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Short-term toxicity to aquatic invertebrates

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short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: October 27, 2019; Experimental completion date: December 19, 2019
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on sampling:
- Concentrations: Control and 100 mg/L treatment
- Sampling method: For the determination of the test item concentrations, duplicate samples were taken from the test medium and the control at the start and at the end of the test. For sampling at the end of the test, the contents of the replicate test vessels were combined prior to sampling.
- Sample storage conditions before analysis: All samples were stored frozen (-20 ± 5 °C) immediately after sampling until analysis.
- Analysed samples: The concentrations of the test item were measured in one of the duplicate test medium samples of the single test concentration and the control, taken at the start and end of the test.
Details on test solutions:
- Method: The test medium was prepared by dissolving 100.13 mg of test item completely in 1000 mL of test water using ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature.
- Evidence of undissolved material: No remarkable observations were made concerning the appearance of the test medium. The test medium was a clear solution throughout the entire test duration. The test medium was prepared just before the introduction of the daphnids (i.e., the start of the exposure).
Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Water flea
- Strain/clone: A clone of the species Daphnia magna (originally from the Daphnia Collection of the University of Basel/Switzerland in 2015) is successfully bred in IES Ltd Laboratories.
- Source: Daphnia Collection of the University of Basel/Switzerland in 2015
- Age at study initiation: At the start of the test, the test organisms were ≤ 24 hours old and were not first brood progeny.
- Feeding during test: The daphnids were not fed during the test.

- Acclimation period: No acclimatisation was necessary, since the bred of the daphnia was under identical conditions to those of the test.
- Type and amount of food during the breeding: An algal suspension of the green algae Desmodesmus subspicatus CHODAT, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen/Germany) and cultivated at IES Ltd Laboratories under standardized conditions or a mixture of this algal suspension and a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-GmbH, 49324 Melle/Germany).
- Feeding frequency: During breeding, daphnids were generally fed three times a week.

The cultivation of the parental daphnids is performed in reconstituted water of the quality identical to the water quality used in the tests (in respect to pH, temperature of 21°C, main ions, and total hardness). Each stock animal was maintained separately in a 100-mL glass beaker filled with about 80 mL culture medium and was transferred twice a week to fresh medium. The condition of the stock animals was frequently checked. No signs of stress were observed and the brood stock was healthy. The culture contains no ephippia, the first offspring were produced before day 12, the cultured daphnids did not shown any signs of stress during the 48 hours prior to test start (e.g., abnormal behavior or >5 % mortality). Additionally, the cultured daphnids produced an average of at least 3 offspring per female per day over a 7-day period prior to test initiation.
Test type:
Water media type:
Reconstituted test water (ISO Test water) according to the OECD Guideline No. 202 was used in the study.
Limit test:
Total exposure duration:
48 h
2.5 mmol/L = 250 mg/L as CaCO3
Test temperature:
The test was performed under temperature-controlled conditions (climate room with continuous monitoring of the room temperature and the water temperature in a separate vessel placed in the test area). The test water temperature was maintained between 20 and 21 °C.
At the beginning and end of the test period, the pH value of the test medium was between 6.5 and 7.9.
Dissolved oxygen:
At the beginning and end of the test period, the dissolved oxygen concentration in the test medium and control was in the range of 8.5 to 8.8 mg/L.
Nominal and measured concentrations:
- Nominal concentrations: 0 and 100 mg/L.
- Measured concentrations: 102 mg/L at the start of the test and 110 mg/L at the end, 102 % and 110 % of nominal, respectively. Thus, the analytical results confirm the correct dosage and the stability of the test item during the exposure period. Therefore, the endpoint values were reported on the nominal concentration of the test item
Details on test conditions:
- Test vessel: Beakers
- Type: Open. The test vessels were loosely covered with glass sheets to reduce the loss of water by evaporation and to avoid the entry of dust into the solutions.
- Material, size, headspace, fill volume: Glas, 100 mL, open - thus unlimited headspace, 50 mL
- Aeration: No. The test vessels were not aerated during the test.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates):4
- No. of vessels per control (replicates):4
- Biomass loading rate: 1 Daphnid / 10 mL test media

- Source/preparation of dilution water: ISO Medium
- Ingredients / [mmol/L] / Concentration [mg/L]
• CaCl2 × 2H2O / 2.0 / 294
• MgSO4 × 7H2O / 0.5 / 123
• NaHCO3 / 0.75 / 65
• KCl / 0.075 / 5.8
- Alkalinity: 0.8 mmol/L
- Ca/Mg ratio (molar): 2.0÷0.5 = 4

- Adjustment of pH: No. The pH of the test water after equilibration was at 7.9 thus no adjustment was required.
- Photoperiod:16-hour light to 8-hour dark cycle with a 30-minute transition period
- Light intensity: Light intensity during the light period was 17 µmol/(m²·s).

