solvent control	10	0	0	0	0	0	0	0	0	0	0	0
2	1.0	10	0	0	0	0	0	0	0	0	0	0	0	3.6
3	1.3	10	0	0	0	0	0	0	0	0	0	0	0		3.4
4	1.7	10	0	0	0	0	1	0	0	0	10	10	10			3.2
5	2.2	10	0	0	0	1	0	0	0	10	10	10	10				3.2
6	3.0	10	0	0	0	1	2	1	0	10	30	40	40
7	4.0	10	0	0	0	7	0	1	0	70	70	80	80
8	5.0	10	0	0	0	9	1	-	-	90	100	100	100

- : no surviving fish

Table 2: Acute toxicity to rainbow trout of positive control

Group	Concentration (mg/L)	No. of fish	No. of dead fish (after x hrs)							Mortality (%) after x hrs				LC50 (mg/L) after x hrs
			1	3	6	24	48	72	96	24	48	72	96	24	48	72	96
1	control	10	0	0	0	0	0	0	0	0	0	0	0
2	0.06	10	0	0	0	0	0	0	0	0	0	0	0	0.074
3	0.08	10	0	0	0	7	0	0	0	70	70	70	70		0.074
4	0.10	10	0	0	0	9	0	0	0	90	90	90	90			0.074
5	0.13	10	0	0	0	10	-	-	-	100	100	100	100				0.074
6	0.17	10	0	0	0	10	-	-	-	100	100	100	100

Validity criteria fulfilled:

not applicable

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

15 Mar - 19 Sep 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Oxygen concentration decreased with increasing test concentrations and test period. Nevertheless, no mortalities were observed neither in the exposed fish nor in the controls. Thus, oxygen depletion had no effect on the outcome of the study.

Qualifier:

according to guideline

Guideline:

other: Japanese guidelines of November 25, 1965

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Principles of method if other than guideline:

The study follows an outdated guideline and does not meet nowadays guideline requirements. However, the study was basically conducted similar to OECD guideline 203.

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

yes

Remarks:

DMSO

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 1262 mg (active ingredient 1250 mg) were weighed in 100 mL of special grade dimethyl sulfoxide (DMSO) to reach a 1.25% (w/v) stock solution. This stock solution was added to the test water at the concentrations of 1.0, 1.3, 1.7, 2.2, 3.0, 4.0 and 5.0 mg/L. Only 20 mL of DMSO was added to the control water. Positive control: 100 mg of DMSO added to 100 mL pure water to reach a 0.1% (w/w) solution. This stock solution was added to the test water at the concentrations of 0.03, 0.04, 0.06, 0.08, 0.10, 0.13 and 0.17 mg/L. The control was only with the test water.
- Controls: vehicle control, positive control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): special DMSO
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0.4 mL DMSO/L for test concentration 5 mg/L; 0.32 mL DMSO/L for test concentration 4 mg/L; 0.24 mL DMSO/L for test concentration 3 mg/L; 0.17 mL DMSO/L for test concentration 2.2 mg/L; 0.13 mL DMSO/L for test concentration 1.7 mg/L; 0.10 mL DMSO/L for test concentration 1.3 mg/L; 0.08 mL DMSO/L for test concentration 1.0 mg/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

Mugil cephalus

Details on test organisms:

TEST ORGANISM
- Common name: Gray mullet
- Source: Juvenile gray mullet were harvested in Hamanalake, then fishes were maintained in holding tank at Hamana Suisan K. K. (Shizuoka, Japan) and they were acclimatized to the freshwater with subterranean water.
- Length at study termination (length definition, mean, range and SD): 4.7 ± 0.2 cm
- Weight at study termination (mean and range, SD): 1.6 ± 0.2 g

ACCLIMATION
- Acclimation period: 12 days
- Acclimation conditions (same as test or not): same
- Type and amount of food during acclimation: dry food for fish (Hamana Suisan K. K)
- Feeding frequency during acclimation: twice a day

FEEDING DURING TEST: no (not fed 24 hours before the beginning and duration of the test)

Test type:

static

Water media type:

freshwater

Remarks:

