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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 January 2010 to 12 February 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 100, 56, 32, 18, 10, 5.6, 3.2, 18. and 1.0 mg/L.
Water samples were taken fro mthe control, 1.0, 3.2, 10, 32 and 100 mg/L test groups (replicates R1- R2 pooled) at 0 (fresh media), 24 (old and fresh media) and 48 hours (old media) for immediate quantitative analysis. Duplicate samples were taken and stored at approximately 20 °C.
Vehicle:
no
Details on test solutions:
For the purpose of the definitive test the test item was dissolved directly in water.

An amount of test item (200 mg) was dissolved in reconstituted water and the volume adjusted ti 2 litres to give the 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 56, 32, 18, 10, 5.6, 3.2, 18. and 1.0 mg/L.

Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

The concentration, homogeneity and stability of the test item in the test preparations were verified by chemical analysis at 0 (fresh media), 24 (old and fresh media) and 48 hours (old media).
Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using first instar Daphnia magna derived from in-house laboratory cultures.

Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at approximately 20°C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a suspendion of algae (Chlorella sp.) Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were approximately 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
Not applicable
Hardness:
Not reported
Test temperature:
Overall range - 19 - 20 °C
pH:
Overall range 7.6 - 8.0
Dissolved oxygen:
Overall range - 8.8 - 9.7 mg O2/L; 97-107 % dissolved oxygen concentration

At 0-hours, and in some instances at 24 hours the oxygen concetrations in the controls and test vessels was observed to be in excess of 100 % of air saturation. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects of exposure were observed in the control group throughout the duration of the test.
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: control (0), 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/L
Measured concentrations were determiend in the 1.0, 3.2, 10, 32 and 100 mg/L treatments at the start and end of each test medium renewal. Mean measured concentrations were as follows: 0.906, 2.98, 10.1, 32.4 and 103 mg/L.
As the measured concentrations were within 80 to 120% of nominal concentrations athe results are based on the nominal concentrations.
Details on test conditions:
250 mL glass jars containing approximately 200 mL of test preparation were used. At the start of the test 10 daphnids were placed in each test and control vessel at random, in the test preparations. Duplicate test vessels were used for each test and control group. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at approximately 20 °C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.

The control group was maintained under identical conditions but not exposed to the test item.

Given that the preliminary stability analyses conducted indicated that the test item was unstable over a 48-hour period semi-static test conditions were employed in the test in an effort to maintain near nominal test concentrations. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferrred by wide bore pipette from the 24-hour old test media into the fresh test media.

Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

Reference substance (positive control):
yes
Remarks:
Potassium Dichromate
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % CL: 20 - 30 mg/L
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
17 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % CL: 14 - 20 mg/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Observation on test item solubility:
The test preparations were obvserved to be clear, colourless solutions throughout the duration of the test.

Verification of test concentration:
Analysis of the test preparations at 0 (new), 24 (new and old) and 48 hours (old) showed measured test concentrations to range from 81 % to 106 % of nominal value and so it was considered justifiable to calculate the EC50 values in terms of the nominal test concentrations only.

Results with reference substance (positive control):
Analysis of the immobilisation data by the probit method (Finney 1971) at 24 hours and the trimmed Spearman-karber method (Hamilton et al 1977) at 48 hours based on the nominal test concentrations gave the following results:
EC50 (24 h): 0.84 mg/L; 95 % CL: 0.72 - 0.97 mg/L
EC50 (48 h): 0.65 mg/L; 95 % CL: 0.58 - 0.72 mg/L
The NOEC after 24 and 48 hours was 0.32 mg/L.
The slope and its standard error of the response curve at 24 hours was 7.7 (SE = 1.6). Due to the unsuitable nature of the data it was not possible to calculate the slope and error of response curve at 48 hours.
The results from the positive control with potassium dichromate were within the normal range for this reference item. The mean 48-hour EC50 value calculated from all positive controls was 0.77 mg/L (sd = 0.20).
Reported statistics and error estimates:
Analysis of the immobilisation data by the probit method (Finney 1971) at 24 and 48 hours based on the nominal test concentrations gave the followin results:
24-hour EC50: 25 mg/L; 95 % CL: 20 - 30 mg/L
48-hour EC50: 17 mg/L; 95 % CL: 14 - 20 mg/L
The NOEC after 24 and 48 hours exposure was 10 mg/L. The NOEC is based upon no significant (<10%) immobilisation at this concentration.

