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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:
From August 26 to September 23, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
Two minor deviations: Water hardness was 252 mg/L as CacO3, temperature situated between 21.4 and 22.7°C
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Qualifier:
according to
Guideline:
ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.5, 0.8, 1.3, 2.0, 3.2, 5 mg/L
The concentrations of the test item were determined by chemical analyses at the start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h).
- Sampling method: The samples were injected directly after a dilution by a factor of two with acetonitrile (500 μL sample + 500 μL acetonitrile). If the sample concentration was too high and not included in the concentration range of the calibration, the samples were diluted appropriately with a test water/acetonitrile (50/50 v/v) mixture. The samples were injected as soon as possible after their preparation.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The standard test procedures require preparation of test solutions, which contain completely dissolved test substance concentrations or stable and homogeneous mixtures. Two stock solutions (one for the fresh medium at t=0h and another for the fresh medium at 24h) were prepared by slow-stirring. 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 stock solution. The volume of the mixing vessel was approximately 1 L. A magnetic stirring bar was placed in the vessel and test water (2.2.) was added. Then 10 mg (nominal) of the test item (actual measured amount: 11.18 mg for the fresh medium at t=0h and 12.43 mg for the fresh medium at t=24h) were weighed on a weighing boat that afterwards was placed above the mixing vessel and rinsed with test water. The mixing vessel was then carefully filled with the remaining volume of test water to obtain 1L of stock solution and thereafter was closed immediately. The mixing was initiated with the vortex in the centre extending maximally around 10-20% vessel depth from the top to the bottom of the vessel. The stirring speed was kept 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 at room temperature, the content of the vessel was allowed to stand undisturbed for ca. 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 extracted via the drain port and diluted with test water as necessary into 200-mL volumetric flasks to obtain the required test concentrations based on the measured concentration of the stock solution (estimated concentration for the fresh medium at t=0h: 10.8 mg.L-1 and for the fresh medium at t=24h: 11.2 mg.L-1). Each prepared concentration was inverted several times before filling test tubes to ensure adequate mixing and homogeneity. After filling and introduction of daphnids the vessels were sealed immediately. No bubbles were observed in the test tubes. The test solution was observed to be clear and colourless at all test concentrations. The test was carried out without adjustment of the pH.
- Controls : yes
- Reference substance : Potassium dichromate
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia Magna
- 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.
- Age at study initiation: < 24 hours old
- Method of breeding: Daphnids were cultured in the Laboratoires des Pyrénées et des Landes under similar temperature and light conditions as used in the test.
- No feeding


ACCLIMATION
At least 48 hours prior to the start of the test, gravid daphnids were transferred to OECD test water and held at similar temperature and light conditions as used in the test. During this period, daphnids were fed in the same manner as that of the stock population. Only daphnids up to 24 hours old were used for the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
252 mg/L as CaCO3
Test temperature:
21.4-22.7 °C, average: 22.1 °C
pH:
pH: 7.71- 8.04
Dissolved oxygen:
≥ 60% of the air-saturation value at the end of the test
Nominal and measured concentrations:
Nominal (measured) concentrations: 0.5 (0.48), 0.8 (0.70), 1.3 (1.13), 2.0 (1.82), 3.2 (3.15), 5 (4.86) mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: All-glass test tubes of approximately 20 mL capacity sealed with screw caps. 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.
- Semi-static test.
- Daily renewal.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Dilution water: deionised water.
- Test medium: reconstituted water, as prescribed by the OECD Guideline 202
- CaCl2.2H2O 11.76 g.L-1
- MgSO4.7H2O 4.93 g.L-1
- NaHCO3 2.59 g.L-1
- KCl 0.23 g.L-1
- Conductivity: < 10 μS.cm-1
- Culture medium different from test medium: Yes
- Intervals of water quality measurement: t0, t24h, t48h, (temperature measured continuously)