Test species is euryhaline, it has the capability to acclimate to different levels of salinity

Limit test:

no

Total exposure duration:

96 h

Test temperature:

21 - 25 °C

pH:

6.5 - 6.9 (for details see Table 1 under "Any other information on materials and methods incl. tables" below)

Dissolved oxygen:

2.8 - 8.2 mg/L (for details see Table 2 under "Any other information on materials and methods incl. tables" below)

Nominal and measured concentrations:

solvent control, 1.0, 1.3, 1.7, 2.2, 3.0, 4.0, and 5.0 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel:
Material, size, headspace, fill volume: All-glass test chambers (600 mm width x 300 mm depth x 360 mm height); fill volume: 50 L
- Aeration: yes
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per vehicle control (replicates): 1
- Biomass loading rate: 0.3 g/L
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Well activated carbon-filtered tap water was used as the test water.
- Intervals of water quality measurement: The dissolved oxygen and pH levels were measured before the beginning of the test and at 24, 48, 72, and 96 hours, thereafter.

OTHER TEST CONDITIONS
- Photoperiod: 13 hrs light (5 a.m. to 6 p.m.), 11 hrs dark
- Light: Fluorescent lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The number of dead or affected fishes in each test chamber was counted at 1, 3, 6, 24, 48, 72, and 96 hours after the beginning of the test.

Reference substance (positive control):

yes

Remarks:

Pentachlorphenol-sodium (PCP-Na)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities:
Control group:
Neither dead nor affected fish in general conditions was observed throughout the test period.
1.0 and 1.3 mg/L groups:
After 24 hours from the beginning of the test, loss of equilibrium, depression of spontaneous motor activity and body color turning dark were apparent in some fishes. There was no dead fish in these groups.

1.7 and 2.2 mg/L groups:
After 3 hours from the beginning of the test, body color turn dark was apparent in some fishes. At 24 hours, loss of equilibrium, depression of spontaneous motor activity and lying on one side were apparent in some fishes. There was one and 2 dead fishes at 72 hours in these groups, respectively.

3.0 mg/L group: After 3 hours from the beginning of the test, loss of equilibrium, depression of spontaneous motor activity, body color turning dark and lying on one side were apparent in some fishes. There was one dead fish at 48 and 72 hours, respectively and 2 dead fishes at 96 hours in this group.

4.0 mg/L group:
After one hour from the beginning of the test, loss of equilibrium was apparent in some fishes. At 3 hours, depression of spontaneous motor activity, body color turning dark and lying on one side were apparent in some fishes. There were 2 dead fishes at 48 hours and 3 dead fishes at 96 hours in this group.

5.0 mg/L group:
After hour from the beginning of the test, loss of equilibrium was apparent in some fishes. At 3 hours, depression of spontaneous motor activity, body color turning dark and lying on one side were apparent in some fishes. There were 9 dead fishes at 24 hours and one dead fish at 48 hours in this group.

At 96 hours, the dissolved oxygen concentrations of the test water were decreased in control and 4.0 mg/L groups.

Results with reference substance (positive control):

LC50 (96 h) = 0.086 mg/L

Reported statistics and error estimates:

The 50% lethal concentrations (LC50) were calculated on the basis of mortality at 24, 48, 72, and 96 hours after the beginning of the test by the Doudroff method.

Sublethal observations / clinical signs:

Table 1: Acute toxicity to rainbow trout of test substance

Group	Concentration (mg/L)	No. of fish	No. of dead fish (after x hrs)							Mortality (%) after x hrs				LC50 (mg/L) after x hrs
			1	3	6	24	48	72	96	24	48	72	96	24	48	72	96
1	Solvent control	10	0	0	0	0	0	0	0	0	0	0	0
2	1.0	10	0	0	0	0	0	0	0	0	0	0	0	4.5
3	1.3	10	0	0	0	0	0	0	0	0	0	0	0		4.4
4	1.7	10	0	0	0	0	0	1	0	0	0	10	10			4.4
5	2.2	10	0	0	0	0	0	2	0	0	0	20	20				4.0
6	3.0	10	0	0	0	0	1	1	2	0	10	20	40
7	4.0	10	0	0	0	0	2	0	3	0	20	20	50
8	5.0	10	0	0	0	9	1	-	-	90	100	100	100

