2'-acetonaphthone

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study Initiation Date: November 24, 2020 to Study Completion Date: April 02, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Test chemical weighed individually for all concentrations & formulation was prepared in glass vial, then respective volumes of solution were transferred from vial to aquarium to obtain the required concentrations for the experiment

Vehicle:

yes

Remarks:

Acetone

Details on test solutions:

33.3 mg, 39.8 mg, 47.9 mg, 57.1 mg, 68.6 mg of testchemical were weighed individually and dissolve in 500 µL of acetone then transferred into respective glass aquariums and made up to 5000 mL in using natural water. Hence concentrations of 6.61, 7.93, 9.52, 11.42, 13.71 mg/L were achieved.
Natural water (control),
Acetone + natural water (vehicle control).
- Controls: Natural water (control),
Acetone + natural water (vehicle control).
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone
concentration of acetone 500µl/5000ml

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: Tarun Fish Farm, India
- Age at study initiation (mean and range, SD): Juveniles (originate from same source and population)
- Length at study initiation (length definition, mean, range and SD): 1.4 cm on average
- Weight at study initiation (mean and range, SD): 0.012 g on average

ACCLIMATION
- Acclimation period: 7 days
- Acclimation conditions (same as test or not): same as test
- Type and amount of food during acclimation: Commercial fish food.at ad libiitum
- Feeding frequency during acclimation: Daily
- Health during acclimation (any mortality observed): healthy


FEEDING DURING TEST
NO feeding during test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

165 mg CaCO3/L

Test temperature:

22.1-22.7 °C

pH:

6.4 to 7.1

Dissolved oxygen:

6.8-8.5 mg/L

Salinity:

-

Conductivity:

-

Nominal and measured concentrations:

Nominal Concentration - 0, 6.61, 7.93, 9.52, 11.42, 13.71 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquarium
- Material, size, headspace, fill volume: Glass aquarium 5 liters
- Aeration: no aeration duration test
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1 replicate
- No. of vessels per control (replicates): 1 replicate
- No. of vessels per vehicle control (replicates): 1 replicate
- Biomass loading rate: 0.012 g fish/L

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod: 16 h light- 8 h dark
- Light intensity: 732 to 804 Lux


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.2
- Range finding study : yes
- Test concentrations: 0 (control), 0 (vehicle control), 2.35, 4.23, 7.61, 13.71, 24.67 mg/L
- Results used to determine the conditions for the definitive study: No mortality or abnormality was found in control groups and 2.35, 4.23, 7.61 mg/L conc., but 100% mortality were found in 13.71, 24.67 mg/L concentrations. Hence, a main study was conducted using 0 (control), 0 (vehicle control), 6.61, 7.93, 9.52, 11.42, 13.71 mg/L concentrations.

Reference substance (positive control):

not required

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

10.44 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CI 10.09-10.97 mg/l

Details on results:

- Behavioural abnormalities: Head shaking, loss of buoyancy of control, corkscrew swimming
- Mortality of control: No
- Other adverse effects control: No
- Abnormal responses: Observed in 9.52, 11.42, 13.71 mg/L concentrations.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Based on the mortality data, LC50 value was determined statistically by probit analysis using NCSS Software 2019, Version 19.0.3.

Sublethal observations / clinical signs:

Table1: Percentage of Fish Mortality

Concentration (mg/L)	Mortality (%)
	6 hours	24 hours	48 hours	72 hours	96 hours
Range finding test
Control	0	0	0	0	0
Solvent control	0	0	0	0	0
2.35	0	0	0	0	0
4.23	0	0	0	0	0
7.61	0	0	0	0	0
13.71	0	100	100	100	100
24.67	0	100	100	100	100
Definitive test
Control	0	0	0	0	0
Solvent control	0	0	0	0	0
6.61	0	0	0	0	0
7.93	0	0	0	14.29	28.57
9.52	0	0	0	14.29	28.57
11.42	0	0	14.29	14.29	42.86
13.71	0	42.86	100	100	100

: LC₅₀Value of test item

Parameters	48 hours	72 hours	96 hours
LC50	12.07	11.59	10.44
95% confidence interval- Lower Fiducial limit	11.83	11.20	10.09
95% confidence interval - Upper Fiducial limit	12.31	11.98	10.79

Key:LC₅₀- Median Lethal Concentration

Appendix 1: Individual Fish Mortality

Concentration (mg/L)	Mortality (number)									Cumulative Mortality
	2.5h	5h	24h	30h	48h	54h	72h	78h	96h
Range finding test
Control	0	0	0	0	0	0	0	0	0	0
Solvent control	0	0	0	0	0	0	0	0	0	0
2.35	0	0	0	0	0	0	0	0	0	0
4.23	0	0	0	0	0	0	0	0	0	0
7.61	0	0	0	0	0	0	0	0	0	0
13.71	0	0	6	1	-	-	-	-	-	7
24.67	0	0	7	-	-	-	-	-	-	7
Definitive test
Control	0	0	0	0	0	0	0	0	0	0
Solvent control	0	0	0	0	0	0	0	0	0	0
6.61	0	0	0	0	0	0	0	0	0	0
7.93	0	0	0	0	0	0	1	0	1	2
9.52	0	0	0	0	0	0	1	0	1	2
11.42	0	0	0	0	1	0	0	0	2	3
13.71	0	0	3	0	3	1	-	-	-	7

