Diphenylacetonitrile

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 0 (vehicle control), 0.25, 0.5, 1, 2, and 4 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 M7 medium. Volumetric flaks and measuring cylinder were used for solution preparation.
- Sample storage conditions before analysis: sample was analysed immediately after preperation

Vehicle:

yes

Remarks:

Acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
-Method: 10 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 M7 medium (stock). From the stock solution 12.5 ml, 25 ml, 50 ml, 100 ml, 200 mL were made up to 1 litre in a 1000 mL measuring cylinder using natural water. Thus, concentrations of 0.25, 0.5, 1.0, 2.0 and 4.0 mg/L were achieved.
- Controls: M7 medium (Control), Acetone + M7 medium (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: Daphnia obtained from DTU. 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:

166 mg CaCO3/L

Test temperature:

20-21 °C

pH:

7.2-7.8

Dissolved oxygen:

6.8-7.6 mg/L

Salinity:

-

Conductivity:

-

Nominal and measured concentrations:

Nominal concentrations: Control, solvent control, 0.25, 0.5, 1, 2 and 4 mg/L

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): 4 (main study)
- No. of vessels per control (replicates): 4 (main study)
- No. of vessels per vehicle control (replicates): 4 (main study)
- Biomass loading rate: 5 daphnids/20 mL test medium

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod: 16 h light- 8 h dark
- Light intensity: 1330-1352 Lux

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

RANGE-FINDING STUDY
- Test concentrations: Control, solvent control, 0.25, 0.5, 1, 2 and 4 mg/L ( main stuyd study concentrations)
- Results used to determine the conditions for the definitive study: The definitive study was conducetd based on the available information on the test substance.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % CI 1.86 - 2.33

Details on results:

- Mortality of control: No mortality in control and solvent 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

Table1: Percentage ofDaphniaImmobilization

Concentration (mg/L)	Daphnia Immobilization
	24 hours	48 hours	Cumulative Immobilization(%)
Definitive test
Control	0.0	0.0	0.0
Solvent control	0.0	0.0	0.0
0.25	0.0	0.0	0.0
0.5	1	1	10
1	1	2	15
2	3	3	30
4	10	8	90

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
0.25	R1	0	0	0
	R2	0	0
	R3	0	0
	R4	0	0
0.5	R1	1	0	2
	R2	0	1
	R3	0	0
	R4	0	0
1	R1	0	2	3
	R2	0	0
	R3	0	0
	R4	1	0
2	R1	0	0	6
	R2	1	2
	R3	1	1
	R4	1	0
4	R1	2	2	18
	R2	0	4
	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
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	7.6	7.2	20	20	6.9	6.9
8.4	7.6	7.5	20	21	6.9	6.8
11.8	7.6	7.4	20	21	7.6	6.9
16.5	7.6	7.3	21	20	6.9	6.8
23.0	7.6	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 (6.8-7.6 mg/L) in control and test vessels

Conclusions:

The 48-h EC50 of the test chemical was reported to be 2.10 mg/l with 95% CI- 1.86 - 2.33 mg/L

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. M7 medium was used as control, acetone with M7 medium was used as vehicle control and the same was used for test item formulation and test medium (50 µL acetone/500 mL medium concentration was used). 25 mL glass beakers having a solution volume of 20 mL were used in the test. A main study (using a spacing factor of 2) was conducted using 0 (control), 0 (vehicle control), 0.25, 0.5, 1, 2, 4 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 0.25 conc. but 10%, 15%, 30, and 90% mortality were observed in the test concentrations of 0.5, 1, 2, 4 mg/L, respectively. The analytial monitoring was done, and the test chemicals were maintained within the acceptable range i.e., 80 -120% of the nominal concentrations. 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 (7.2-7.8), temperature (20-21 °C), dissolve oxygen (6.8-7.6 mg/L), hardness (166 mg CaCO₃/L), conductivity (0.227 µS/cm), photoperiod (16 h light- 8 h dark) and light intensity (1330-1352 Lux) was maintained in acceptable range throughout the test. Feed was not provided during the test. The 48-h EC₅₀of test chemical to daphnid,Daphnia magnaare 2.10mg/L. The 48-h EC₅₀of 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.

Thus, based on the EC50 value, test chemical can considered as toxic and hazardous to aquatic invertebrates. Thus, chemical can be classified into aquatic chronic category 2 as per CLP classification criteria