- : no surviving fish

Table 2: Acute toxicity to rainbow trout of positive control

Group	Concentration (mg/L)	No. of fish	No. of dead fish (after x hrs)							Mortality (%) after x hrs				LC50 (mg/L) after x hrs
			1	3	6	24	48	72	96	24	48	72	96	24	48	72	96
1	control	10	0	0	0	0	0	0	0	0	0	0	0
2	0.03	10	0	0	0	0	0	0	0	0	0	0	0	0.14
3	0.04	10	0	0	0	0	0	0	0	0	0	0	0		0.11
4	0.06	10	0	0	0	0	0	1	0	0	0	10	10			0.09
5	0.08	10	0	0	0	0	2	1	0	0	20	30	30				0.086
6	0.10	10	0	0	0	0	4	3	1	0	40	70	80
7	0.13	10	0	0	0	2	7	1	-	20	90	100	100
8	0.17	10	0	0	0	10	-	-	-	100	100	100	100

- : no surviving fish

Validity criteria fulfilled:

not applicable

Reference 3

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

15 Mar - 19 Sep 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Oxygen concentration decreased with increasing test concentrations and test period. Nevertheless, no mortalities were observed neither in the exposed fish nor in the controls. Thus, oxygen depletion had no effect on the outcome of the study.

Qualifier:

according to guideline

Guideline:

other: Japanese guidelines of November 25, 1965

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Principles of method if other than guideline:

The study follows an outdated guideline and does not meet nowadays guideline requirements. However, the study was basically conducted similar to OECD guideline 203.

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

yes

Remarks:

DMSO

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 505 mg (active ingredient 500 mg) were weighed in 50 mL of special grade dimethyl sulfoxide (DMSO) to reach a 1.0% (w/v) stock solution. This stock solution was added to the test water at the concentrations of 0.6, 0.8, 1.0, 1.3, 1.7, and 2.2 mg/L. Only 1 mL of DMSO was added to the control water. Positive control: 100 mg of DMSO added to 100 mL pure water to reach a 0.1% (w/w) solution. This stock solution was added to the test water at the concentrations of 0.10, 0.13, 0.17, 0.22, and 0.3 mg/L. The control was only with the test water.
- Controls: vehicle control, positive control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): special DMSO
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0.22 mL DMSO/L for test concentration 2.2 mg/L; 0.17 mL DMSO/L for test concentration 1.7 mg/L; 0.13 mL DMSO/L for test concentration 1.3 mg/L; 0.10 mL DMSO/L for test concentration 1.0 mg/L; 0.08 mL DMSO/L for test concentration 0.8 mg/L; 0.06 mL DMSO/L for test concentration 0.6 mg/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

other: Chrysophrys major

Details on test organisms:

TEST ORGANISM
- Common name: Red seabream
- Source: Obtained from Shizuoka Prefectural Thermal Effluent Utilization Research Center (Shizuoka, Japan)
- Length at study termination (length definition, mean, range and SD): 4.3 ± 0.1 cm
- Weight at study termination (mean and range, SD): 2.0 ± 0.2 g

ACCLIMATION
- Acclimation period: 12 days
- Acclimation conditions (same as test or not): same
- Type and amount of food during acclimation: dry food for Red sea bream (Chubu Shiryo Co., Ltd., Japan)
- Feeding frequency during acclimation: twice a day

FEEDING DURING TEST: no (not fed 24 hours before the beginning and duration of the test)

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

21 - 25 °C

pH:

6.7 - 7.2 (for details see Table 1 under "Any other information on materials and methods incl. tables" below)

Dissolved oxygen:

4.0 - 8.0 (for details see Table 1 under "Any other information on materials and methods incl. tables" below)

Nominal and measured concentrations:

solvent control, 0.6, 0.8, 1.0, 1.3, 1.7, and 2.2 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel:
Material, size, headspace, fill volume: All-glass test chambers (600 mm width x 300 mm depth x 360 mm height); fill volume: 50 L
- Aeration: yes
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per vehicle control (replicates): 1
- Biomass loading rate: 0.4 g/L
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Natural sea water was used as the test water
- Intervals of water quality measurement: The dissolved oxygen and pH levels were measured before the beginning of the test and at 24, 48, 72, and 96 hours, thereafter.