Fish body weight and length record

S. No.	Body weight (g)	Length (cm)
Range finding test
1	0.011	1.2
2	0.010	1.3
3	0.014	1.2
4	0.013	1.2
5	0.012	1.3
6	0.035	1.1
7	0.018	1.2
8	0.010	1.4
9	0.023	1.2
10	0.012	1.2
Average	0.016	1.2
Definitive test
1	0.009	1.5
2	0.011	1.6
3	0.016	1.6
4	0.008	1.4
5	0.009	1.4
6	0.009	1.3
7	0.010	1.3
8	0.011	1.4
9	0.021	1.3
10	0.019	1.5
Average	0.012	1.4

Validity criteria fulfilled:

yes

Remarks:

No mortality in control or vehicle control were found throughout test duration. Dissolve oxygen concentration was maintained above 60% in all test vessels throughout the test. The recovery active ingredient content was found between 80-120% up to 96h.

Conclusions:

The 96 h LC50 of test chemical to zebrafish (Danio rerio) was determined to be 10.44 mg/L with 95% confidence upper and lower limit were found to be 10.79 and 10.09 mg/L, respectively.

Executive summary:

This study was conducted as per OECD 203 (2019) to assess the acute toxicity effects of test chemical on zebrafish (Danio rerio) following exposure up to 96 h under static condition. Juvenile fish of same age and normal in appearance were used in this (originate from same source and population). The average length and weight (10 fish) were observed 1.4 cm and 0.012g, respectively. Fish were fed (commercial fish food) daily during acclimatization. Photoperiod and light intensity were maintained such as16 h light- 8 h dark, 732-804 Lux during experiment. Hardness of water was measured once during acclimatization and found to be 165 mg CaCO3/L, temperature, pH and dissolve oxygen were maintained between 22.1-22.7 °C,6.4-7.1,6.8-8.5 mg/L, respectively, throughout the test. Fish were acclimatized for 7 days prior dosing. Natural water was used as dilution control and acetone in natural water (500µL/5000 mL conc) was used as vehicle control and the same concentration was used for formulation of test chemical. The study was initiated with a range finding test by using following concentrations of 0 (control), 0 (vehicle control), 2.35, 4.23, 7.61, 13.71, 24.67 mg/L. During range finding test, no mortality or abnormality was found in control groups and 2.35, 4.23, 7.61 mg/L conc., but 100% mortality were found in 13.71, 24.67 mg/L concentrations. Hence, a main study was conducted using 0 (control), 0 (vehicle control), 6.61, 7.93, 9.52, 11.42, 13.71 mg/L concentrations. No mortality or abnormality was found in control groups and 6.61, 7.93 mg/L concentration but abnormal behavioural responses like head shaking, loss of buoyancy of control, corkscrew swimming were observed in 9.52, 11.42, 13.71 mg/L conc. 7 fish were used/concentration during range finding and main study. HPLC method was used for method validation and active ingredient analysis along with stability of the test item in the test medium. Test item was found to be stable in the test medium. The active ingredient content results were considered acceptable (80-120%) as during main study 0 h and 96 h avg. recovery for 6.61, 9.52, 13.71 mg/L conc were 96.80%, 95.72%, 95.72% & 96.92%, 95.83%, 95.58%, respectively, were found in acceptable range. The test is valid as all the validity criteria are fulfilled: No mortality in control or vehicle control were found throughout the 96-h test duration; Dissolve oxygen conc. was maintained above 60% in all test vessels throughout the test; The recovery active ingredient content was found between 80-120% up to 96h. The 96 h LC50 of test chemical to zebrafish (Danio rerio) was determined to be 10.44 mg/L with 95% confidence upper and lower limit were found to be 10.79 and 10.09 mg/L, respectively. 96h LC50 value with 95% confidence limits (upper limit, lower limit) were calculated by probit analysis using NCSS Software 2019, version 19.0.3. Hence, as per CLP classification category, the test chemical can be categorized as aquatic chronic category 3.

