ethyl 5,5-diphenyl-2-isoxazoline-3-carboxylate

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:

key study

Study period:

16 Mar - 22 May 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

adopted in 1989

Qualifier:

according to guideline

Guideline:

other: EPA OPP 72-2 EPA 540/9-82-024, adopted in 1982

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all replicate test concentrations were analyzed at test start and after 96 h
- Sampling method: 20 mL samples were taken at mid-depth for each time point
- Sample storage conditions before analysis: samples were immediately analyzed

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances):
A primary stock solution of the test substance in acetone was prepared at a nominal concentration of 10.1 mg/mL by addition of 10.1 g of neat material to a 1.0 Liter Class A glass volumetric flask and filling it to volume with acetone.
During each diluter cycle, a Hamilton Microlab 440 syringe pump injected 0.46 mL of the primary stock solution into 4.645 liters of dilution water in the diluter mix tank. This secondary stock concentration was the highest test concentration of 1.0 mg/L. The 1.0 mg/L solution was further diluted by the diluter system to produce the nominal test concentrations of 0.13, 0.22, 0.36 and 0.60 mg/L.
A control of dilution water and a solvent control (acetone at 0.1 mL/L) were also tested.
The diluter cycled approximately 6 cycles/hour (144 cycles per day). The diluter delivered approximately 1.0 L of test solution per replicate tank per cycle, or approximately 0.066 volume additions per cycle and approximately 9.5 volume additions per day.

Test organisms (species):

Lepomis macrochirus

Details on test organisms:

TEST ORGANISM
- Common name: Bluegill sunfish
- Source: North Eastern Aquatics, Rhinebeck, NY
- Age at study initiation (mean and range, SD): juvenile
- Length at study initiation (length definition, mean, range and SD): no data
- Length of control fish at the end of the study (length definition, mean, range and SD): 2.0 cm± 1.0 cm, 2.0 cm, 1.8 cm - 2.2 cm, 0.14 cm
- Weight at study initiation (mean and range, SD): no data
- Weight of control fish at the end of the study (mean and range, SD): 0.143 g, 0.092 g - 0.202 g, 0.0309 g

ACCLIMATION
- Acclimation period: 13 days
- Acclimation conditions (same as test or not): 20 °C - 25.4 °C, 16:8 light:dark, approx. 150 foot candles
- Type and amount of food: Rangen Salmon Starter
- Feeding frequency: ad libitum
- Health during acclimation (any mortality observed): There were 3 mortalities during the acclimation period, which represents less than 1% of the population (i.e., 371 fish). There were 2 mortalities during the 48 hours immediately prior to testing, which represents less than 1% of the population (i.e., 365 fish).

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

118 mg/L CaCO3

Test temperature:

21.0 - 22.0 °C

pH:

7.2 - 7.8

Dissolved oxygen:

6.1 - 8.3 ppm
69 - 95%

Nominal and measured concentrations:

nominal: 0, 0.13, 0.22, 0.36, 0.60 and 1.0 mg/L
mean measured: 0.086, 0.12, 0.22, 0.37 and 0.61 mg/L (maximum solubility)

Details on test conditions:

TEST SYSTEM
- Test vessel: 18 L fish tanks
- Type (delete if not applicable): closed
- Material, size, fill volume: glass, 24.4 cm x 30.8 cm x 24.4 cm, fill volume 15.2 L
- Aeration: none
- Type of flow-through (e.g. peristaltic or proportional diluter): proportional diluter
- Renewal rate of test solution (frequency/flow rate): The diluter cycled approximately 6 cycles/hour (144 cycles per day). The diluter delivered approximately 1.0 L of test solution per replicate tank per cycle, or approximately 0.066 volume additions per cycle and approximately 9.5 volume additions per day.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2
- Biomass loading rate: 0.094 g/L static and 0.010 g/L*day