OTHER TEST CONDITIONS
- Photoperiod: 13 hrs light (5 a.m. to 6 p.m.), 11 hrs dark
- Light: Fluorescent lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The number of dead or affected fishes in each test chamber was counted at 1, 3, 6, 24, 48, 72, and 96 hours after the beginning of the test.

Reference substance (positive control):

yes

Remarks:

Pentachlorphenol-sodium (PCP-Na)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

1.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities:
Control group:
Neither dead nor affected fish in general conditions was observed throughout the test period.
0.6 mg/L group:
Neither dead nor affected fish was observed in this group throughout the test period.

0.8 mg/L group:
After 24 hours from the beginning of the test, loss of equilibrium and body color turning dark were apparent in some fishes. There was no dead fish in this group.

1.0 mg/L group:
After 6 hours from the beginning of the test, loss of equilibrium, depression of spontaneous motor activity were apparent in some fishes. There was no dead fish in this group.

1.3 mg/L group:
After one hour from the beginning of the test, loss of equilibrium, depression of spontaneous motor activity and body color turning dark were apparent in some fishes. At 3 hours, lying on one side was apparent in some fishes. There was one dead fish at 96 hours in this group.

1.7 mg/L group:
After one hour from the beginning of the test, loss of equilibrium, depression of spontaneous motor activity and body color turning dark were apparent in some fishes. At 3 hours, lying on one side was apparent in some fishes. There were 2 dead fishes at 24 hours, 4 dead fishes at 48 hours and one dead fish at 72 hours in this group.

2.2 mg/L group:
After one hour from the beginning of the test, loss of equilibrium, depression of spontaneous motor activity, body color turn dark and lying on one side were apparent in some fishes. There were 8 dead fishes at 24 hours and 2 dead fishes at 48 hours in this group.

The dissolved oxygen concentrations in the test water are presented in Table 2. At 48 hours, the dissolved oxygen concentrations of the test water were decreased in all test groups. The Red sea bream are known to have little resistance to the low dissolved oxygen concentration, so each test water was aerated gently.

Results with reference substance (positive control):

LC50 (96 h) = 0.19 mg/L

Reported statistics and error estimates:

The 50% lethal concentrations (LC50) were calculated on the basis of mortality at 24, 48, 72, and 96 hours after the beginning of the test by the Doudroff method.

Sublethal observations / clinical signs:

Table 1: Acute toxicity to rainbow trout of test substance

Group	Concentration (mg/L)	No. of fish	No. of dead fish (after x hrs)							Mortality (%) after x hrs				LC50 (mg/L) after x hrs
			1	3	6	24	48	72	96	24	48	72	96	24	48	72	96
1	Solvent control	10	0	0	0	0	0	0	0	0	0	0	0
2	0.6	10	0	0	0	0	0	0	0	0	0	0	0	1.9
3	0.8	10	0	0	0	0	0	0	0	0	0	0	0		1.6
4	1.0	10	0	0	0	0	0	0	0	0	0	0	0			1.5
5	1.3	10	0	0	0	0	0	0	1	0	0	0	10				1.5
6	1.7	10	0	0	0	2	4	1	0	20	60	70	70
7	2.2	10	0	0	0	8	2	-	-	80	100	100	100

- : no surviving fish

Table 2: Acute toxicity to rainbow trout of positive control

Group	Concentration (mg/L)	No. of fish	No. of dead fish (after x hrs)							Mortality (%) after x hrs				LC50 (mg/L) after x hrs
			1	3	6	24	48	72	96	24	48	72	96	24	48	72	96
1	control	10	0	0	0	0	0	0	0	0	0	0	0
2	0.10	10	0	0	0	0	0	0	0	0	0	0	0	0.25
3	0.13	10	0	0	0	0	0	0	0	0	0	0	0		0.20
4	0.17	10	0	0	0	0	0	3	0	0	0	30	30			0.19
5	0.22	10	0	0	0	1	6	1	0	10	70	80	80				0.19
6	0.30	10	0	0	0	10	-	-	-	100	100	100	100

