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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:
2012-11-09 to 2013-01-22 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study in accordance with guidance with no deviations
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: The concentrations of the test item were analyzed in one of the duplicate test media samples from the dilution 1:10, 1:3.2 and the undiluted filtrate from both sampling times (0 and 48 hours). The samples of the dilutions 1:32 and 1:100 were not analyzed since these concentrations were below the 48-hour NOEC determined in this test and, thus, were not relevant for the interpretation of the biological results. From the control, one of the duplicate samples was analyzed per sampling time, too.
- Sampling method: For the determination of the actual test item concentrations, duplicate samples were taken from each treatment at the start and at the end of the test (after 48 hours). For the 48-hour stability samples, the contents of the respective replicates were combined prior to sampling.
- Sample storage conditions before analysis: Immediately after sampling, acetonitrile (9 mL acetonitrile per mL sample volume) was added to each sample to stabilize the latter during the storage period. Thereafter, all samples were deep-frozen (at about -20 °C). Based on pre-experiments for investigation of the storage stability, the test item was found to be stable in the test water under these storage conditions.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low water solubility of the test item, a dispersion with the loading rate of 100 mg/L was prepared at the start of the test by dispersing 108.0 mg of the test item in 1080 mL of test water in a completely filled and tightly closed stirring vessel. This preparation was supported by ultrasonic treatment for 15 minutes and intense stirring on a magnetic stirrer over 3 hours in the dark, to dissolve a maximum amount of the test item in the dispersion. No auxiliary solvent or emulsifier was used. The stirring period of 3 hours was chosen according to the results of a pre-experiment (GLP) which showed that the solution equilibrium was reached after this time.
After the 3-hour stirring period, the dispersion of the test item was filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 μm) after preconditioning of the filter with about 200 mL dispersion to avoid losses of the test item. The negative pressure of the filtration unit was reduced as far as possible to avoid losses of volatile components of the test item during filtration. The undiluted filtrate was used as the highest concentrated test medium and as a stock solution for preparation of the test media with lower test concentrations. For this preparation, the filtrate was serially diluted with test water. The test media were prepared just before the start of the test (= addition of daphnids). The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.
- Controls: test medium without substance
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the entire test duration.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain: Daphnia magna Straus
- Source: A clone of this species (defined by the supplier as clone 5) was originally supplied by the University of Sheffield / UK in 1992. Since that time, the clone has been bred at Harlan Laboratories in reconstituted water of the quality identical to the water quality used in the tests (in respect to pH, main ions, and total hardness) and under temperature and light conditions identical to those of the test.
- Age at study initiation (mean and range, SD): 6 to 24 hours
- Method of breeding: not reported
- Feeding during test: not applicable
- Feeding during breeding
- Food type: algal suspension of the green algae Desmodesmus subspicatus CHODAT, Strain No. 86.81 SAG or a mixture of this algal suspension and a commercial fish diet
- Amount: not reported
- Frequency: 3 times a week
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
250 mg/L as CaCO3
Test temperature:
20-21 °C
pH:
7.4 to 7.7
Dissolved oxygen:
7.7 to 8.8 mg/L
Nominal and measured concentrations:
nominal: undiluted filtrate (based on loading rate 100 mg/L), 1:3.2, 1:10, 1:32, 1:100, 0 (control)
mean measured: 3.7 mg/L, 0.68 mg/L, 0.24 mg/L, not measured, not measured
Details on test conditions:
TEST SYSTEM
- Test vessel: all
- Type (delete if not applicable): closed, tightly sealed with glass stoppers
- Material, size, headspace, fill volume: 60 mL glass tubes, completely filled with test medium
- Aeration: test water was aerated prior to the start of the test
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water according to ISO 6341 was used in the study. It consisted of analytical grade salts dissolved in purified water at the following nominal concentrations.
- Total organic carbon: Not relevant as medium is based on reconstituted test water.
- Particulate matter: Not relevant as medium is based on reconstituted test water.
- Metals: Detailed mineral composition given in study report. Toxic metals not relevant as medium is based on reconstituted test water.
- Pesticides: Not relevant as medium is based on reconstituted test water.
- Chlorine: Not relevant as medium is based on reconstituted test water.
- Alkalinity: 0.8 mmol/L
- Ca/Mg ratio: 4:1
- Na/K ratio: 10:1
- Conductivity: not reported
- Culture medium different from test medium: no
- Intervals of water quality measurement: At the start and end of the test, the pH values, dissolved oxygen concentrations and water temperature were determined at each treatment. The appearance of the test media was visually recorded at the start of the test and after 24 and 48 hours.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: A 16-hour light to 8-hour dark cycle with a 30-minute transition period was used.
- Light intensity: Light intensity during the light period was approximately between 400 and 540 Lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): immobilisation

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2

- Range finding study
- Test concentrations: undiluted filtrate (based on loading rate of 100 mg/L)
- Results used to determine the conditions for the definitive study: The selection of the test concentrations were based on a first definitive Limit test and in consultation with the Sponsor.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
0.68 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
1.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: not determined due to inappropriate data
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: 0.68-3.7 mg/L
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
3.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.68 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
The test is considered to be valid, as in the control no daphnids showed immobilization or other signs of disease or stress (e.g., discoloration or unusual behavior such as trapping at the surface water). Furthermore, the dissolved oxygen concentration at the end of the test was ≥3 mg/L in the control and test vessels.
Results with reference substance (positive control):
For evaluation of the quality of the daphnia clone and the experimental conditions, potassium dichromate is tested as a positive control twice a year. The result of the latest positive control test in September 2012 (48-hour EC50: 0.94 mg/L, Harlan Laboratories Study D64333) showed that the sensitivity of the test organisms was within the internal historical range (48-hour EC50 from 2000 to 2012: 0.43-1.1 mg/L).