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: filtered well water
- Total organic carbon: < 1.0 mg/L
- Total suspended solids: < 9.0 mg/L
- Metals (mg/L): Ba 0.22, Ca 28.5, Fe 0.16, Mg 3.54, Mn 0.03, K 2.26, Na 5.58; 19 other metals were shown to be below detection limit
- Pesticides: all 34 pesticides analyzed were below detection limit
- Chloride: 10.8 mg/L
- Alkalinity: 90 mg/L
- Conductivity: 220 µmhos/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: Dissolved oxygen, pH, conductivity and temperature once per day

OTHER TEST CONDITIONS
- Photoperiod: 16:8, light:dark photoperiod, with gradual intensity changes at dawn and dusk
- Light intensity: 115 foot candles

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Observations for death, and for abnormal appearance and behavior were performed at 3, 6, 24, 48, 72, and 96 hours (± 1 hour).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.67 - 1.69
- Range finding study
- Test concentrations: 1.0 mg/L
- Results used to determine the conditions for the definitive study: Thirty fish were exposed to a nominal concentration of 1.0 mg/L. Within 24 hours approx. 77% mortality was observed in the treated test chambers. Since there was mortality at this one dose, the study was repeated with a concentration range to calculate LC50 values.

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.22 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95%, CL = 0.20 to 0.25 mg/L

Details on results:

- Behavioural abnormalities: Lethargy and immobility were observed at the three highest concentrations
- Mortality of control: No mortality or sublethal effects were observed in the control or solvent control treatments during the study.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: A precipitate was observed in the diluter mix box and on the walls of the "E" chambers (toxicant delivery) during the study. The mean measured concentration of 0.61 mg/L represents the maximum solubility of the test substance.

Sublethal observations / clinical signs:

Table 1: Cumulative Mortality, days 0 - 4 for replicates

Nominal Concentr mg/L	Contr (1)	Contr (2)	Solv Contr (1)	Solv Contr (2)	0.13 (1)	0.13 (2)	0.22 (1)	0.22 (2)	0.36 (1)	0.36 (2)	0.60 (1)	0.60 (2)	1.00 (1)	1.00 (2)
Day 0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Day 1	0	0	0	0	0	0	0	0	0	0	3	3	10	10
Day 2	0	0	0	0	0	0	0	1	2	1	9	6	10	10
Day 3	0	0	0	0	0	0	0	1	3	3	10	9	10	10
Day 4	0	0	0	0	0	0	0	1	4	4	10	10	10	10

Table 2: Effect concentrations for 24 h, 48 h and 72 h and 96 h NOEC/LOEC

Duration	Endpoint	Effect conc.	Nom./Meas.	Conc. based on	Basis for effect	Remarks
24 h	LC50	0.41 mg/L	meas. (arithm. mean)	test mat.	mortality	95% CL = 0.37 to 0.45 mg/L
48 h	LC50	0.29 mg/L	meas. (arithm. mean)	test mat.	mortality	95% CL = 0.25 to 0.34 mg/L
72 h	LC50	0.24 mg/L	meas. (arithm. mean)	test mat.	mortality	95% CL = 0.21 to 0.28 mg/L
96 h	NOEC	0.086 mg/L	meas. (arithm. mean)	test mat.	mortality
96 h	LOEC	0.12 mg/L	meas. (arithm. mean)	test mat.	mortality

Table 3: Validity criteria for OECD 203.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality in the control(s) should not exceed 10% (or one fish if less than ten are used) at the end of the test	No mortality or sublethal effects were observed in the control or solvent control treatments	yes
The dissolved oxygen concentration must have been at least 60 per cent of the air saturation value throughout the test	69 - 95%	yes
There must be evidence that the concentration of the substance being tested has been satisfactorily maintained, and preferably it should be at least 80 per cent of the nominal concentration throughout the test. If the deviation from the nominal concentration is greater than 20 per cent, results should be based on the measured concentration.	55 - 66% Analytical measurement has been performed. Results are based on the measured concentration.	yes

