2'-acetonaphthone

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study Initiation Date: November 24, 2020 to Study Completion Date: April 23, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 0 (vehicle control), 6.0, 8.4, 11.8, 16.5, 23.0 mg/L.
- Sampling method: Test item was weighed and dissolved in acetone, then transferred into a volumetric flask and made up to the required volume using natural water and coded as stock. Test solutions of the selected concentrations were prepared by dilution of the stock solution. The solutions were prepared in natural water. Volumetric flaks and measuring cylinder were used for solution preparation.
- Sample storage conditions before analysis: sample was analysed immediately after preparation

Vehicle:

yes

Remarks:

Acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
-Method: 12.1 mg of test item was dissolve in 50 µL of acetone in glass vial then transferred and made up to 500 mL in a volumetric flask using natural water (stock). From the stock solution 37.19, 52.07, 73.14, 102.27, 142.56 mL were made up to 150 mL in a 250 mL measuring cylinder using natural water. Thus, concentrations of 6.0, 8.4, 11.8, 16.5, 23.0 mg/L were achieved.
- Controls: Natural water (Control), Acetone + natural water (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)): 0.1ml/L

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Source: Initially procured from “Microworms”. Later test system was sub-cultured in the test facility and same was used for the study
- Age at study initiation (mean and range, SD): <24 h
- Feeding during test : No

ACCLIMATION
- Acclimation period: 48 hours prior test
- Acclimation conditions (same as test or not): Yes (healthy gravid females were acclimatized)
- Type and amount of food: Food was provided during acclimatization.
- Health during acclimation (any mortality observed): healthy no mortality was observed

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

165 mg CaCO3/L

Test temperature:

20-21 °C

pH:

7.3-7.9

Dissolved oxygen:

6.8-7.2 mg/L

Salinity:

-

Conductivity:

-

Nominal and measured concentrations:

Nominal concentrations

Details on test conditions:

TEST SYSTEM
- Test vessel: 25 mL Beakers
- Type (delete if not applicable): open / closed Loosely covered with glass lid to reduce the loss of water due to evaporation and to avoid the entry of dust into the solutions.
- Material, size, headspace, fill volume: Glass, volume 20 mL
- Volume of solution: 20 ml
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 2 (Range Finding), 4 (main study)
- No. of vessels per control (replicates): 2 (Range Finding), 4 (main study)
- No. of vessels per vehicle control (replicates): 2 (Range Finding), 4 (main study)
- Biomass loading rate: 5 daphnids/20 mL test medium

OTHER TEST CONDITIONS
- Photoperiod: 16 h light- 8 h dark
- Light intensity: 1337-1346 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobilisation
VEHICLE CONTROL PERFORMED: yes/
- Spacing factor for test concentrations:2 (range finding study); 1.4 (main study)

RANGE-FINDING STUDY
- Test concentrations: 0 (control), 0 (vehicle control), 1.5, 3, 6, 12, 24 mg/L.
- Results used to determine the conditions for the definitive study:No immobilization or abnormality was found in control groups and 1.5, 3, 6.0 mg/L conc., but 20% and 100% mortality were found in 12, 24 mg/L concentrations, respectively. Hence, a main study was conducted using 0 (control), 0 (vehicle control), 6.0, 8.4, 11.8, 16.5, 23.0 mg/L concentrations.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

17.13 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % CI 16.21-18.05

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

20.34 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI - 18.89-21.79

Details on results:

- Mortality of control: No mortality in control
- Other adverse effects control: None
- Immobilisation of control: No immobilisation
- Abnormal responses: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes valid
EC50: 0.690 mg/L
95% confidence limits: Upper limit = 0.749 mg/L
Lower limit = 0.631 mg/L

Reported statistics and error estimates:

48h EC50 value with 95% confidence limits (upper limit, lower limit) were calculated by probit analysis using NCSS Software 2019, version 19.0.3.