Description of key information

Short term toxicity to aquatic invertebrates

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. M7 medium was used as control, acetone with M7 medium was used as vehicle control and the same was used for test item formulation and test medium (50 µL acetone/500 mL medium concentration was used). 25 mL glass beakers having a solution volume of 20 mL were used in the test. A main study (using a spacing factor of 2) was conducted using 0 (control), 0 (vehicle control), 0.25, 0.5, 1, 2, 4 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 0.25 conc. but 10%, 15%, 30, and 90% mortality were observed in the test concentrations of 0.5, 1, 2, 4 mg/L, respectively. The analytial monitoring was done, and the test chemicals were maintained within the acceptable range i.e., 80 -120% of the nominal concentrations. 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 (7.2-7.8), temperature (20-21 °C), dissolve oxygen (6.8-7.6 mg/L), hardness (166 mg CaCO₃/L), conductivity (0.227 µS/cm), photoperiod (16 h light- 8 h dark) and light intensity (1330-1352 Lux) was maintained in acceptable range throughout the test. Feed was not provided during the test. The 48-h EC₅₀of test chemical to daphnid,Daphnia magnaare 2.10mg/L. The 48-h EC₅₀of 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. Thus, test chemical is considered as toxic and hazardous to aquatic invertebrates. thus chemical can be classified into aquatic chronic category 2 as per CLP classification criteria

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

2.1 mg/L

Additional information

Experimental study of the test chemical and various supporting weight of evidence studies for its read across chemical were reviewed for the short term toxicity to aquatic invertebrates end point which are summarized as below:

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. M7 medium was used as control, acetone with M7 medium was used as vehicle control and the same was used for test item formulation and test medium (50 µL acetone/500 mL medium concentration was used). 25 mL glass beakers having a solution volume of 20 mL were used in the test. A main study (using a spacing factor of 2) was conducted using 0 (control), 0 (vehicle control), 0.25, 0.5, 1, 2, 4 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 0.25 conc. but 10%, 15%, 30, and 90% mortality were observed in the test concentrations of 0.5, 1, 2, 4 mg/L, respectively. The analytial monitoring was done, and the test chemicals were maintained within the acceptable range i.e., 80 -120% of the nominal concentrations. 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 (7.2-7.8), temperature (20-21 °C), dissolve oxygen (6.8-7.6 mg/L), hardness (166 mg CaCO₃/L), conductivity (0.227 µS/cm), photoperiod (16 h light- 8 h dark) and light intensity (1330-1352 Lux) was maintained in acceptable range throughout the test. Feed was not provided during the test. The 48-h EC₅₀of test chemical to daphnid,Daphnia magnaare 2.10mg/L. The 48-h EC₅₀of 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. Thus, test chemical is considered as toxic and hazardous to aquatic invertebrates. thus chemical can be classified into aquatic chronic category 2 as per CLP classification criteria

 

In an experimental study from study report (2017), an acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on Daphnia magna. The test was performed in accordance to OECD guideline No. 202 “Daphnia sp., Acute Immobilization Test”. Own breeding stock at University of Chemistry and Technology, Prague of Daphnia magna was used as a test organism for the study. The stock solution 10 g/l was prepared by dissolving white powder in acetone. Test solutions of required concentrationas were prepared by mixing the stock solution of the test sample in reconstituted test water. Nominal test chemical conc. used for the study were 0, 0.25, 0.5, 1, 2, 4 and 8 mg/L, respectively. Study was performed using total 5 organisms per vessel/replicates in a static fresh water system. Daphnids were exposed to test chemical in 50 ml glass vessel in a volume of 25 ml of liquid solution containing both the chemical and media. The beakers were placed in a room at a temperature of 20±1°C. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. On the basis of the effect of test chemical on the mobility of the test organism Daphnia magna, the 48 hr EC50 value was determined to be 2.27 mg/l with a 95% confidence interval value ranging from 1.64 to 3.14 mg/l, respectively.

 

Another short term toxicity to aquatic invertebrate study was conducted for 48 hrs for assessing the effect of test chemical (Experimental study report, 2017). The test was performed in accordance to OECD guideline No. 202 “Daphnia sp.,Acute Immobilization Test’’ using the procedure as mentioned above under the same test conditions . The stock solution 100 g/l was prepared by dissolving white powder in acetone. Test solutions of required concentrations was prepared by mixing the stock solution of the test sample in reconstituted test water. Nominal test chemical conc. used for the study were 0, 1.25, 2.5, 5, 10 and 20 mg/L, respectively. On the basis of the effect of test chemical on the mobility of the test organism Daphnia magna, the 48 hr EC50 value was determined to be 5.7 mg/l with a 95% confidence interval value ranging from 4.1 to 8.1 mg/l, respectively.

 

For the test chemical, short term toxicity to aq. Invertebrate study was conducted for 48hrs for assessing the effect of test chemical (Secondary source, 2018). Ceriodaphnia dubia (Water flea) (< 8 hr (neonates)) was used as a test organism for the study. Test was performed in 4 replicates under static conditions which involve pH 7.9 to 8.4, hardness 80-90 mg/l CaCO3 and alkalinity 60-80 mg/l CaCO3, respectively. On the basis of mobility of the test organism Ceriodaphnia dubia, the 48 hrs LC50 value was determined to be 3.94 mg/l (95% C. I. – 3.77 to 4.15 mg/l).

 

On the basis of the above results, it can be concluded that the test chemical can be considered as toxic and hazardous to aquatic invertebrate. Thus, test chemical is considered as toxic and hazardous to aquatic invertebrates. thus chemical can be classified into aquatic chronic category 2 as per CLP classification criteria