- Immobility of the daphnids and other non-lethal effects (e.g. abnormal behavior or appearance) after 24 and 48 hours of exposure.

- Test concentrations: 1.0 / 10 / 100 mg/L
- Results used to determine the conditions for the definitive study: 48-hour immobilization: 1.0 mg/L / 0%; 10 mg/L / 0%; 100 mg/L / 0%
Reference substance (positive control):
Potassium dichromate (CAS 7778-50-9), Batch No. MKBR3876V) obtained from Sigma-Aldrich
Key result
48 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Key result
48 h
Dose descriptor:
Effect conc.:
>= 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Details on results:
- Behavioural abnormalities: No adverse effects were observed in the test animals.
- Mortality of control: None
- Other adverse effects control: None
- Any observations: None
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: The 24-hour EC50 of 1.8 mg/L showed that the sensitivity of the test organisms was within the range given by the guideline (OECD 202: 24-hour EC50: 0.60-2.1 mg/L).
- Dose-response test: The reference test was carried out to check the sensitivity of the test system as used by IES Ltd. Daphnia were exposed for a period of 48 hours to potassium dichromate concentrations of 0.19, 0.38, 0.75, 1.5 and 3.0 mg/L and a control (test water without test item). Twenty daphnia were exposed per concentration.
- ECx: The 24-hour EC50 was 1.8 mg/L with a 95 % confidence interval between 1.6 and 2.0 mg/L. The 48-hour EC50 was 1.4 mg/L with 95 % confidence limits of 1.2 and 1.6 mg/L.
- Other:
•Potassium Dichromate - Acute Toxicity to Daphnia magna in a 48-Hour Immobilization Test (IES Ltd Study 20190394).
•Experimental Starting Date: October 28, 2019
•Experimental Completion Date: October 31, 2019
•The reference test was based on the OECD Guideline No. 202 (2004) and the Commission Regulation (EC) No. 440/2008, Part C.2.
•The reference test was carried out to check the sensitivity of the test system as used by IES Ltd.
•Effect of Potassium Dichromate on the Mobility of Daphnia magna:
• Nominal Test Item Concentration [mg/L] / No. of Daphnids Tested / Immobilized Daphnids after 24 Hours / Immobilized Daphnids after 48 hours
• Control / 20 / 0 / 0
• 0.19 / 20 /0 / 0
• 0.38 / 20 / 0 / 0
• 0.75 / 20 / 0 / 0
• 1.5 / 20 / 5 (25 %) / 12 (60 %)
• 3.0 / 20 / 20 (100 %) / 20 (100 %)
Reported statistics and error estimates:
No statistics needed due to the absence of toxicity of the test item. The 24 and 48-hour EC50 of the test item could not be calculated.

The test is considered valid as in the control no daphnids showed immobilization (should be ≤10%) or other signs of disease or stress (e.g. discoloration or unusual behavior such as trapping at the water surface). Furthermore, the dissolved oxygen concentration at the end of the test was 8.8 mg/L in the control and test vessels (should be ≥3 mg/L).

Validity criteria fulfilled:
The test item was not toxic to aquatic freshwater invertebrates (Daphnia magna) in a 48-hour static test up to a concentration of 100 mg/L.
Executive summary:

The acute toxicity of the test item to young (≤24 hours old) water flea (Daphnia magna) was determined under GLP in a 48 hour static test according to OECD TG 202, adopted 2004. The validity criteria were met. On the basis of the pre-test results, a limit test was performed in accordance with the test guideline to demonstrate that the test item has no toxic effect on the test organisms up to and including the nominal concentration of 100 mg/L during an exposure period of 48 hours. A control was tested in parallel. A static test design was applied. For each treatment, 20 daphnids were used divided into four replicates of five daphnids each. The immobility of the daphnids was determined by visual inspection after 24 and 48 hours of exposure. Observations were also performed for other non-lethal effects (e.g. abnormal behaviour or appearance). No remarkable observations were made concerning the appearance of the test medium. The test medium was a clear solution throughout the entire test duration. The measured concentration of the test item in the test medium was 102 mg/L at the start of the test and 110 mg/L at the end, 102% and 110% of nominal, respectively. No adverse effects were observed in the test animals.


Description of key information

48-hour EC50 >100 mg/L, water flea (Daphnia magna)

Key value for chemical safety assessment

Additional information

The acute toxicity of the substance to the freshwater invertebrate water flea (Daphnia magna) was investigated under GLP to OECD TG 202. The substance had no adverse effects on the test organisms over the exposure period of 48 hours, and the EC50 was greater than 100 mg/L.