Table1: Percentage ofDaphniaImmobilization

Concentration (mg/L)	Daphnia Immobilization (%)
	24 hours	48 hours	Cumulative Immobilization(%)
Range finding test
Control	0.0	0.0	0.0
Solvent control	0.0	0.0	0.0
1.5	0.0	0.0	0.0
3	0.0	0.0	0.0
6	0.0	0.0	0.0
12	0.0	20.0	20.0
24	50.0	50.0	100.0
Definitive test
Control	0.0	0.0	0.0
Solvent control	0.0	0.0	0.0
6.0	0.0	0.0	0.0
8.4	0.0	0.0	0.0
11.8	10.0	10.0	20.0
16.5	25.0	10.0	35.0
23.0	65.0	20.0	85.0

Table 2: LC₅₀Value of test item

Parameters	24 hours	48 hours
EC50	20.34 mg/L	17.13 mg/L
95% confidence interval- Lower Fiducial limit	18.89 mg/L	16.21 mg/L
95% confidence interval - Upper Fiducial limit	21.79 mg/L	18.05 mg/L

Key:LC₅₀- Median Lethal Concentration

Appendix 1: Individual Daphnia Immobilization – Range finding test

Concentration (mg/L)	Replication	Immobilization (Numbers)		Cumulative Immobilization (Numbers)  for period of 48 hours
		24 hours	48 hours
Control	R1	0	0	0
	R2	0	0
Solvent Control	R1	0	0	0
	R2	0	0
1.5	R1	0	0	0
	R2	0	0
3	R1	0	0	0
	R2	0	0
6	R1	0	0	0
	R2	0	0
12	R1	0	0	2
	R2	0	2
24	R1	2	3	10
	R2	3	2

Key: - Not applicable

Appendix 2: Individual Daphnia Immobilization – Definitive test

Concentration (mg/L)	Replication	Immobilization (Numbers)		Cumulative Immobilization (Numbers)  for period of 48 hours
		24 hours	48 hours
Control	R1	0	0	0
	R2	0	0
	R3	0	0
	R4	0	0
Solvent Control	R1	0	0	0
	R2	0	0
	R3	0	0
	R4	0	0
6.0	R1	0	0	0
	R2	0	0
	R3	0	0
	R4	0	0
8.4	R1	0	0	0
	R2	0	0
	R3	0	0
	R4	0	0
11.8	R1	0	1	4
	R2	1	0
	R3	0	1
	R4	1	0
16.5	R1	2	1	7
	R2	1	0
	R3	1	1
	R4	1	0
23.0	R1	2	1	17
	R2	3	1
	R3	5	0
	R4	3	2

Physicochemical Parameters (pH, Temperature (^oC) and DO (mg/L)

Concentration (mg/L)	pH		Temperature (⁰C)		Dissolved oxygen (mg/L)
	0 hour	48 hour	0 hour	48 hour	0 hour	48 hour
Range finding test
Control	7.1	7.0	20	20	8.5	8.2
Solvent control	7.0	7.0	20	20	8.8	8.0
1.5	6.9	6.8	20	21	8.2	7.6
3	7.0	6.9	21	21	7.9	7.8
6	7.0	6.9	20	20	8.0	7.5
12	6.8	6.7	20	21	8.1	7.0
24	6.9	6.8	21	21	8.3	6.8
Definitive test
Control	7.4	7.3	21	20	7.1	7.0
Solvent control	7.6	7.4	20	20	7.2	7.0
6.0	7.7	7.2	20	20	6.9	6.9
8.4	7.8	7.5	20	21	6.8	6.8
11.8	7.9	7.4	20	21	7.0	6.9
16.5	7.9	7.3	21	20	6.9	6.8
23.0	7.9	7.5	21	20	6.9	6.8

Validity criteria fulfilled:

yes

Remarks:

In the control, there was no daphnid found immobilized or other clinical signs throughout the test. The dissolved oxygen concentration at the end of the test was ≥ 3 mg/ L (6.8-7.2 mg/L) in control and test vessels

Conclusions:

The 24-h and 48-h EC50 of test chemical to daphnid, Daphnia magna are 20.34 and 17.13 mg/L, respectively. The 48-h EC50 of reference item to daphnid, Daphnia magna (found to be in acceptable range) is 0.690 mg/L. Hence, the results of the test with reference item establish the acceptability of the test system response, test procedures followed and results obtained with test item.

