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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997-01-20 till 1997-10-06
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Klimisch 1a: GLP guideline study (OECD guideline 202, part I)

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
according to
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
yes (incl. certificate)

Test material

Details on test material:
- Name of test material (as cited in study report): phenylacetonitrile
- Physical state: liquid (colorless to yellow)
- Analytical purity: 99.6%
- Purity test date: 1995-06-13
- Lot/batch No.: 950602
- Other:
Date of receipt: 1995-06-22

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Concentrations:100, 58.82, 34.6, 20.35, 11.97, 7.04 and 4.14 mg/L
- Sampling: 40 µL of the test solutions were injected as they were used in the test

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:As the substance is poorly soluble, a saturated stock test solution was made by adding 100 mg of phenylacetonitrile to the test medium (1L). This mixture was agitated during 23h at room temperature under a hood and this resulted in a colorless, homogenous and transparant saturated stock solution from which the test solutions could be prepared.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
- Strain: clone 5 (also known as clone A)
- Source: The laboratory where the study was performed
- Age at study initiation: Less than 24h
- Feeding during test: No

No further data

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
48 h

Test conditions

No data
Test temperature:
20.5 - 21.5 °C
Time 0h: 7.94 (for the highest concentration)
Time 48h: 7.87 - 8.02
Dissolved oxygen:
Time 48h: 7.6 - 8.2 mg/L
Nominal and measured concentrations:
- Nominal concentrations:
100, 58.82, 34.60, 20.35, 11.97, 7.04, 4.14 mg/L

- Measured concentrations:
At time 0: 100.54, 60.58, 35.76, 21.21, 12.31, 7.01 and 3.81 mg/L
At time 48h: 91.4, 53.24 and 31.35 mg/L
Details on test conditions:
- Test vessel: flasks
- Type: closed (volatile test substance)
- Material, size, headspace, fill volume: 120 mL flasks entirely filled and closed with butyl rubber caps covered with PTFE
- Renewal rate of test solution: Not done
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 5 daphnids per 120 mL of test solution

- As test medium ISO test water was prepared as described in Annex 3 of the OECD guideline 202
- Source/preparation of dilution water: Ultrapure water (Ultrafiltration, active charcoal, ion exchange, filter 0.22 µm)
- Culture medium different from test medium: Yes, culture medium Elendt M7

- Spacing factor for test concentrations: 1.7
- Range finding study
Test concentrations: 0.1% to 100% (vol)
Results from the range finding study were used to determine the conditions for the definitive study
Reference substance (positive control):
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
24 h
Dose descriptor:
Effect conc.:
48 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: 95% CL of 26 to 94 mg/L; r² = 0.944
48 h
Dose descriptor:
Effect conc.:
57 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: r² = 0.963
Details on results:
- Immobilisation of control: after 48h 0% of the daophnids were immobilized
Results with reference substance (positive control):
- EC50(24h): 0.83 mg/L

Any other information on results incl. tables

- Detailed results of the acute immobilisation test:

Concentration     Immobilisation
Nominal Measured     
Volume of the standard stock solution(%) Initial (time 0h)(mg/L) Final (time 48h)(mg/L) Final/Initial(%)  24h(%) 48h(%)
100 100.54 91.40 90.91 100 100
58.82  60.58 53.24 87.88 90 80
34.60 35.76 31.25 87.67 5 0
20.35  21.21 NA NA 0 0
11.97  12.31 NA NA 0
7.04 7.01 NA NA 0
4.14 3.81 NA NA 0
0  0 0 / 0

NA: Not analysed

Applicant's summary and conclusion

Validity criteria fulfilled:
Under the test conditions described an EC50(24h) of 48 mg/L (95% CL 26 to 94 mg/L) and EC50(48h) of 57 mg/L were observed. Thus, phenylacetonitrile can be considered non-hazardous to Daphnids according to the CLP regulation (EC) N° 1272/2008.
Executive summary:

The authors tested the acute toxicity of phenylacetonitrile (CAS n° 140-29-4) to Daphnia magna in a 48h immobilization test according to the OECD guideline 202 (Daphnia sp., Acute Immobilisation Test) and to the EU Method C.2 (Acute Toxicity for Daphnia). The study was carried out in compliance with the principles of OECD Good Laboratory Practices (GLP). A range finding study was performed and based on these results a static test was performed with nominal concentrations of 100, 58.82, 34.60, 20.35, 11.97, 7.04 and 4.14 mg/L (geometric progression with a factor 1.7).

A total of 20 daphnids (i.e. 5 animals, 4 replicates) were exposed to the test substance and as negative control an equal amount of daphnids were tested in parallel. The test vessels were 120 mL flasks which were entirely filled with the test solutions and closed with butyl rubber caps covered with PTFE to avoid volatilisation of the test substance. Total exposure duration was 48h and at the start and end of the testing period different parameters were measured (pH, dissolved oxygen). After 24h and 48h immobility was recorded. Samples were taken to confirm the concentration of the test substance in the test medium with HPLC at the beginning and end of the test.

Under these test conditions, immobility was observed and EC50(24h) and EC50(48h) were calculated with measured initial concentrations by regression analysis using the Probit/log model. The EC50(24h) was 48 mg/L with 95% confidence interval ranging from 26 to 94 mg/L and the EC50(48h) was 57 mg/L.

In conclusion, the EC50(48h) of phenylacetonitrile was 57 mg/L based on an acute immobilisation test, thus the test substance can be considered non-hazardous to Daphnids according to the CLP regulation (EC) N° 1272/2008.

The study was performed according to OECD guideline 202 in compliance to GLP standards (certificate included) and all validity criteria were fulfilled. Therefore, the study can be considered a Klimisch 1 study (reliable without restrictions).