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16h light/8h dark

EFFECT PARAMETERS MEASURED :
- Immobility and abnormal behaviour recorded at 24 and 48h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.58
- Range finding study
- Nominal test concentrations: 0.5, 1.0, 2.0, 5.0 and 10 mg test item/L
- Results used to determine the conditions for the definitive study: After 48 hours of exposure, immobilisations were 0% at 0.5 mg/L, 10% at 1.0 mg/L, 40% at 2.0 mg/L and 100% at 5.0 and 10 mg/L.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
2.59 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 2.26 - 2.94 mg/L
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.34 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 1.18 - 1.54 mg/L
Results with reference substance (positive control):
On August 14, 2015 (most recent test), the 24h-EC50 was 0.91 mg.L-1. Hence, the sensitivity of the clone of Daphnia magna was in agreement with the OECD 202 (1) (expected 24h-EC50: 0.6 mg.L-1 to 2.1 mg.L-1) at this time.
Reported statistics and error estimates:
The 24 and 48-hour EC50 including the 95% confidence interval using Probit-analysis were determined by the computer program ToxRat.

Function : S= 10^(1/b)

- Parameters of the probit analysis for Mobility at 24h:
Results of the regression analysis
Computation runs: 9
Slope b: 8,81630
Intercept a: -3,64079
Variance of b: 2,97347
Goodness of Fit
Chi²: 0,27193
Degrees of freedom: 4
p(Chi²): 0,99155
Log EC50: 0,41296
SE Log EC50: 0,02672
g-Criterion: 0,14696
F: 384,507
p(F) (df: 1;4): 0,000
- Parameters of the probit analysis for Mobility at 48h:
Computation runs: 8
Slope b: 7,44517
Intercept a: -0,95445
Variance of b: 2,03278
Goodness of Fit
Chi²: 0,99924
Degrees of freedom: 4
p(Chi²): 0,90991
Log EC50: 0,12820
SE Log EC50: 0,02798
g-Criterion: 0,14088
F: 109,157
p(F) (df: 1;4): 0,000

Acute Immobilisation of Daphnids after 24 and 48 Hours in the Final Test:

Nominal Concentration (mg test item.L-1) Average Measured Concentration (mg test item.L-1) Replicate Number of Daphnids Exposed Response at 24h  Response at 48h
Number  Total % Number  Total %
Control 0 1 5 0 0 0 0
2 5 0 0
3 5 0 0
4 5 0 0
0.5 0.48 1 5 0 0 0 0
2 5 0 0
3 5 0 0
4 5 0 0
0.8 0.7 1 5 0 0 0 0
2 5 0 0
3 5 0 0
4 5 0 0
1.3 1.13 1 5 0 0 0 35
2 5 0 1
3 5 0 3
4 5 0 3
2 1.82 1 5 1 10 4 80
2 5 0 4
3 5 0 4
4 5 1 4
3.2 3.15 1 5 2 75 5 100
2 5 4 5
3 5 4 5
4 5 5 5
5 4.86 1 5 5 100 5 100
2 5 5 5
3 5 5 5
4 5 5 5
Validity criteria fulfilled:
yes
Conclusions:
The toxic effect of the test item to the freshwater invertebrate Daphnia magna was investigated in a semi-static test. Under the experimental conditions and based on the average of measured exposure concentrations, the 48-hour EC50 value estimated was 1.34 mg/L.
Executive summary:

An acute aquatic toxicity study was performed on Daphnia magna in accordance with GLP and OECD Guideline 202. Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids/replicate) were exposed to an aqueous solution of the test item, at the required nominal test concentrations of 0.5, 0.8, 1.3, 2.0, 3.2 and 5 mg/L. The immobility of the daphnids was determined in a semi-static 48h test by visual observation after 24 and 48 h. The concentrations of the test item were determined by chemical analyses at the start (t = 0 h), at t = 24 h (new and old solutions) and at the end of the test (t = 48 h). Although concentrations measured in freshly prepared solutions and in 24-hour old solutions revealed that the concentration of the test item was satisfactorily maintained within ± 20% of the initial concentration during the first day, losses of ca. 30% were recorded during the second exposure period (t=24hFresh-t=48h) at 1.3 and 3.2 mg/L (nominal).Therefore, since the deviation of the exposure concentrations of the test substance was greater than 20% of the initial concentrations only for two test concentrations, it was decided to express the biological results in terms of geometric means of the exposure concentrations for the present study: 0.48, 0.70, 1.13, 1.82, 3.15, and 4.86 mg/L. After 24 hours of exposure, immobilisations were 0% at 0.48, 0.70 and 1.13 mg/L 10% at 1.82 mg/L, 75% at 3.15 mg/L and 100% at 4.86 mg test item/L. After 48 hours of exposure, immobilisations were 0% at 0.48 and 0.70 mg/L, 35% at 1.13 mg/L, 80% at 1.82 mg/L and 100% at 3.15 and 4.86 mg test item/L. The EC50 at each of the observation times was 2.59 mg/L (24h) and 1.34 mg/L (48h).