- : no surviving fish

Validity criteria fulfilled:

not applicable

Reference 4

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

6 Jun - 24 Jun 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Principles of method if other than guideline:

Although no guideline was followed and the study does not meet nowadays guideline requirements, the study was basically conducted similar to OECD guideline 203. No analytical monitoring.

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 1.6 mg test substance dissolved in 200 mL of acetone. This stock solution was then further diluted with acetone for each stock solution
- Controls: control, vehicle control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Test media were made by adding 100 mL of stock solution into 100 L bioassay water, i.e. 1 mL acetone/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): yes (in the 8.0 and 4.0 mg/L groups unhomogeneous test medium and test substance at the surface was observed)

Test organisms (species):

other: Plecoglossus altivelis

Details on test organisms:

TEST ORGANISM
- Common name: Ayu-fish
- Source: Yabe Pisciculturing Farm (Tyofu-City, Tokyo, Japan)
- Length at study initiation (length definition, mean, range and SD): mean 9.7 cm (8.5 - 11.0 cm)
- Weight at study initiation (mean and range, SD): mean 8.6 g (6.8 - 11.9 g)

ACCLIMATION
- Acclimation period: 40 days
- Acclimation conditions: fish kept in rearing pond (vinyl) at a temperature of 17 - 20 °C
- Feeding frequency during acclimation: fed until 48 hrs before testing
- Health during acclimation: only healthy fish used for experiment

FEEDING DURING TEST : no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

18 - 20.3 °C (for details see Table 1 under "Any other information on materials and methods incl. tables" below)

pH:

7.5 - 8.0 (for details see Table 1 under "Any other information on materials and methods incl. tables" below)

Dissolved oxygen:

93 - 100% (for details see Table 1 under "Any other information on materials and methods incl. tables" below)

Nominal and measured concentrations:

control, vehicle control, 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel:
Type (delete if not applicable): open
Material, size, headspace, fill volume: glass aquaria, ca. 180 L (900 mm width x 450 mm depth x 450 mm height); fill volume: 100 L
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Filtrated underground water was used
- Intervals of water quality measurement: temperature, pH, dissolved oxygen were monitored (no details on interval). Fish were examined daily for general behavior and mortality.

OTHER TEST CONDITIONS
- Photoperiod: 16 hrs lighting, 8 hrs darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Fish were examined daily for general behavior and mortality.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

LC50

Remarks:

Originally reported as TLM

Effect conc.:

2.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Remarks:

Originally reported as TLM

Effect conc.:

1.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: Symptoms observed were lying on side at the bottom and surface and irregular behavior of swimming in the test medium. These appeared within 3 hrs in the 4.0 and 8.0 mg/L concentration groups, within 24 hrs in the 1.0 and 2.0 mg/L groups, and within 48 hrs in the 0.5 mg/L group. The appearance time of the symptoms correlated to the test concentrations.
- Mortality of control: none
- Mortality of concentration groups: Mortalities occured within 3 hrs in the 8.0 mg/L concentration group, and within 24 to 48 hrs in the 0.5 mg/L groups and more. No mortality was observed in the 0.25 mg/L group.
- Any observations (e.g. precipitation): in the 8.0 and 4.0 mg/L groups unhomogeneous test medium and test substance at the surface was observed

Reported statistics and error estimates:

LC50 values were calculated at 24, 48, 72 and 96 hrs by aid of the method of Doudroff.