Executive summary:

This study was designed (as per OECD 202, adopted in 2004) to assess the acute toxicity of test chemical following exposure of daphnids up to 48h by static method.The brood daphnids were acclimatized 48 hours prior to the test item exposure. Less than 24 h old daphnids were collected from the acclimatized gravid females and exposed to the test item. After exposure on day 0, daphnids were observed for immobilization at 24 and 48 h. Natural water was used as control, acetone with natural water was used as vehicle control and the same was used for test item formulation and test medium (15 µL acetone/150 mL natural water concentration was used). 25 mL glass beakers having a solution volume of 20 mL were used in the test. A range finding test (2 replicates/concentration having 5 daphnids/replicate) with the test concentrations of 0 (control), 0 (vehicle control), 1.5, 3, 6, 12, 24 mg/L was done prior to main study. No immobilization or abnormality was found in control groups and 1.5, 3, 6.0 mg/L conc., but 20% and 100% mortality were found in 12, 24 mg/L conc, respectively. Hence, a main study (using a spacing factor of 1.4) was conducted using 0 (control), 0 (vehicle control), 6.0, 8.4, 11.8, 16.5, 23.0 mg/L concentrations. 4 replicates/concentration having 5 daphnids/replicate was used for the main study. Normal behavioural response and no immobilization (0% mortality) were observed up to 48 h followed by control groups and 6.0, 8.4 mg/L but 20%, 35% and 85% mortality were observed in the test concentrations of 11.8, 16.5, 23.0 mg/L, respectively. 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 48 h avg. recovery for 6.0, 11.8, 23.0 mg/L conc were 95.80%, 95.30%, 95.70% & 93.93%, 95.13%, 95.52%, respectively, were found in acceptable range. Hence the results were based on nominal concentration, since the deviation in the initial measured concentration didn’t exceed 20%. Environmental parameters such as pH (7.3-7.9), temperature (20-21 °C), dissolve oxygen (6.8-7.2 mg/L), hardness (165 mg CaCO3/L), conductivity (0.227 µS/cm), photoperiod (16 h light- 8 h dark) and light intensity (1337-1346 Lux) was maintained in acceptable range throughout the test. Feed was not provided during the test. The 24-h and 48-h EC50 of test chemical to daphnid,Daphnia magnaare 20.34 and 17.13 mg/L, respectively. The 48-h EC50 of reference item (Potassium dichromate) to daphnid,Daphnia magna(found to be in acceptable range) is 0.690 mg/L. Hence, the results of the test with reference item establish the acceptability of the test system response, test procedures followed and results obtained with test item. 48h EC50 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 designed (as per OECD 202, adopted in 2004) to assess the acute toxicity of test chemical following exposure of daphnids up to 48h by static method.The brood daphnids were acclimatized 48 hours prior to the test item exposure. Less than 24 h old daphnids were collected from the acclimatized gravid females and exposed to the test item. After exposure on day 0, daphnids were observed for immobilization at 24 and 48 h. Natural water was used as control, acetone with natural water was used as vehicle control and the same was used for test item formulation and test medium (15 µL acetone/150 mL natural water concentration was used). 25 mL glass beakers having a solution volume of 20 mL were used in the test. A range finding test (2 replicates/concentration having 5 daphnids/replicate) with the test concentrations of 0 (control), 0 (vehicle control), 1.5, 3, 6, 12, 24 mg/L was done prior to main study. No immobilization or abnormality was found in control groups and 1.5, 3, 6.0 mg/L conc., but 20% and 100% mortality were found in 12, 24 mg/L conc, respectively. Hence, a main study (using a spacing factor of 1.4) was conducted using 0 (control), 0 (vehicle control), 6.0, 8.4, 11.8, 16.5, 23.0 mg/L concentrations. 4 replicates/concentration having 5 daphnids/replicate was used for the main study. Normal behavioural response and no immobilization (0% mortality) were observed up to 48 h followed by control groups and 6.0, 8.4 mg/L but 20%, 35% and 85% mortality were observed in the test concentrations of 11.8, 16.5, 23.0 mg/L, respectively. 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 48 h avg. recovery for 6.0, 11.8, 23.0 mg/L conc were 95.80%, 95.30%, 95.70% & 93.93%, 95.13%, 95.52%, respectively, were found in acceptable range. Hence the results were based on nominal concentration, since the deviation in the initial measured concentration didn’t exceed 20%. Environmental parameters such as pH (7.3-7.9), temperature (20-21 °C), dissolve oxygen (6.8-7.2 mg/L), hardness (165 mg CaCO3/L), conductivity (0.227 µS/cm), photoperiod (16 h light- 8 h dark) and light intensity (1337-1346 Lux) was maintained in acceptable range throughout the test. Feed was not provided during the test. The 24-h and 48-h EC50 of test chemical to daphnid,Daphnia magnaare 20.34 and 17.13 mg/L, respectively. The 48-h EC50 of reference item (Potassium dichromate) to daphnid,Daphnia magna(found to be in acceptable range) is 0.690 mg/L. Hence, the results of the test with reference item establish the acceptability of the test system response, test procedures followed and results obtained with test item. 48h EC50 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 invertebrates

