(2Z)-2-phenylhex-2-enenitrile

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:

11-05-2018 to 03-09-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: July 2016; signature: January 2017

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Definitive test: nominal concentrations: 0 (control), 1.0, 1.6, 2.6, 4.3 and 7.0 mg/L
Spacing factor (1.63) determined from range-finding test and physico-chemical properties of the substance.
Controls: test water without test item but treated in the same way as the test item solutions.
- Sampling method: Single samples for analysis were taken from the control and all test concentrations at the start and at the end of the test (t=48h)
- Sample storage conditions before analysis: Samples were used on day of sampling and analysed as soon as possible after their preparation.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock preparation: The mixing vessel was a cylindrical glass bottle sealed with screw cap and fitted with a drain port near the bottom for drawing off the saturated stock solution. The volume of the mixing vessel was approximately 1 L. A magnetic stirring bar was placed in the vessel and approx. 1 L test water was added. Then an excess of the test item (approx. 100 mg) was carefully added directly to the surface of the test water. The mixing vessel was thereafter closed immediately. The mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 24 hours of gentle stirring in the dark at room temperature, the contents of the vessel was allowed to stand undisturbed for at least 1 hour before use. The first 100 mL were discarded via the drain port. This was subject to analysis. The stock solution was diluted with test water as necessary to obtain the required test concentrations into 500 mL volumetric flasks based on measured concentration of the stock solution (ca. 22 mg/L). Each flask was inverted several times before filling the test vessels (without headspace) and introduction of the daphnids.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): stock-solution: ca. 22 mg/L ; final test solutions (range finder): nominal concentrations: 0 (control), 0.32, 1.0, 3.2, 10.0 and 21.0 mg/L ; final test solutions (definitive): nominal concentrations: 0 (control), 1.0, 1.6, 2.6, 4.3 and 7.0 mg/L.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None. Turbidity testing indicated absence of undissolved test item.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphinds
- Strain/clone: Daphnia magna (Straus), clone 5
- Justification for species other than prescribed by test guideline: Not applicable.
- Source: in-house laboratory cultures ; origin: LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine Campus Bridoux
- Age of parental stock (mean and range, SD): < 24 hours.
- Method of breeding: Parthenogenesis
- Feeding during test: No.
- Food type: Not applicable
- Amount: Not applicable.
- Frequency: Not applicable.

ACCLIMATION
- Acclimation period: None.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

Elendt M4 medium hardness: 250 mg/L expressed as CaCO3 and the pH: 6 to 9

Test temperature:

The temperature continuously measured in a temperature control vessel varied between 20.8 and 20.9°C during the test, and complied with the requirements as laid down in the protocol (18-22°C, constant within 1°C).

pH:

Test conditions remained within the limits prescribed by the guideline; pH: 7.90-8.20, not varying by more than 1.5 unit at the end of the test.

Dissolved oxygen:

Test conditions remained within the limits prescribed by the guideline; oxygen: > 3 mg/L (or > 60% ASV) at the end of the test.

Nominal and measured concentrations:

Range-finding test: nominal concentrations: 0 (control), 0.32, 1.0, 3.2, 10.0, and 21.0 mg/L
Definitive test: nominal concentrations: 0 (control), 1.0, 1.6, 2.6, 4.3 and 7.0 mg/L
Corresponding: geometric mean measured concentrations: 0 (control), 1.03, 1.72, 2.97. 4.54. 7.41 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL
- Type (delete if not applicable): closed without headspace; static
- Material, size, headspace, fill volume: Glass, no headspace.
- Aeration: No aeration of the test solutions.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable.
- Renewal rate of test solution (frequency/flow rate): Not applicable.
- No. of organisms per vessel: Control and test item: 20 per concentration, 5 per vessel containing 60 mL of test solution
- No. of vessels per concentration (replicates): 4 (four replicates, 5 daphnia per vessel).
- No. of vessels per control (replicates): 4 (four replicates, 5 daphnia per vessel).
- No. of vessels per vehicle control (replicates): Not applicable.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Not reported. Reconstituted water (Elendt M4 medium), as prescribed by OECD Guideline 202.
- Culture medium different from test medium: No.

OTHER TEST CONDITIONS
- Adjustment of pH: None.
- Photoperiod: 16 hours light; 8 hours dark photoperiod daily
- Light intensity: Not reported.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility (including mortality): 24 hours and at 48 hours.

VEHICLE CONTROL PERFORMED: Not applicable.

RANGE-FINDING STUDY
- Test concentrations: nominal concentrations: 0 (control), 0.32, 1.0, 3.2, 10.0 and 21.0 mg/L (highest concentration was maximum water solubility).
- Results used to determine the conditions for the definitive study: Yes.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

4.037 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 2.064 - 9.862 mg/L

Remarks:

based on geometric mean measured concentrations

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

3.046 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 2.575 - 3.590 mg/L

Remarks:

based on geometric mean measured concentrations

Details on results:

- Behavioural abnormalities: None
- Observations on body length and weight: Not applicable.
- Other biological observations: Not applicable.
- Mortality of control: No mortalities.
- Other adverse effects control: None.
- Abnormal responses: None.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Absence of undissolved test item in the stock solution was checked by determination of turbidity (ISO 7027, Water quality - Determination of turbidity). Turbidity analysis was not performed in compliance with the OECD GLP principles but in accordance with ISO 17025.Turbidity measurements revealed the absence of undissolved material in the stock solution, represented by turbidity analysis of the highest test concentration, in comparison with the turbidity value of the control.
- Effect concentrations exceeding solubility of substance in test medium: No.