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to table 3 at “Any other information on results incl. tables”.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

02 Jun - 06 Jun 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

adopted in 1989

Deviations:

yes

Remarks:

Fish were slightly shorter than recommended guideline values

Qualifier:

according to guideline

Guideline:

other: EPA OPP 72-2 EPA 540/9-82-024, adopted in 1982

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all replicate test concentrations were analyzed at test start and after 96 h
- Sampling method: 20 mL samples were taken at mid-depth for each time point
- Sample storage conditions before analysis: samples were immediately analyzed

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances):
A primary stock solution of the test substance in acetone was prepared at a nominal concentration of 8.2 mg/mL by addition of 4.1 g of neat material to a 500 ml volumetric flask and bringing to volume with acetone. During each diluter cycle, a Hamilton Microlab 400 syringe pump injected 0.40 ml of the primary stock solution into 4.675 liters of dilution water in the diluter mix tank. This secondary stock concentration was the highest test concentration of 0.70 mg/L. The 0.70 mg/L solution was further diluted by the diluter system to produce the nominal test concentrations of 0.09, 0.15, 0.25 and 0.42 mg/L. A control of dilution water and a solvent control (acetone at 0.10 mL/L) were also tested.
The diluter cycled approximately 6 cycles/hour (144 cycles per day). The diluter delivered approximately 1.0 L of test solution per replicate tank per cycle, or approximately 0.066 volume additions per cycle and approximately 9.5 volume additions per day.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Source: Mount Lassen Trout Farm, Red Bluff CA
- Age at study initiation (mean and range, SD): juvenile
- Length at study initiation (length definition, mean, range and SD): no data
- Length of control fish at the end of the study: mean 3.9 cm, range 3.5 cm to 4.4 cm, SD 0.293 cm
- Weight at study initiation (mean and range, SD): no data
- Weight of control fish at the end of the study: mean 0.832 g, range 0.528 g - 1.203 g, SD 0.1994 g
- Feeding during test: none

ACCLIMATION
- Acclimation period: 14 days
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: Rangen Salmon Starter
- Feeding frequency: ad libitum
- Health during acclimation (any mortality observed): There were no mortalities during the acclimation period, and no mortalities occurred during the 48 hours immediately prior to testing.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

86 mg/L CaCO3

Test temperature:

12.4 - 13.3 °C

pH:

7.0 - 7.3

Dissolved oxygen:

9.1 - 10.8 ppm
87 - 102 %

Nominal and measured concentrations:

nominal: 0.09, 0.15, 0.25, 0.42 and 0.70 mg/L
mean measured: 0.08, 0.12, 0.18, 0.30 and 0.52 mg/L (maximum solubility)

Details on test conditions:

TEST SYSTEM
- Test vessel: 18 L fish tanks
- Type (delete if not applicable): closed
- Material, size, fill volume: glass, 24.4 cm x 30.8 cm x 24.4 cm, fill volume 15.2 L
- Aeration: none
- Type of flow-through (e.g. peristaltic or proportional diluter): proportional diluter
- Renewal rate of test solution (frequency/flow rate): The diluter cycled approximately 6 cycles/hour (144 cycles per day). The diluter delivered approximately 1.0 L of test solution per replicate tank per cycle, or approximately 0.066 volume additions per cycle and approximately 9.5 volume additions per day.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2
- Biomass loading rate: 0.547 g/L static and 0.0578 g/L*day

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: filtered well water
- Total organic carbon: < 1.0 mg/L
- Total suspended solids: < 9.0 mg/L
- Metals (mg/L): Ba 0.23, Ca 16.8, Fe 0.47, Mg 3.84, Mn 0.043, K 2.57, Na 5.96, Zn 0.045, Cu 0.0066; 17 other metals were shown to be below detection limit
- Pesticides: all 25 pesticides analyzed were below detection limit
- Chloride: 8.2 mg/L
- Alkalinity: 58 mg/L
- Conductivity: 160 µmhos/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: Dissolved oxygen, pH, conductivity and temperature once daily