Effect of (ethoxymethoxy)cyclododecane on the mobility of Daphnia magna

Treatment/Dilution

Mean measured concentration (mg/L)

Number of daphnids tested

Immobilised daphnids after 24 hours

Immobilised daphnids after 48 hours

Number

%

Number

%

Control

---

20

0

0

0

0

1:100

---

20

0

0

0

0

1:32

---

20

0

0

0

0

1:10

0.24

20

0

0

0

0

1:3.2

0.68

20

0

0

0

0

Undiluted filtrate based on loading rate of 100 mg/L

3.7

20

20

100

20

100

Conditions during the test

Treatment/Dilution

Start (0 hours)

End (48 hours)

pH

Oxygen (mg/L)

Temperature (°C)

pH

Oxygen (mg/L)

Temperature (°C)

Control

7.7

8.8

20

7.5

7.9

21

1:100

7.7

8.7

20

7.5

7.7

21

1:32

7.7

8.7

20

7.5

7.7

21

1:10

7.7

8.6

20

7.4

7.8

21

1:3.2

7.7

8.5

20

7.4

7.8

21

Undiluted filtrate based on loading rate 100 mg/L

7.7

8.1

20

7.5

7.7

21

Validity criteria fulfilled:
yes
Conclusions:
The substance (ethoxymethoxy)cyclododecane was acutely toxic to the freshwater invertebrate Daphnia magna. The test was conducted with filtrates based on loading rates of 100 mg/L and serial dilutions of these filtrates. The mean measured concentration determined for the undiluted filtrate was 3.7 mg/L and hundred percent of daphnids were immobile at this concentration after 24 hours. The mean measured concentration of the first 1:3.2 dilution was 0.68 mg/L and no immobilisation was observed over the testing period of 48 hours at that concentration. The EC50 value over the period of 48 hours was 1.6 mg/L and the NOEC was 0.68 mg/L.
Executive summary:

The acute toxicity of the test substance to the freshwater invertebrate Daphnia magna was tested under GLP in accordance with OECD TG 202. The study was conducted under static conditions for a period of 48 hours in tightly closed vessels, which were completely filled with 60 mL of test medium. Five daphnids were tested per vessel and four replicates were investigated per test concentration. In addition, 4 replicates with test medium only were studied as control. Due to the low solubility of the test substance, a dispersion with the loading rate of 100 mg/L was prepared in tightly closed stirring vessels. After a 3 -hour stirring period, the dispersion was filtered through a membrane filter (pore size 0.45 µm). The undiluted filtrate was the highest concentrated test medium and the lower test concentrations were obtained by serial dilution (1:3.2, 1:10, 1:32, 1:100) of this stock solution. The concentrations of test substance at the two highest test concentration were determined by means of GC since these test concentrations were above the NOEC determined in the study. The mean measured concentration was 3.7 mg/L (74% of the initially measured concentration) for the undiluted filtrate, 0.68 for the 1:3.2 dilution and 0.24 mg/L for the 1:10 dilution. Hundred percent of the daphnids were immobile after 24 hours at the test concentration of 3.7 mg/L (undiluted filtrated based on loading rate of 100 mg/L). No immbolisation was observed at the tested dilutions of the filtrate. The EC50 value over the testing period of 48 hours was 1.6 mg/L and the NOEC was 0.68 mg/L. The validity criteria described in the test guideline were fulfilled and the suitability of the test system was confirmed in a test with the positive test substance potassium dichromate. The study therefore was considere to be valid.

Description of key information

The substance was acutely toxic to the freshwater invertebrate Daphnia magna over a testing period of 48 hours.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
1.6 mg/L

Additional information

The acute toxicity of the substance was studied in a valid GLP test according to OECD TG 202. The test was performed under static conditions in closed vessels over a period of 48 hours. Due to the low solubility of the test substance, the test was carried out with the undiluted filtrated obtained from a dispersion of the substance based on a loading rate of 100 mg/L. Additional test concentrations were obtained by serial dilution. Test concentrations were determined by GC and mean measured concentrations were 3.7 mg/L for the undiluted filtrate, 0.68 mg/L for a 1:3.2 dilution and 0.24 mg/L for a 1:10 dilution. Hundred percent of daphnids were immobile after 24 hours at the highest test concentration, whereas no immobilisation was observed over the period of 48 hours at the other two measured concentrations. The 48-hour EC50 value was calculated to be 1.6 mg/L and the NOEC was 0.68 mg/L.