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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:
2020-12-19 to 2021-02-13
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:
2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))
Version / remarks:
2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Analysis of the test item concentration directly after each preparation (fresh medium) and after 24 h (old medium) was performed. The samples were analyzed undiluted due to the results of the method implementation. The concentrations of the test item were determined using the calibration curve. One replicate was given to the analytical laboratory for analysis. The second replicate was deep-frozen (< -20 °C) and used as backup.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock preparation with a test item concentration of 100 mg/L was freshly prepared. For that purpose, the test item was weighed in a calibrated flask and reconstituted water added. The preparation was stirred with a magnetic stirrer for 24 hours. Then the formulation was passed through a filter membrane (pore size: 0.2 µm). The filtrate was used for the study. The pH was not adjusted.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Age at study initiation: Offspring less than 24 hours old were used for the study
- Method of breeding: Daphnids are kept individually in 100 mL glass vessels containing approximately 60 mL reconstituted water (ELENDT M4 medium) at a water temperature of 20 ± 2 °C and a 16 hour light and 8 hour dark regime to ensure similar conditions as in the experiment.
- Source: IBACON GmbH (Roßdorf, Germany)
- Feeding during test: no

ACCLIMATION
- Acclimation period: same as test
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
234 – 239 mg CaCO3/L
Test temperature:
20.2 to 20.9 °C
pH:
7.80 – 7.96
Dissolved oxygen:
8.13 – 8.37 mg/L
Conductivity:
594 – 597 µS/cm
Nominal and measured concentrations:
Nominal conc.: 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: glass vessels
- Type: open
- Material, size, headspace, fill volume: 20 mL glass vessels containing approximately 20 mL reconstituted water (control) or medium (test item group)
- Aeration: none
- Renewal rate of test solution: the test item was renewed after 24 hours
- No. of organisms per vessel: 5
- No. of vessels per concentration: 4 replicates
- No. of vessels per control: 4 replicates

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (ELENDT M4 medium)
- Conductivity: 594 – 597 µS/cm
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hour light and 8 hour dark regime
- Light intensity: 587 – 588 Lux

EFFECT PARAMETERS MEASURED: immobilisation after 24 and 48 h
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
Remarks on result:
other: LOD: > 0.545 mg/L
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: LOD: > 0.545 mg/L
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: The 24 h EC50: 1.05 mg/L, (95 % confidence limits: 0.93 – 1.20 mg/L)
- Limit test: no
- Dose-response test: yes

Validity of the Test


In the control group, no daphnids were immobilized. The dissolved oxygen concentration at the end of the exposure period was > 3 mg/L. Thus, the validity criteria were fulfilled.


 


Biological Results


A nominal concentration of 100 mg/L of the test item revealed no toxicity to daphnids as given in the following table:































Nominal Test Item Concentration [mg/L]



Number of daphnids immobilized / exposed



% of daphnids immobilized



24 hours



48 hours



24 hours



48 hours



Control



0/20



0/20



0 %



0 %



100



0/20



0/20



0 %



0 %



 


Analytical result


Concentrations of the Test Item in Control and Test Medium: Measured concentrations of the test item in control and daphnia test medium are given in the following table. Each value represents the mean concentration of three measurements.


 



































Nominal Test Item Concentration


[mg/L]



0 hours



24 hours



Fresh medium



Old medium



Meas. Cone. [mg/L]



Recovery from nom. [%]



Meas. Cone. [mg/L]



Recovery from nom. [%]



0



<LOD



-



<LOD



-



100



<LOD



-



<LOD



-



LOD = 0.545 mg/L


 



































Nominal Test Item Concentration


[mg/L]



24 hours



48 hours



Fresh medium



Old medium



Meas. Cone. [mg/L]



Recovery from nom. [%]



Meas. Cone. [mg/L]



Recovery from nom. [%]



0



<LOD



-



<LOD



-



100



<LOD



-



<LOD



-



LOD = 0.545 mg/L


The measured concentrations of the test item in the control and daphnia test medium (100 mg/L) were below the LOD of 0.545 mg/L during the experimental phase.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study, the 48 h EC50 was > 0.545 mg/L (nominal > 100 mg/L) and, thus, could not be determined in this study.
Executive summary:

The aquatic toxicity of the test item to Daphnia magna was assessed in a 48 h Acute Immobilisation Test according to OECD TG 202 and under GLP conditions. For this purpose, juvenile daphnids aged less than 24 hours old at the start of the study, were exposed for a period of 48 hours to a nominal test item concentration of 100 mg/L (limit test) in an open semi-static system. 20 daphnids, divided into four replicates, each with five animals were used in the test item group and the control group. The test item preparation (test item group) and the vehicle (control group) were renewed after 24 hours. Immobilization was recorded at 24 and 48 hours and compared with control values. The results were analyzed in order to calculate the EC50 at 24 h and 48 h. Concentrations of the test item in the test medium were analyzed via HPLC with UV-detection directly after each preparation (fresh medium) and 24 h after each preparation (old medium). The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium. The measured concentrations of the test item were < LOD (LOD = 0.545 mg/L) during the experimental phase. No test item was measured in the control medium. Therefore, it was confirmed that daphnia in the control group were not exposed to the test item. Under the conditions of the present study, an aqueous solution of nominal 100 mg/L of  the test item revealed no toxicity to daphnids. The 48 h EC50 was > 0.545 mg/L (nominal > 100 mg/L) and, thus, could not be determined in this study.

Description of key information

Under the conditions of the present study, the 48 h EC50 was > 0.545 mg/L (nominal > 100 mg/L) and, thus, could not be determined in this study (reference 6.1.3-1).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
EC50
Effect concentration:
> 100 mg/L

Additional information

The aquatic toxicity of the test item to Daphnia magna was assessed in a 48 h Acute Immobilisation Test according to OECD TG 202 and under GLP conditions. For this purpose, juvenile daphnids aged less than 24 hours old at the start of the study, were exposed for a period of 48 hours to a nominal test item concentration of 100 mg/L (limit test) in an open semi-static system. 20 daphnids, divided into four replicates, each with five animals were used in the test item group and the control group. The test item preparation (test item group) and the vehicle (control group) were renewed after 24 hours. Immobilization was recorded at 24 and 48 hours and compared with control values. The results were analyzed in order to calculate the EC50 at 24 h and 48 h. Concentrations of the test item in the test medium were analyzed via HPLC with UV-detection directly after each preparation (fresh medium) and 24 h after each preparation (old medium). The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium. The measured concentrations of the test item were < LOD (LOD = 0.545 mg/L) during the experimental phase. No test item was measured in the control medium. Therefore, it was confirmed that daphnia in the control group were not exposed to the test item. Under the conditions of the present study, an aqueous solution of nominal 100 mg/L of  the test item revealed no toxicity to daphnids. The 48 h EC50 was > 0.545 mg/L (nominal > 100 mg/L) and, thus, could not be determined in this study.