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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:
2018/11/05 - 2018/11/09
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:
yes
Remarks:
The temperature during the test was 20.4 - 21.6°C and therefore fluctuated by more than 1°C. Since there was no mortality in the controls, this deviation can be considered uncritical.
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, 55116 Mainz (15.05.2018)
Specific details on test material used for the study:
The test item was stored in a closed vessel dark and dry at room temperature (20 ± 5°C).
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 100; 46; 22; 10; 4.6; 2.2 mg/L; The concentrations to be tested are based on the result of a non-GLP pre-test.
- Sampling method: The content of the test item in the test solutions was measured at the beginning of the test and every 24 hours.
- Sample preparation:
Sample Preparation for treatment 100 mg/L:
10 mL of the sample was mixed with 2 g NaCl, then the solution was extracted two times with the solvent dichloromethane (2 x 4 mL) and the organic phase was collected into a 10 mL flask, after drying with Na2SO4. The flask was filled up to 10 mL with dichloromethane after addition of 500 μL ISTD stock solution (1000 mg/L in dichloromethane) and the solution was measured via GC/FID.
Sample Preparation for treatments 22 - 46 mg/L (twofold enrichment):
20 mL of the sample was mixed with 3 g NaCl, then the solution was extracted two times with the solvent dichloromethane (2 x 4 mL) and the organic phase was collected into a 10 mL flask, after drying with Na2SO4. The flask was filled up to 10 mL with dichloromethane after addition of 500 μL ISTD stock solution (1000 mg/L in dichloromethane) and the solution was measured via GC/FID. Twofold enrichment was achieved during extraction.
Sample Preparation for treatments 4.6 – 10 mg/L (fivefold enrichment):
50 mL of a solution in media was mixed with 3 g NaCl, then the solution was extracted two times with the solvent dichloromethane (2 x 4 mL) and the organic phase was collected into a 10 mL flask, after drying with Na2SO4. The flask was filled up to 10 mL with dichloromethane after addition of 500 μL ISTD stock solution (1000 mg/L in dichloromethane) and the solution was measured via GC/FID. Fivefold enrichment was achieved during extraction.
Sample Preparation for treatment Control and 2.2 mg/L (tenfold enrichment):
100 mL of a solution in media was mixed with 6 g NaCl, then the solution was extracted two times with the solvent dichloromethane (2 x 4 mL) and the organic phase was collected into a 10 mL flask, after drying with Na2SO4. The flask was filled up to 10 mL with dichloromethane after addition of 500 μL ISTD stock solution (1000 mg/L in dichloromethane) and the solution was measured via GC/FID. Tenfold enrichment will be achieved during extraction.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution containing 100 mg/L test item in dilution water was prepared. This was done by mixing 123.3 μL/L test item (based on a density of 0.81 g/mL, given by the sponsor) with the corresponding amount of dilution water. To guarantee complete dissolution, the solution was treated in an ultrasonic bath for 55 minutes followed by shaking vigorously for 1 hour. The lower treatments were prepared by dilution of this stock solution with dilution water.
- Controls: Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a current reference study (201714R201). The 24h-EC50i value was determined as 1.9 mg/L. The value was within the requested range for the 24h-EC50 between 0.6 - 2.1 mg/L of potassium dichromate K2Cr2O7 as specified in the OECD guideline.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Berlin
- Source: Umweltbundesamt Berlin
- Age of parental stock (mean and range, SD): between 0 and 24 hours
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
After 24 and 48 hours, the immobilised daphnia were counted.
Hardness:
2.502 mmol/L
Test temperature:
20.4-21.6 °C
pH:
7.5-7.7
Dissolved oxygen:
7.5 - 8.9 mg/L
Nominal and measured concentrations:
please refer to table 1a
Details on test conditions:
TEST SYSTEM
- Test vessel: Closed glass flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, total volume 65 mL
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Daphnia medium
CaCl2*2 H2O = 293.80 mg/L
MgSO4*7H2O = 123.30 mg/L
NaHCO3 = 64.80 mg/L
KCl = 5.80 mg/L

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : After 24 and 48 hours, the immobilised daphnia were counted. The pH and the concentration of dissolved oxygen in the test vessels were measured at the beginning and at the end of the test. The content of the test item in the test solutions and the control was measured at the beginning of the test and every 24 hours. The samples for analytical measurement were taken from the extra vessels

RANGE-FINDING STUDY
- Test concentrations: 100; 46; 22; 10; 4.6; 2.2 mg/L
- Results used to determine the conditions for the definitive study: The concentrations to be tested are based on the result of a non-GLP pre-test.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
7.16 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
1.43 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
3.32 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
2.79 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC10
Effect conc.:
2.35 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
8.89 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities:
Immobilisation was recorded at 24 hours and 48 hours and compared with control values. Not more than 10% of the control daphnia should show immobilisation or other signs of disease or stress, for example, discoloration or unusual behaviour such as trapping at surface of water. The results were analysed in order to calculate the EC50 at 24 h and at 48 h.
No observations were made which might cause doubts concerning the validity of the study outcome. All validity criteria were met.
- Other biological observations:
Immobilisation in the controls was 0 %.
Results with reference substance (positive control):
- Results with reference substance valid? yes, Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) in reference study (201714R201)
- Relevant effect levels: 24h-EC50
- EC50: determined as 1.9 mg/L; between 0.6 - 2.1 mg/L as specified in the guideline
Reported statistics and error estimates:
The estimation of the biological results was accomplished using the software ToxRat® Professional, version 3.2.1