Fresh water invertebrates

Effect concentration:

17.13 mg/L

Additional information

This study was designed (as per OECD 202, adopted in 2004) to assess the acute toxicity of test chemical following exposure of daphnids up to 48h by static method.The brood daphnids were acclimatized 48 hours prior to the test item exposure. Less than 24 h old daphnids were collected from the acclimatized gravid females and exposed to the test item. After exposure on day 0, daphnids were observed for immobilization at 24 and 48 h. Natural water was used as control, acetone with natural water was used as vehicle control and the same was used for test item formulation and test medium (15 µL acetone/150 mL natural water concentration was used). 25 mL glass beakers having a solution volume of 20 mL were used in the test. A range finding test (2 replicates/concentration having 5 daphnids/replicate) with the test concentrations of 0 (control), 0 (vehicle control), 1.5, 3, 6, 12, 24 mg/L was done prior to main study. No immobilization or abnormality was found in control groups and 1.5, 3, 6.0 mg/L conc., but 20% and 100% mortality were found in 12, 24 mg/L conc, respectively. Hence, a main study (using a spacing factor of 1.4) was conducted using 0 (control), 0 (vehicle control), 6.0, 8.4, 11.8, 16.5, 23.0 mg/L concentrations. 4 replicates/concentration having 5 daphnids/replicate was used for the main study. Normal behavioural response and no immobilization (0% mortality) were observed up to 48 h followed by control groups and 6.0, 8.4 mg/L but 20%, 35% and 85% mortality were observed in the test concentrations of 11.8, 16.5, 23.0 mg/L, respectively. 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 48 h avg. recovery for 6.0, 11.8, 23.0 mg/L conc were 95.80%, 95.30%, 95.70% & 93.93%, 95.13%, 95.52%, respectively, were found in acceptable range. Hence the results were based on nominal concentration, since the deviation in the initial measured concentration didn’t exceed 20%. Environmental parameters such as pH (7.3-7.9), temperature (20-21 °C), dissolve oxygen (6.8-7.2 mg/L), hardness (165 mg CaCO3/L), conductivity (0.227 µS/cm), photoperiod (16 h light- 8 h dark) and light intensity (1337-1346 Lux) was maintained in acceptable range throughout the test. Feed was not provided during the test. The 24-h and 48-h EC50 of test chemical to daphnid,Daphnia magnaare 20.34 and 17.13 mg/L, respectively. The 48-h EC50 of reference item (Potassium dichromate) to daphnid,Daphnia magna(found to be in acceptable range) is 0.690 mg/L. Hence, the results of the test with reference item establish the acceptability of the test system response, test procedures followed and results obtained with test item. 48h EC50 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.