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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:
20 June 2016 to 12 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Reference:
Composition 0
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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
05 March 2015
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
- Chemical analyses: Single samples for analysis were taken from the control and all test concentrations.
- Frequency of sampling: At the start (t=0h) and the end of the test (t=48h).
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- The mixing vessel was a cylindrical glass bottle sealed with a screw cap and fitted with a drain port near the bottom for drawing off the saturated solution. The volume of the mixing vessel was approximately 5 L. A magnetic stirring bar was placed in the vessel and 5 L of test water (2.2.) was added in order to use a maximum volume and to minimise headspace. Then an excess of the test item (ca. 2 g) was carefully added directly to the surface of the test water and the mixing vessel was thereafter closed. 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. The stirring speed was as low as possible to maintain mixing of the water phase without dispersing the test substance in the water phase. After 24 +/- 2 hours of gentle stirring, the content of the vessel was allowed to stand undisturbed for at least 1 hour before use. The first 100 mL were removed via the drain port and samples were taken from the remaining stock solution and chemically analysed. Then the stock solution was diluted with test water as necessary into 500-mL volumetric flasks to obtain the required test concentrations into test vessels based on the measured concentration of the stock solution (24.3 mg.L-1). After filling (without headspace) and introduction of daphnids the test vessels were sealed immediately with screw caps. No small bubble was observed in the test vessels. The test solution was observed to be clear and colourless. The test was carried out without adjustment of the pH.

- Controls: Test water without test substance but treated in the same way as the test substance solutions.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain: Daphnia magna (Straus), clone 5
- Source: LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine Campus Bridoux - Bât. IBISE, 8, rue du Général Delestraint - 57070 METZ, bred in the Laboratoires des Pyrénées et des Landes.
- Daphnids originated from a healthy stock, showing no signs of stress such as mortality, presence of males, ephippia or discoloured animals.
- Age at study initiation: < 24 h
- Breeding Conditions: Daphnids were cultured in the Laboratoires des Pyrénées et des Landes under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in all-glass vessel containing test water. Cultures were maintained at a density of 1 adult daphnid per 25 mL of culture medium. Daphnids were fed at least three times a week with a suspension of algal cells (Pseudokirchneriella subcapitata) up to 0.1-0.2 mg C.Daphnia.-1day.-1. The water was changed at least once per week. These culture conditions maintained the daphnids in the parthenogenetic reproductive stage.
- Feeding during test: No feeding
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
None
Post exposure observation period:
None
Hardness:
Total water hardness was approximately 250 mg/L (as CaCO3)
Test temperature:
Between 20.7 and 20.8 °C throughout the test (average value: 20.8°C)
pH:
7.24 - 7.76
requirement: pH: 6.0-9.0, not varying by more than 1.5 units
Dissolved oxygen:
7.34 - 8.75 mg O2/L
Dissolved oxygen concentration at the end of the test was ≥ 60% of the air-saturation value in controls and test vessels
Salinity:
No data
Conductivity:
No data
Nominal and measured concentrations:
Nominal: 4.00; 5.20; 6.90; 9.00; 11.80; 15.40 and 20.20 mg test item.L-1
Corresponding average exposure concentrations (geometric means): 1.24; 2.32; 3.63; 4.94; 8.46; 12.80 and 18.81 mg Neryl Acetate.L-1
Details on test conditions:
TEST SYSTEM
- Test vessel: 60-mL glass flasks sealed with assembled screw cap with hole and PTFE/silicone septum. Each test vessel was uniquely identified with study code, replicate number, date of experimentation and concentration.
- Aeration: No aeration of the test solutions occurred throughout the test.
- Test type: static
- No. of daphnids: 20 per control and test concentration, divided into 4 groups of 5 animals
- Loading: 5 daphnids per vessel each completely filled with test solution and without headspace
- Introduction of Daphnids: Daphnids were introduced into the test vessel each completely filled with test solution and without headspace immediately after filling the test vessels with test solutions.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water as prescribed by the OECD Guideline 202.