Description of key information

This study was conducted as per OECD 203 (2019) to assess the acute toxicity effects of test chemical on zebrafish (Danio rerio) following exposure up to 96 h under static condition. Juvenile fish of same age and normal in appearance were used in this (originate from same source and population). The average length and weight (10 fish) were observed 1.4 cm and 0.012g, respectively. Fish were fed (commercial fish food) daily during acclimatization. Photoperiod and light intensity were maintained such as16 h light- 8 h dark, 732-804 Lux during experiment. Hardness of water was measured once during acclimatization and found to be 165 mg CaCO3/L, temperature, pH and dissolve oxygen were maintained between 22.1-22.7 °C,6.4-7.1,6.8-8.5 mg/L, respectively, throughout the test. Fish were acclimatized for 7 days prior dosing. Natural water was used as dilution control and acetone in natural water (500µL/5000 mL conc) was used as vehicle control and the same concentration was used for formulation of test chemical. The study was initiated with a range finding test by using following concentrations of 0 (control), 0 (vehicle control), 2.35, 4.23, 7.61, 13.71, 24.67 mg/L. During range finding test, no mortality or abnormality was found in control groups and 2.35, 4.23, 7.61 mg/L conc., but 100% mortality were found in 13.71, 24.67 mg/L concentrations. Hence, a main study was conducted using 0 (control), 0 (vehicle control), 6.61, 7.93, 9.52, 11.42, 13.71 mg/L concentrations. No mortality or abnormality was found in control groups and 6.61, 7.93 mg/L concentration but abnormal behavioural responses like head shaking, loss of buoyancy of control, corkscrew swimming were observed in 9.52, 11.42, 13.71 mg/L conc. 7 fish were used/concentration during range finding and main study. HPLC method was used for method validation and active ingredient analysis along with stability of the test item in the test medium. Test item was found to be stable in the test medium. The active ingredient content results were considered acceptable (80-120%) as during main study 0 h and 96 h avg. recovery for 6.61, 9.52, 13.71 mg/L conc were 96.80%, 95.72%, 95.72% & 96.92%, 95.83%, 95.58%, respectively, were found in acceptable range. The test is valid as all the validity criteria are fulfilled: No mortality in control or vehicle control were found throughout the 96-h test duration; Dissolve oxygen conc. was maintained above 60% in all test vessels throughout the test; The recovery active ingredient content was found between 80-120% up to 96h. The 96 h LC50 of test chemical to zebrafish (Danio rerio) was determined to be 10.44 mg/L with 95% confidence upper and lower limit were found to be 10.79 and 10.09 mg/L, respectively. 96h LC50 value with 95% confidence limits (upper limit, lower limit) were calculated by probit analysis using NCSS Software 2019, version 19.0.3. Hence, as per CLP classification category, the test chemical can be categorized as aquatic chronic category 3.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

10.44 mg/L

Additional information

This study was conducted as per OECD 203 (2019) to assess the acute toxicity effects of test chemical on zebrafish (Danio rerio) following exposure up to 96 h under static condition. Juvenile fish of same age and normal in appearance were used in this (originate from same source and population). The average length and weight (10 fish) were observed 1.4 cm and 0.012g, respectively. Fish were fed (commercial fish food) daily during acclimatization. Photoperiod and light intensity were maintained such as16 h light- 8 h dark, 732-804 Lux during experiment. Hardness of water was measured once during acclimatization and found to be 165 mg CaCO3/L, temperature, pH and dissolve oxygen were maintained between 22.1-22.7 °C,6.4-7.1,6.8-8.5 mg/L, respectively, throughout the test. Fish were acclimatized for 7 days prior dosing. Natural water was used as dilution control and acetone in natural water (500µL/5000 mL conc) was used as vehicle control and the same concentration was used for formulation of test chemical. The study was initiated with a range finding test by using following concentrations of 0 (control), 0 (vehicle control), 2.35, 4.23, 7.61, 13.71, 24.67 mg/L. During range finding test, no mortality or abnormality was found in control groups and 2.35, 4.23, 7.61 mg/L conc., but 100% mortality were found in 13.71, 24.67 mg/L concentrations. Hence, a main study was conducted using 0 (control), 0 (vehicle control), 6.61, 7.93, 9.52, 11.42, 13.71 mg/L concentrations. No mortality or abnormality was found in control groups and 6.61, 7.93 mg/L concentration but abnormal behavioural responses like head shaking, loss of buoyancy of control, corkscrew swimming were observed in 9.52, 11.42, 13.71 mg/L conc. 7 fish were used/concentration during range finding and main study. HPLC method was used for method validation and active ingredient analysis along with stability of the test item in the test medium. Test item was found to be stable in the test medium. The active ingredient content results were considered acceptable (80-120%) as during main study 0 h and 96 h avg. recovery for 6.61, 9.52, 13.71 mg/L conc were 96.80%, 95.72%, 95.72% & 96.92%, 95.83%, 95.58%, respectively, were found in acceptable range. The test is valid as all the validity criteria are fulfilled: No mortality in control or vehicle control were found throughout the 96-h test duration; Dissolve oxygen conc. was maintained above 60% in all test vessels throughout the test; The recovery active ingredient content was found between 80-120% up to 96h. The 96 h LC50 of test chemical to zebrafish (Danio rerio) was determined to be 10.44 mg/L with 95% confidence upper and lower limit were found to be 10.79 and 10.09 mg/L, respectively. 96h LC50 value with 95% confidence limits (upper limit, lower limit) were calculated by probit analysis using NCSS Software 2019, version 19.0.3. Hence, as per CLP classification category, the test chemical can be categorized as aquatic chronic category 3.