OTHER TEST CONDITIONS
- Photoperiod: 16:8, light:dark photoperiod, with gradual intensity changes at dawn and dusk
- Light intensity: 120 foot candles

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Observations for death, and for abnormal appearance and behavior were performed at 3, 6, 24, 48, 72, and 96 hours (± 1 hour).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.67
- Range finding study
- Test concentrations: 0.13, 0.22, 0.36, 0.60, and 1.0 mg/L
- Results used to determine the conditions for the definitive study: No mortality was observed in the control or solvent control test chambers. Total mortality was observed in the 0.60 and 1.0 mg/L treatments.

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.34 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95%, CL = 0.30 to 0.38

Details on results:

- Behavioural abnormalities: Loss of equilibrium, lethargy and immobility were observed at the three highest concentrations
- Mortality of control: No mortality or sublethal effects were observed in the control or solvent control treatments during the study.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: A precipitate was observed in the diluter mix box, on the walls of the "E" chambers, and floating on the surface of the treated test chambers during the study. The mean measured concentration of 0.52 mg/L represents the maximum solubility of the test substance.

Sublethal observations / clinical signs:

Table 1: Cumulative Mortality, days 0 - 4 for replicates

Nominal Concentr mg/L	Contr (1)	Contr (2)	Solv Contr (1)	Solv Contr (2)	0.09 (1)	0.09 (2)	0.15 (1)	0.15 (2)	0.25 (1)	0.25 (2)	0.42 (1)	0.42 (2)	0.70 (1)	0.70 (2)
Day 0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Day 1	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Day 2	0	0	0	0	0	0	0	0	0	0	0	0	9	10
Day 3	0	0	0	0	0	0	0	0	0	0	0	4	10	10
Day 4	0	0	0	0	0	0	0	0	0	0	2	4	10	10

Table 2: Additional information: Effect concentrations for 48 h and 72 h, 96 h NOEC/LOEC

Duration	Endpoint	Effect conc.	Nom./Meas.	Conc. based on	Basis for effect	Remarks
48 h	LC50	0.41 mg/L	meas. (arithm. mean)	test mat.	mortality	95% CL = 0.30 to 0.52 mg/L
72 h	LC50	0.36 mg/L	meas. (arithm. mean)	test mat.	mortality	95% CL = 0.32 to 0.39 mg/L
96 h	NOEC	0.12 mg/L	meas. (arithm. mean)	test mat.	mortality
96 h	LOEC	0.18 mg/L	meas. (arithm. mean)	test mat.	mortality

Table 3: Validity criteria for OECD 203.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality in the control(s) should not exceed 10% (or one fish if less than ten are used) at the end of the test	No mortality or sublethal effects were observed in the control or solvent control	yes
The dissolved oxygen concentration must have been at least 60 per cent of the air saturation value throughout the test	87 - 102 %	yes
There must be evidence that the concentration of the substance being tested has been satisfactorily maintained, and preferably it should be at least 80 per cent of the nominal concentration throughout the test. If the deviation from the nominal concentration is greater than 20 per cent, results should be based on the measured concentration.	71 - 86% Analytical measurement has been performed. Results are based on the measured concentration.	yes

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Description of key information

LC50 (96 h):0.22 mg/L (mean measured, Lepomis macrochirus, OECD 203)

Additional information

Two short term studies with fish were carried out for the evaluation of short term effects to freshwater fish in cold and warm water. The studies referred to herein were conducted according to the prevailing OECD testing guidelines (OECD 203) and GLP.

Based on analytically verified test concentrations in flow-through systems, the short term effects of the substance towards fish were found in the same range with an LC50 (96 h) of 0.34 mg/L for the rainbow trout (Oncorhynchus mykiss) and an LC50 (96 h) of 0.22 mg/L for the bluegill sunfish (Lepomis macrochirus).