Sublethal observations / clinical signs:

Table 1: Toxicological findings (Tests on 0.5 and 1.0 mg/L concentration groups were repeated and the results were reported)

Concentration (mg/L)	No. of fish	Duration (hrs)					Findings
		3	24	48	72	96	Symptoms	Test medium
8.0	10	6/4*	6/4	8/2	10/0	10/0	A, B**	1, 2***
4.0	10	0/5	0/10	6/4	10/0	10/0	A, B	1, 2
2.0	10	0/0	2/4	5/5	8/2	8/2	A, B, C	0
1.0	20	0/0	0/2	1/5	4/4	4/4	B, C	0
0.5	20	0/0	0/0	1/1	3/0	3/0	B, C	0
0.25	10	0/0	0/0	0/0	0/0	0/0	-	0
Vehicle control	20	0/0	0/0	0/0	0/0	0/0	-	-
Control	10	0/0	0/0	0/0	0/0	0/0	-	-

* Cumulative No. of death / No. of sympton

** A: lying on side, B: fish mainly at bottom / surface, C: irregular behavior of swimming (apathy)

*** 0: clear, 1: unhomogeneous, 2: test substance at the surface

Table 2: TLM values

	24 hrs	48 hrs	72 hrs	96 hrs
TLM value (mg/L)	5.5	2.7	1.3	1.3
NOEC (mg/L)	0.5	0.25	0.25	0.25

Validity criteria fulfilled:

not applicable

Reference 5

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2 - 6 Aug 1993

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

abstract

Qualifier:

no guideline followed

Principles of method if other than guideline:

No details on the test procedure are given

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solution containing 100 mg of a.i. in 10 mL of acetone was added to the bioassay water in respective amounts to yield the corresponding test concentration.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0.5 mL acetone/L for test concentration 5 mg a.i./L; 0.2 mL acetone/L for test concentration 2 mg a.i./L; 0.1 mL acetone/L for test concentration 1 mg a.i./L; 0.05 mL acetone/L for test concentration 0.5 mg a.i./L

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: Common carp
- Source: Suzuki Fish Farm, Saitama Pref., Japan
- Length (length definition, mean, range and SD): 3.5 cm (mean total)
- Weight (mean and range, SD): 0.3 g (mean total)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

22.0 - 24.1 °C

pH:

7.13 - 7.38

Dissolved oxygen:

50 - 100%

Nominal and measured concentrations:

control, 0.5, 1, 2, and 5 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel:
Material, size, headspace, fill volume: 15 L fill volume
- No. of organisms per vessel: 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: filtered underground water

OTHER TEST CONDITIONS
- Photoperiod: 16 hrs light, 8 hrs dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): mortality and signs of toxicity (swimming behaviour, respiration, colour, morphological abnormalities) at 24, 48, 72, and 96 h

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Remarks:

Originally reported as TLM

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Reported statistics and error estimates:

The LC50 values were determined at 24, 48, 72 and 96 hrs by Doudroff's method.

Sublethal observations / clinical signs:

Table 1: Toxicological findings

Concentration (mg/L)	No. of fish	Cumulative mortality / Signs of toxicity
		24 h	48 h	72 h	96 h
5	10	10/0	10/0	10/0	10/0
2	10	0/2	0/0	0/10	0/10
1	10	0/0	0/4	0/8	0/10
0.5	10	0/0	0/0	0/8	0/8
Control	10	0/0	0/0	0/0	0/0
LC50 (mg/L)		3.2	3.2	3.2	3.2

Validity criteria fulfilled:

not applicable

Reference 6

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

19 - 23 Jul 1993

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

abstract

Qualifier:

no guideline followed

Principles of method if other than guideline:

No details on the test procedure are given

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

yes

Remarks:

Acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solution containing 30 mg of a.i. in 30 mL of acetone was added to the bioassay water in respective amounts to yield the corresponding test concentration.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 10 mL acetone/L for test concentration 10 mg a.i./L;5 mL acetone/L for test concentration 5 mg a.i./L; 2 mL acetone/L for test concentration 2 mg a.i./L; 1 mL acetone/L for test concentration 1 mg a.i./L; 0.5 mL acetone/L for test concentration 0.5 mg a.i./L

Test organisms (species):

Poecilia reticulata

Details on test organisms:

TEST ORGANISM
- Common name: Guppy
- Source: Laboratory of NBA, Hino Institute bred in 1993
- Length (length definition, mean, range and SD): 1.2 cm (mean total)
- Weight (mean and range, SD): 0.05 g (mean total)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

23.0 - 23.4 °C

pH:

7.20 - 7.39

Dissolved oxygen:

50 - 100%

Nominal and measured concentrations:

control, 0.5, 1, 2, 5, and 10 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel:
Material, size, headspace, fill volume: 1 L fill volume
- No. of organisms per vessel: 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: filtered underground water

OTHER TEST CONDITIONS
- Photoperiod: 16 hrs light, 8 hrs dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): mortality and signs of toxicity (swimming behaviour, respiration, colour, morphological abnormalities) at 24, 48, 72, and 96 h

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Remarks:

Originally reported as TLM

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Reported statistics and error estimates:

The LC50 values were determined at 24, 48, 72 and 96 hrs by Dourdroff's method.

Sublethal observations / clinical signs:

Table 1: Toxicological findings

Concentration (mg/L)	No. of fish	Cumulative mortality / Signs of toxicity
		24 h	48 h	72 h	96 h
10.0	10	10/0	10/0	10/0	10/0
5.0	10	1/9	10/0	10/0	10/0
2.0	10	1/4	2/7	5/2	5/5
1.0	10	0/0	0/4	2/3	2/1
0.5	10	0/0	0/0	0/0	0/0
control	10	0/0	0/0	0/0	0/0
LC50 (mg/L)		6.8	2.8	2.0	2.0

 

Validity criteria fulfilled:

not applicable

Description of key information

LC50 (96 h) = 1.3 mg/L (nominal, similar to OECD 203, Plecoglossus altivelis)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

1.3 mg/L

Additional information

Six experimental studies are available testing the acute toxicity of the test substance towards fish. Five studies were conducted with five different species under freshwater conditions, i.e. Plecoglossus altivelis (Ayu fish; amphidromous species, moving between coastal marine waters and freshwater lakes and rivers), Oncorhynchus mykiss (reported as Salmo gairdneri irideus), Mugil cephalus (Gray mullet; an euryhaline species, which has the capability to acclimate to different levels of salinity and naturally occurs in coastal waters and freshwater rivers), and Cyprinus carpio (Common carp). A further study was conducted with the marine species Chrysophrys major (Red seabream).

None of the studies was conducted according to GLP. Three studies (1996a, 1996b, and 1996c) followed Japanese guidelines, dating to 1965 which were not further specified. All studies were conducted under static test conditions without replacement of test medium and either acetone or DMSO was used as a solvent. Test concentrations were not analytically monitored. Two reports (1993a, 1993b) provide only short summaries. The studies do not fulfill nowadays guideline testing requirements and none of them can be considered as a key study. However, in sum all of the studies provide basic data sufficient for hazard assessment purposes and therefore a weight-of-evidence (WoE) approach is followed here. Details are discussed in the following.

The study with Plecoglossus altivelis (1994) was basically performed similar to OECD guideline 203, although there are restrictions. Ten fish per concentration were exposed to nominal test concentrations of 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 mg/L. Acetone was used as a vehicle and therefore a vehicle control as well as an untreated control were run in parallel. The concentration of the solvent in bioassay water was 1 mL/L, and thus above the recommendation of 0.1 mL/L as given by the OECD (2018). However, no symptoms or mortalities were observed in the vehicle control and dissolved oxygen remained between 93 to 100% in the vehicle control and 94 to 100% in the test concentrations of 0.25 mg/L and 0.2 mg/L. Thus, there is no indication neither for direct mortality nor for bacterial growth causing oxygen depletion and subsequently lethal or sublethal effects. The study resulted in a LC50 (96 h) (originally reported as TLM) of 1.3 mg/L (nominal). In the 8.0 and 4.0 mg/L groups unhomogeneous test medium and test substance at the surface was observed. Effects were observed within and above the water solubility (2.5 mg/L). Thus, mortalities and effects in the higher test concentrations could be of physical nature and the result of the study can be considered as worst case.