 

This toxicity study is considered acceptable and satisfies the guideline requirement for acute daphnid toxicity study.

Description of key information

The substance exhibits an EC50 for freshwater invertebrates of 1.34 mg/L.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
1.34 mg/L

Additional information

Two experimental data and one QSAR data are available to determine the short-term toxicity to aquatic invertebrates.

Delpit, 2015

The 48–hr acute toxicity of 2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-(2E)-buten-1-ol to Daphnia magna was studied under semi-static conditions. Daphnids were exposed to the test chemical at nominal test concentrations of 0, 0.5, 0.8, 1.3, 2.0, 3.2, 5.0 mg/L for 48 h. Immobility of daphnids was determined by visual observation after 24 and 48 hours. Concentrations of the test item were determined by chemical analyses at the start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h). After 48 hours of exposure, immobilisation rates were 0% at 0, 0.48 and 0.70 mg/L, 35% at 1.13 mg/L, 80% at 1.82 mg/L, 100% at 3.15 mg/L and 4.86 mg/L (measured concentrations). The calculated 48–hour EC50 was 1.34 mg/L. Based on the results of this study, 2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-(2E)-buten-1-ol has to be classified as hazardous to the aquatic environment (chronic 2 – H411 Toxic to aquatic life with long lasting effects) in accordance with the EU classification system EU.

This toxicity study is considered as acceptable and satisfies the guideline requirement for acute daphnid toxicity study.

This study is given a reliability score of 1 (reliable without restrictions) since the test method meets the requirement of the EU test method and the OECD test guidelines with consideration of the “difficult to test” nature of the test substance.

Hahne, 1999

In an acute aquatic toxicity study performed according to the EPA OPPTS 850.1010 guideline, groups of Daphnia magna were exposed to Super Radjanol at test concentrations of 0, 0.65, 1.3, 2.5, 5.0 and 10.0 mg/L (nominal) for 48 hours under flow-through conditions. Mobility/mortality was observed after 48 hours.

Mobility/mortality (48 hours) at 0, 0.38, 0.80, 1.7, 3.4 or 8.1 mg/L (measured) were 0, 0, 5, 75, 100 and 100%, respectively. Calculated EC50 at 24 and 48 hours were 2.6 and 1.4 mg/L respectively, based on the measured concentrations of the test item.

This toxicity study is classified as acceptable and satisfies the guideline requirements for an acute daphnid toxicity study.

This study is given a reliability score of 2 (reliable with restrictions) since the test method is considered as an alternative to the EU test method and the OECD test guideline.

Thomas, 2015

A QSAR prediction was performed to assess the acute toxicity of the test item 2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-(2E)-buten-1-ol to daphnid. This QSAR has been validated to be compliant with the OECD recommendations for QSAR modeling (OECD, 2004) and predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following Guideline for Testing of Chemicals No. 202, "Daphniasp., Acute Immobilisation Test" (1), referenced as Method C.2 of Commission Regulation No. 440/2008 (2). The criterion predicted 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.

The QSAR determination of the daphnids immobility was based on validated data derived from 48-hour tests on daphnids, for which the concentrations of the test item had been determined by chemical analyses over the test periods.

The 48h-EC50 was predicted as 1.6 mg/L. This toxicity study is considered acceptable and satisfies the guideline requirements for acute daphnid toxicity study.