Results with reference substance (positive control):

- Results with reference substance valid?: Yes.
- Mortality: None. Acute immobilisation observed only.
- EC50/LC50: 24h-EC50 was 1.31 mg/L (expected 24h-EC50 0.6 to 2.1 mg/L).
- Other: The sensitivity of this batch of test organism was in agreement with the historical data based on the most recent sensitivity test using potassium dichromate reference substance.

Reported statistics and error estimates:

Statistical analyses were performed by the computer program ToxRat® Professional, version 3.2.1. The probit model was used for estimation of the EC50 including the 95% confidence interval.

Table 1. pH and oxygen (mg/L) concentrations during the definitive test

		pH values		Dissolved oxygen concentration (mg/L)
Nominal Concentration (mg/L)	Geometric Mean Measured Concentration (mg/L)	Initial 0 hours	End 48 hours	Initial 0 hours	End 48 hours
Control	0 (Control)	8.20	7.90	8.34	8.24
1.0	1.03	8.11	8.00	8.44	8.28
1.6	1.72	8.10	8.04	8.42	8.35
2.6	2.97	8.15	8.09	8.43	8.30
4.3	4.54	8.16	8.05	8.44	8.12
7.0	7.41	8.11	8.09	8.36	8.26

All dissolved oxygen concentrations are > 60% air-saturation

 

Table 2. Number of introduced daphnids and incidence of immobility in the definitive test

Nominal concentration (mg test item/L)	Geometric Mean Measured Concentration (mg/L)	Replicate	Number of daphnids exposed	Response at 24h		Response at 48h
				Number	Total %	Number	Total %
Control	0 (Control)	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
1.0	1.03	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
1.6	1.72	1 2 3 4	5 5 5 5	2 0 0 0	10	2 1 1 0	20
2.6	2.97	1 2 3 4	5 5 5 5	2 0 0 2	20	2 1 2 2	35
4.3	4.54	1 2 3 4	5 5 5 5	1 1 4 3	45	5 3 5 3	80
7.0	7.41	1 2 3 4	5 5 5 5	5 5 5 5	100	5 5 5 5	100

Validity criteria fulfilled:

yes

Conclusions:

The test item 48h-EC50 was 3.046 mg/L (95% CL: 2.575 – 3.590 mg/L) based on geometric mean measured concentrations.

Executive summary:

The acute toxicity to Daphnia magna was examined according to OECD TG 202 Daphnia Sp., Acute Immobilisation Test and EU Method C.2 guidelines under GLP. Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed under close-static conditions to an aqueous solution of the test item over 48 hours at the required nominal test concentrations 1.0, 1.6, 2.6, 4.3 and 7.0 mg/L and to a control. The immobilisation of the daphnids was determined in a closed static 48-hour test by visual observation after 24 and 48 hours. Samples taken from the control and all test concentrations and subject to analysis at the start and end of the test. The test item levels were found to be relatively stable throughout the test, with test item losses within ± 20% of the initial and nominal concentrations, except for the measured concentration of 2.6 mg/L that was slightly higher at t=0h compared to its corresponding nominal concentration. Therefore, the evaluation of the effects on Daphnia magna was based on the geometric mean measured concentrations which were 1.03, 1.72, 2.97, 4.54 and 7.41 mg/L. After 48 hours of exposure, immobilisation rates were 0% at 1.03 mg/L, 20% at 1.72 mg/L, 35% at 2.97 mg/L, 80% at 4.54 mg/L and 100% at 7.41 mg/L, respectively. Under the conditions of this study, the EC50 values at each observation time were 24h: 4.037 (C.I. 2.064 – 9.862) mg/L and 48h: 3.046 (C.I. 2.575 – 3.590) mg/L based on geometric mean measured concentrations.

Description of key information

EC50-48h (invertebrates) = 3.046 mg/L (95% CL: 2.575 – 3.590 mg/L) based on geometric mean measured concentrations, 48-hour, freshwater, OECD 202, 2018

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

3.046 mg/L

Additional information

Key data : OECD TG 202, 2018 : The acute toxicity to Daphnia magna was examined according to OECD TG 202 Daphnia Sp., Acute Immobilisation Test and EU Method C.2 guidelines under GLP. Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed under close-static conditions to an aqueous solution of the test item over 48 hours at the required nominal test concentrations 1.0, 1.6, 2.6, 4.3 and 7.0 mg/L and to a control. The immobilisation of the daphnids was determined in a closed static 48-hour test by visual observation after 24 and 48 hours. Samples taken from the control and all test concentrations and subject to analysis at the start and end of the test. The test item levels were found to be relatively stable throughout the test, with test item losses within ± 20% of the initial and nominal concentrations, except for the measured concentration of 2.6 mg/L that was slightly higher at t=0h compared to its corresponding nominal concentration. Therefore, the evaluation of the effects on Daphnia magna was based on the geometric mean measured concentrations which were 1.03, 1.72, 2.97, 4.54 and 7.41 mg/L. After 48 hours of exposure, immobilisation rates were 0% at 1.03 mg/L, 20% at 1.72 mg/L, 35% at 2.97 mg/L, 80% at 4.54 mg/L and 100% at 7.41 mg/L, respectively. Under the conditions of this study, the EC50 values at each observation time were 24h: 4.037 (C.I. 2.064 – 9.862) mg/L and 48h: 3.046 (C.I. 2.575 – 3.590) mg/L based on geometric mean measured concentrations.