Three additional studies are available and were conducted in the same manner using Oncorhynchus mykiss (reported as Salmo gairdneri irideus), Mugil cephalus, and Chrysophrys major as test species (1996a, 1996b, 1996c). These studies were conducted according to a Japanese guideline dating 1965 without giving further bibliographic details. The test procedure was in principle similar to OECD guideline 203, although there are restrictions. Ten fish per concentration were exposed to 1.0, 1.3, 1.7, 2.2, 3.0, 4.0, and 5.0 mg/L (1996a, 1996b) and 0.6, 0.8, 1.0, 1.3, 1.7, and 2.2 mg/L (1996c) nominal test concentration. DMSO was used as a vehicle and therefore a vehicle control was run in parallel. The concentration of the solvent in bioassay water was in part above the recommendation of 0.1 mL/L as given by the OECD (2018): 0.2 and 0.16 mL/L in the two highest test concentrations (1996a), 0.4 to 0.13 mL/L in the five highest test concentrations (1996b), and 0.22 to 0.13 mL/L in the three highest test concentrations (1996c). However, no symptoms or mortalities were observed in the vehicle control. A decrease of oxygen was observed at different time intervals in these studies so the test water was aerated. Therefore, there is no indication neither for direct mortality nor for oxygen depletion and subsequent lethal or sublethal effects. The studies resulted in nominal LC50 (96 h) values of 3.2 mg/L (1996a), 4.0 mg/L (1996b), and 1.5 mg/L (1996c), respectively. No indication of undissolved test item is mentioned. Effects were observed within (all three studies) and above the water solubility (2.5 mg/L) (1996a, 1996b). Thus, mortalities and effects above the water solubility could be of physical nature and the results of these studies can be considered as a worst case.

Only short summaries are available for two further studies conducted with Cyprinus carpio and Poecilia reticulata (1993a, 1993b). Ten fish per concentration were exposed to 0.5, 1.0, 2.0, and 5.0 mg/L (1993a) and 0.5, 1.0, 2.0, 5.0, and 10 mg/L (1993b) nominal test concentration. Acetone was used as a vehicle. A control was run in parallel the type of which is not indicated. The concentration of the solvent in bioassay water was too high in all concentrations (1993b) or in part above the recommendation of 0.1 mL/L as given by the OECD (2018): 0.5 and 0.2 mL/L in the two highest test concentrations (1993a). No symptoms or mortalities were observed in the control. Dissolved oxygen was in the range between 50 to 100%. The studies resulted in nominal LC50 (96 h) values (reported as TLM) of 3.2 mg/L (1993a) and 2.0 mg/L (1993b), respectively. Within and above the water solubility mortalities were observed in one study (1993b), whereas mortalities only occurred above the water solubility in the second study (1993a). Sublethal effects were observed in both studies within and above the water solubility. Thus, mortalities and effects above the water solubility could be of physical nature and the results of these studies can be considered as a worst case.

All determined LC50 values are in the same level of magnitude and range between 1.3 to 4.0 mg/L. The lowest determined LC50 value of 1.3 mg/L was derived in the test with Plecoglossus altivelis (1994). As discussed above, solvents were used in all studies and in several cases with concentrations above the recommendation of 0.1 ml/L given by the OECD (2018). However, in none of the studies mortalities or sublethal effects were observed in the vehicle controls. Also no oxygen depletion based on bacteria growth induced by a solvent can be seen. The water solubility of the test item was determined to be 2.5 mg/L and mortalities and sublethal effects were observed within and above the water solubility limit, indicating that also physical effects might have occurred. Thus, the LC50 values determined rather represent overestimations and a worst case.

Based on the restrictions of the study reports discussed above a weight-of-evidence (WoE) approach was chosen for the hazard evaluation, which demonstrates a consistent pattern of the acute toxicity of the test item to fish. This approach is further justified by the fact, that aquatic invertebrates and algae demonstrated to be by far the most sensitive organisms, resulting in the most stringent classification of aquatic acute and chronic 1, and that results with organisms from those trophic levels furthermore are used for deriving PNECs and conducting the risk assessment.