Table 2: Immobility

Nominal Concentration in mg/L Immobility 24 hours Immobility 48 hours
absolute in % absolute in %
Blank control 0 0 0 0 0 0 0 0 0 0
2.2 0 0 0 0 0 0 0 0 0 0
4.6 1 1 1 1 20 1 1 1 1 20
10 1 4 3 2 50 1 4 3 2 50
22 5 4 3 3 75 5 4 5 3 85
46 4 5 4 4 85 5 5 5 5 100
100 5 5 5 5 100 5 5 5 5 100

Table 3: pH and O2 values

Nominal Concentration in mg/L pH O2-Concentration in mg/L
0 h 48 h 0 h 48 h
Blank control 7.7 7.7 8.8 7.9
2.2 7.7 7.6 8.8 8.0
4.6 7.7 7.6 8.9 7.9
10 7.7 7.5 8.9 7.7
22 7.7 7.5 8.9 7.5
46 7.7 7.6 8.7 8.1
100 7.7 7.6 8.5 8.1
Validity criteria fulfilled:
yes
Remarks:
Immobilisation in the controls may not exceed 10 %. Immobilisation in the controls was 0 %. The concentration of dissolved oxygen at the end of the test must be at least 3 mg/L. Lowest concentration of dissolved oxygen at the end of the test was 7.5 mg/L.
Conclusions:
The following results were determined for the test item Hexanal (species: Daphnia magna).
48h-NOEC = 1.43 mg/L
48h-LOEC = 3.32 mg/L
48h-EC10 = 2.79 mg/L
48h-EC50 = 7.16 mg/L
Executive summary:

This study was performed in order to evaluate the toxicity of the test item Hexanal in an acute immobilisation test under static conditions towards Daphnia magna, according to the OECD guideline No. 202 and Council Regulation (EC) No. 440/2008, Method C.2. The study was performed under GLP-conditions.

One valid experiment was performed. The study was performed using 6 concentrations ranging from 2.2 to 100 mg/L (nominal). To avoid volatilization of the test item, the study was performed in closed vessels. For each test concentration and the blank control, 20 Daphnia were exposed to the test item for 48 hours in a static test system. After 24 and 48 hours, the immobilised Daphnia were counted. The 5 highest concentrations showed toxicity between 20 and 100% immobilisation. None of the animals was immobilised in the lowest concentration 2.2 mg/L and the blank control. Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a current reference study to assure that the test conditions are reliable. The content of the test item in the test solutions was measured at the beginning of the test and every 24 hours. The measured concentrations were in a range of 61 % and 93 % of the nominal concentration. Therefore, the determination of the results was based on the geometric mean of the measured concentrations. The following results were determined for the test item Hexanal (species: Daphnia magna).

48h-NOEC = 1.43 mg/L

48h-LOEC = 3.32 mg/L

48h-EC10 = 2.79 mg/L

48h-EC50 = 7.16 mg/L

Description of key information

D. magna, 48 h, OECD 202, GLP, EC50 = 7.16 mg/L

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
7.16 mg/L

Additional information

This study was performed in order to evaluate the toxicity of the test item Hexanal in an acute immobilisation test under static conditions towardsDaphnia magna, according to the OECD guideline No. 202 and Council Regulation (EC) No. 440/2008, Method C.2. The study was performed under GLP-conditions.

One valid experiment was performed. The study was performed using 6 concentrations ranging from 2.2 to 100 mg/L (nominal). To avoid volatilization of the test item, the study was performed in closed vessels. For each test concentration and the blank control, 20 Daphnia were exposed to the test item for 48 hours in a static test system. After 24 and 48 hours, the immobilised Daphnia were counted. The 5 highest concentrations showed toxicity between 20 and 100% immobilisation. None of the animals was immobilised in the lowest concentration 2.2 mg/L and the blank control. Potassium dichromate K2Cr2O7(CAS No. 7778-50-9) was used as positive control in a current reference study to assure that the test conditions are reliable. The content of the test item in the test solutions was measured at the beginning of the test and every 24 hours. The measured concentrations were in a range of 61 % and 93 % of the nominal concentration. Therefore, the determination of the results was based on the geometric mean of the measured concentrations. The following results were determined for the test item Hexanal (species:Daphnia magna).

48h-NOEC = 1.43 mg/L

48h-LOEC = 3.32 mg/L

48h-EC10 = 2.79 mg/L

48h-EC50 = 7.16 mg/L