The slopes and their standard errors fo the response curves at 24 and 48 hours were 3.7 (SE = 0.55) and 5.4 (SE = 0.93) respectively.

Analytical results

Nominal conc. (mg/L)

Measured concentration (mg/L)

Mean measured (mg/L)

% of nominal

0 hours (new)

24 hours (old)

24 hours (new)

48 hours (old)

Control

<LOQ

<LOQ

<LOQ

<LOQ

-

-

1.0

0.977

0.806

0.978

0.861

0.906

91

3.2

3.03

2.71

3.25

2.93

2.98

93

10

10.3

9.64

10.4

10.0

10.1

101

32

33.7

31.0

32.4

32.6

32.4

101

100

106

96.0

103

105

103

103

Immobility results

Nominal conc. (mg/L)

Cumulative immobilisedDaphnia

% immobility

24 hours

48 hours

24 hours

48 hours

Control

0

0

0

0

1.0

0

0

0

0

1.8

0

0

0

0

3.2

0

0

0

0

5.6

1

1

5

5

10

0

0

0

0

18

5

11

25

55

32

16

19

80

95

56

17

20

85

100

100

20

20

100

100

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the benzyl acetate to the freshwater invertebrate Daphnia magna was investigated. The 48 -hour EC50 was 17 mg/L (95 % confidence limits 14 -20 mg/L), the corresponding no-observed effect concentration was 10 mg/L.
Executive summary:

A study was performed to assess the acute toxicity of the benzyl acetate to Daphnia magna. The method followed that described in the OECD Guidelines for Testing of Chemicals No. 202.

Following a preliminary range-finding test, twenty daphnids (2 replicates of 10 animals) were exposed for 48 hours at a temperature of approximately 20°C under semi-static conditions. Nominal exposure concentrations were 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/L. Mean measured concentrations in the nominal 1, 3.2, 10, 32 and 100 mg/L treatments were 0.906, 2.98, 10.1, 32.4 and 103 mg/L. As the results were within 80 and 120% of nominal concentrations the results of the study are based on the nominal exposure concentrations. 

The 48 -hour EC50 was 17 mg/L (95% confidence limits 14 - 20 mg/L), the corresponding no-observed effect concentration was 10 mg/L.

Description of key information

A study was performed to assess the acute toxicity of the benzyl acetate to Daphnia magna. The method followed that described in the OECD Guidelines for Testing of Chemicals No. 202.
Following a preliminary range-finding test, twenty daphnids (2 replicates of 10 animals) were exposed for 48 hours at a temperature of approximately 20°C under semi-static conditions. Nominal exposure concentrations were 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/L. Mean measured concentrations in the nominal 1, 3.2, 10, 32 and 100 mg/L treatments were 0.906, 2.98, 10.1, 32.4 and 103 mg/L. As the results were within 80 and 120% of nominal concentrations the results of the study are based on the nominal exposure concentrations. 
The 48-hour EC50 was 17 mg/L (95% confidence limits 14 - 20 mg/L), the corresponding no-observed effect concentration was 10 mg/L.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
17 mg/L

Additional information

The acute toxicity of the benzyl acetate to the freshwater invertebrate Daphnia magna was investigated (Vyrenhoef, H. and Mullee, D.M. 2010). The 48-hour EC50 was 17 mg/L (95% confidence limits 14 - 20 mg/L), the corresponding no-observed effect concentration was 10 mg/L.