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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:
2017-01-10 to 2017-10-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
adopted 13th April 2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
May 30, 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 100 mg/L

- Sampling method: Four replicate samples were taken from the test solution and one from the control solution both at the start and at the end of the experiment. The samples were diluted with the eluent in two consecutive steps. In the second step 0.5 mL of Internal Standard Work Solution was added before the solution was filled up to volume. Then samples were analysed with LC/MS/MS applying MRM assay.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
The test solution used in the test was prepared by mechanical dispersion. An amount of 0.03 g test item was dissolved in 300 mL dilution water (ISO medium) in order to obtain the concentration of 100 mg/L. The test solution was freshly prepared in the testing laboratory just before introduction of test animals (start of the experiment).
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: Straus
- Source: Laboratory of Hydrobiology, 2100 Gödöllö, Kotlan S. u. 3, Hungary
- Feeding during test: no

ACCLIMATION
Test animals were bred under similar (or the same) conditions as that used during the exposure period (holding water, temperature, background colour etc.), therefore additional acclimatization before the test was not necessary. Brood daphnids were maintained in dilution water at the test temperature for at least 48 hours prior to the start of the test.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
249 mg/L (as CaCO3)
Test temperature:
19.8 - 20.1 °C
pH:
7.75 - 8.12
Dissolved oxygen:
7.24 - 8.15 mg/L
Salinity:
not applicable
Conductivity:
not applicable
Nominal and measured concentrations:
Nominal concentration: 100 mg/L
Measured concentration: Concentrations reported as nominal as recovery was 100 - 104 %
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL glass beaker
- Type: open
- Fill volume: 40 mL
- Aeration: none
- 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: reconstituted water (ISO medium acc. to OECD TG 202)

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light and 8 hours darkness

EFFECT PARAMETERS MEASURED: immobilisation at 24 and 48 h

RANGE-FINDING STUDY
- Test concentrations: 0.01, 0.1, 1, 1 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
There was no immobilisation observed in the control and in the 100 mg/L concentration group.
In both groups no abnormal behaviour or appearance of test animals was noted.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
24-h EC50: 1.57 mg/L, (95 % confidence limits: 1.31 – 1.79 mg/L)
- Limit test: no
- Dose-response test: yes

Validity of the Study


Immobilisation was not observed in the control group and the dissolved oxygen concentration at the end of the test in control and test vessels was more than 3 mg/L in all cases. All validity criteria were within acceptable limits and therefore the study was considered as valid.


Table 1: Summary of the Biological Endpoints






















Parameter



Concentration [mg/L] based on nominal concentration



48 h-EC50


95 % conf. limits



> 100


not calculated



48 h-NOEC



100



48 h-LOEC



> 100



 


Analytical Results


A concentration of 100 mg/L (limit concentration) and a concurrent control was tested in the main test. The concentration of the test item was analytically determined at the start and at the end of the experiment. The test item was not detected in the untreated control group. In the treated group measured concentrations of the test item were 97 % of the nominal at the start and 103 % at the end of the test. Since the measured test item concentration remained within ± 20 % of the nominal during the test period of 48 hours, the biological results are given at nominal concentration.


Table 2. Test item’s concentrations measured in the test solutions













































Nominal Concentration (mg/L)



Measured concentrations (mg/L)



In percentage of the nominal



Measured concentrations (mg/L)



In percentage of the nominal



100



98.7



99%



102.0



102%



94.2



94%



102.5



102%



97.0



97%



102.1



102%



96.4



96%



104.7



105%



Mean with 95% confidence interval (mg/L)



96.6 ± 2.9



97%



102.8 ± 2.0



103%



 


Biological Results


There was no immobilisation observed in the control and in the 100 mg/L concentration group. In both groups no abnormal behaviour or appearance of test animals was noted. The 48-h NOEC was determined to be 100 mg/L. The 48-h LOEC and the EC50 values were determined to be > 100 mg/L.


Table 3: Immobilization of the test animals


































































Test Group



Replicate



Number of


treated


animals



Number of immobilised animals



24h



48h



Control



1



5



0



0



2



5



0



0



3



5



0



0



4



5



0



0



100 mg/L



1



5



0



0



2



5



0



0



3



5



0



0



4



5



0



0



 

Validity criteria fulfilled:
yes
Conclusions:
In a GLP-study according to OECD test guideline 202 the test item did show no toxicity to Daphnia magna at the limit test concentration of 100 mg/L. Therefore, the 48-h NOEC was determined to be 100 mg/L and the LOEC and the EC50 values were determined to be > 100 mg/L.
Executive summary:

The aquatic toxicity of the test item was assessed using Daphnia magna in a 48-h Acute Immobilisation Test according to OECD TG 202. For this purpose, young Daphnia were exposed to aqueous test media containing the test item for 48 hours. Based on results obtained in a non-GLP preliminary experiment Daphnia were exposed to a single concentration of 100 mg/L (limit concentration). The reference item potassium dichromate served as the reference control. A static test was performed as the test item was previously shown to be stable in the test medium for 48 hours. The purpose of the analytical part of this study was to verify the concentration of the test item in the test medium. The analytically measured test item concentration remained within ± 20 % of the nominal concentration during the test period of 48 hours; therefore, biological results are based on the nominal concentration. Healthy, young female daphnids with known origin, history and acclimatization conditions were used. The test item and the control were tested using 20 Daphnia (divided into 4 concurrent subgroups) each. Each test vessel contained approximately 50 mL test medium. The immobilisation of the test animals was recorded at 24 and 48 hours after treatment. Environmental conditions were recorded. All measured values remained within acceptable ranges. All validity criteria were met and therefore the study was considered as valid. Under the test conditions, the test item did show no toxicity to Daphnia magna at the limit test concentration of 100 mg/L. Therefore, the 48-h NOEC was determined to be 100 mg/L and the LOEC and the EC50 values were determined to be > 100 mg/L.

Description of key information

In a GLP-study according to OECD test guideline 202 the test item did show no toxicity to Daphnia magna at the limit test concentration of 100 mg/L. Therefore, the 48-h NOEC was determined to be 100 mg/L and the LOEC and the EC50 values were determined to be > 100 mg/L (reference 6.1.3-1).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
100 mg/L

Additional information

The aquatic toxicity of the test item was assessed using Daphnia magna in a 48-h Acute Immobilisation Test according to OECD TG 202. For this purpose, young Daphnia were exposed to aqueous test media containing the test item for 48 hours. Based on results obtained in a non-GLP preliminary experiment Daphnia were exposed to a single concentration of 100 mg/L (limit concentration). The reference item potassium dichromate served as the reference control. A static test was performed as the test item was previously shown to be stable in the test medium for 48 hours. The purpose of the analytical part of this study was to verify the concentration of the test item in the test medium. The analytically measured test item concentration remained within ± 20 % of the nominal concentration during the test period of 48 hours; therefore, biological results are based on the nominal concentration. Healthy, young female daphnids with known origin, history and acclimatization conditions were used. The test item and the control were tested using 20 Daphnia (divided into 4 concurrent subgroups) each. Each test vessel contained approximately 50 mL test medium. The immobilisation of the test animals was recorded at 24 and 48 hours after treatment. Environmental conditions were recorded. All measured values remained within acceptable ranges. All validity criteria were met and therefore the study was considered as valid. Under the test conditions, the test item did show no toxicity to Daphnia magna at the limit test concentration of 100 mg/L. Therefore, the 48-h NOEC was determined to be 100 mg/L and the LOEC and the EC50 values were determined to be > 100 mg/L.