OTHER TEST CONDITIONS
- Light regime: 16 h light : 8 h darkness

EFFECT PARAMETERS MEASURED:
- Immobility: Eventual immobility and abnormal behaviour were determined by visual observation after 24 and 48 hours. Daphnids were considered to be immobile if they were not able to swim within 15 seconds after gentle agitation of test vessels.
- pH and dissolved O2: At the beginning and at the end of the test from all test concentrations and control.
- Temperature of Medium: Measured continuously in a temperature controlled vessel next to the test vessels, over the study period, beginning at the start of the test.
Test conditions were recorded with suitable instruments and documented in the raw data.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 1.31
- Range finding study: Daphnids were exposed to the nominal test concentrations of 0.0; 1.0, 5.0, 10.0, 20.0 mg/L and a concentration close to the solubility limit of test item in test water (maximum solubility).
- Results used to determine the conditions for the definitive study: The percent immobility was 0, 0, 0, 0, 70 and 100 (24 h) and 0, 0, 40, 60, 100 and 100 (48 h) at 0.0; 1.0, 5.0, 10.0, 20.0 mg/L and the maximum solubility respectively. Based on the results of two range-finding tests, the following nominal test concentrations were prepared (spaced by a factor of approximately 1.31): 4.00, 5.20, 6.90, 9.00, 11.80, 15.40 and 20.20 mg test item.L-1.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
10.68 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits: 9.72 – 11.65 mg/L
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
9.97 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
neryl acetate + impurities
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits: 8.97 – 11.03 mg/L
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
9.06 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
neryl acetate only
Basis for effect:
mobility
Details on results:
After 48 hours of exposure, immobilisation rates were 0% at 1.24, 2.32, 3.63 and 4.49 mg.L-1, 20% at 8.46 mg.L-1, 90% at 12.80 mg.L-1 and 100% at 18.81 mg.L-1.
Therefore, the highest concentration without effect was 4.94 mg.L-1 and the lowest concentration with 100% immobilisation was 18.81 mg.L-1.
The 24 and 48-hour EC50 including the 95% confidence interval using Probit-analysis were determined by the computer program ToxRat.
The 48-hour EC50 for the entire test item was estimated to be 9.97 mg test item.L-1.
Results with reference substance (positive control):
On June 3, 2016 (most recent test at this time), the 24h-EC50 was 1.04 mg.L-1. Hence, the sensitivity of the clone of Daphnia magna was in agreement with OECD 202 (expected 24h-EC50: 0.6 mg.L-1 to 2.1 mg.L-1) at this time.
Reported statistics and error estimates:
The evaluation of the effects was based on the measured test item concentrations. The software ToxRat® Professional was used for the determination of the effective concentrations.

Table 6.1.3/1: Acute immobilisation of daphnids after 24 and 48 h in the final test

 

Nominal concentration* (mg/L) [measured concentration (mg Neryl Acetate.L-1)]

Replicate

Number of daphnids exposed

Response at 24 h

Response at 48 h

Number

Total %

Number

Total %

Control [control]

1

5

0

0

0

0

2

5

0

0

3

5

0

0

4

5

0

0

4.00 [1.24]

1

5

0

0

0

0

2

5

0

0

3

5

0

0

4

5

0

0

5.20 [2.32]

1

5

0

0

0

0

2

5

0

0

3

5

0

0

4

5

0

0

6.90 [3.63]

1

5

0

0

0

0

2

5

0

0

3

5

0

0

4

5

0

0

9.00 [4.94]

1

5

0

0

0

0

2

5

0

0

3

5

0

0

4

5

0

0

11.80 [8.46]

1

5

1

5

3

20

2

5

0

0

3

5

0

1

4

5

0

0

15.40 [12.80]

1

5

5

90

5

90

2

5

5

5

3

5

4

4

4

5

4

4

20.20 [18.81]  1  5  5 100  5 100
 2  5  5  5
 3  5  5  5
 4  5  5  5

Water quality parameters and environmental conditions throughout the test:

The results of measurement of pH and oxygen concentrations (mg.L-1) remained within the limits prescribed by the study plan (pH: 6.0-9.0, not varying by more than 1.5 units; oxygen:≥ 60% of the air-saturation valueat the end of the test).Furthermore, the temperature of the test medium was situated between 20.7 and 20.8 °C throughout the test (average value: 20.8°C), and complied with the requirements as laid down in the study plan (20°C ± 2°C, constant within 1°C).

 

Validity criteria of the study:

Controls: In the control, no daphnids became immobilised nor trapped at the surface of the water nor showed signs of stress.

Dissolved [O2]: Dissolved oxygen concentration at the end of the test was ≥ 60% of the air-saturation value in controls and test vessels.

Thus the validity criteria have been fulfilled in the present study.

Validity criteria fulfilled:
yes
Conclusions:
Based on measured exposure concentrations, the 48-hour EC50 value estimated was 9.97 mg test item.L-1 for the test item (neryl acetate + impurities). The 48-hour EC50 value estimated for neryl acetate only was 9.06 mg neryl acetate.L-1.
Executive summary:

A study was performed to assess the acute toxicity of test item NERYL ACETATE to Daphnia magna. The toxic effect of the test item was investigated in a static test, according to Guideline OECD 202, referenced as Method C.2 of Commission Regulation No. 440/2008, and was performed with GLP compliance. The criterion measured was the EC50 (Median Effective Concentration), a statistically derived concentration which is expected to cause immobility in 50% of test animals within a period of 48 hours.

 

Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed to an aqueous solution of the test item at the required nominal test concentrations of 4.00, 5.20, 6.90, 9.00, 11.80, 15.40 and 20.20 mg.L-1and to a control. The immobility 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 were analysed at the start and the end of the test in order to determine maintenance of actual concentrations of the test item (represented by analysis of neryl acetate (90.90% of the mixture)).

 

The analytical results of this test revealed unavoidable test item losses. As observed in the second range-finding test, it was assumed that losses of neryl acetate after 48h were clearly correlated with nerol appearance, one of the degradation products of neryl acetate. Since the deviation of the exposure concentrations of the test substance was greater than ± 20% of the initial concentrations, the results were expressed in terms of geometric means of the exposure concentrations: 1.24, 2.32, 3.63, 4.94, 8.46, 12.80 and 18.81 mg.L-1. After 48 hours of exposure, immobilisation rates were 0% at 1.24, 2.32, 3.63 and 4.94 mg.L-1, 20% at 8.46 mg.L-1, 90% at 12.80 mg.L-1and 100% at 18.81 mg.L-1.

The EC50 at each of the observation times was as follows:

- 24 h EC50: 10.68 mg/L (95% Confidence limits 9.72 - 11.65 mg/L)

- 48 h EC50: 9.97 mg/L (95% Confidence limits 8.97 - 11.03 mg/L)

The 48-hour EC50 for neryl acetate only was estimated to be 9.06 mg neryl acetate.L-1.

 

Therefore, based on measured exposure concentrations, the 48-hour EC50 value estimated was 9.97 mg test item.L-1 for the test item (neryl acetate + impurities). The 48-hour EC50 value estimated for neryl acetate only was 9.06 mg neryl acetate.L-1.

Description of key information

Neryl acetate exhibits a 48h-EC50 for freshwater invertebrates of 9.06 mg/L.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
9.06 mg/L

Additional information

One reliable study is available for the registered substance. The toxicity to freshwater invertebrates was investigated in a static test, according to Guideline OECD 202, referenced as Method C.2 of Commission Regulation No. 440/2008, and under GLP compliance. Daphnids (Daphnia magna) were exposed to an aqueous solution of the test item at nominal test concentrations of 4.00, 5.20, 6.90, 9.00, 11.80, 15.40 and 20.20 mg/L and to a control, over a test period of 48 hours (static conditions). Samples taken from the control and all test concentrations were analysed at the start and the end of the test in order to determine maintenance of actual concentrations of the test item (represented by analysis of Neryl Acetate (90.90% of the mixture)). The analytical results of this test revealed unavoidable test item losses. Since the deviation of the exposure concentrations of the test substance was greater than ± 20% of the initial concentrations, the results were expressed in terms of geometric means of the exposure concentrations: 1.24, 2.32, 3.63, 4.94, 8.46, 12.80 and 18.81 mg/L. Under the experimental conditions and based on measured exposure concentrations, the 48-hour EC50 value determined was 9.97 mg test item/L for the test item (neryl acetate + impurities). The 48-hour EC50 value determined for neryl acetate only was 9.06 mg neryl acetate/L.