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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:
23 - 26 Feb 2016
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)
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
yes (incl. QA statement)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany (18 Nov 2014)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test medium (reconstituted water and test item) was prepared freshly. For that purpose, the calibrated flask with test medium was treated in an ultrasonic device for 1 h. Subsequently, the preparation was stirred with a magnetic stirrer for further 23 h. Afterwards, the formulation was passed through a membrane filter with a pore size of 0.2 µm. The filtrate was used for the study.
- Controls: Yes, there was 1 control group consisting of reconstituted water with daphnids (4 test vessels).
- Evidence of undissolved material: No remarkable observations were made concerning the appearance of the solution of the test item preparation. The test medium was a clear preparation and stayed unchanged throughout the study.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Source: IBACON GmbH, Roßdorf, Germany
- Age of daphnid at test start: < 24 h
- Feeding during test: No
- Breeding: The clone was bred in the laboratories of the test facility at a water temperature of 20 ± 2 °C controlled by MCPS in a separate vessel. Parent animals were used for reproduction until they were about 6 weeks old. Then they were replaced by newborn offspring. They were kept individually in 100 mL test vessels containing approximately 60 mL reconstituted water. During the breeding phase, outside of studies, the offspring were separated from their mothers at least two times per week.
- Food type: Unicellular green algae Desmodesmus subspicatus
- Amount: ad libitum
- Frequency: Three times per week.

ACCLIMATION
To avoid an acclimation phase before start of the study, reconstituted water was used during culture as well as during the study.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
All offspring were separated from their parents less than 24 h before start of the experimental part, i.e. the offspring was not older than 24 h.
Before start of the experimental phase, the young daphnia were separated from their different parent animals and put into a collective test vessel containing reconstituted water. The daphnia were visually inspected for motility. If daphnia did not make any swimming movements within 15 s after their test vessels had been gently agitated, they were considered to be immobile and were not used for the study. At the start of the experimental phase the daphnia were cautiously removed using a pipette, separated from the reconstituted water using a fine-mesh sieve, and placed into the test vessels containing test media (test item group) or reconstituted water (control group).
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Test temperature:
19.3 - 20.0 °C
pH:
0 h: 7.75 (control), 7.85 (treatment)
48 h: 7.86 (control), 7.87 (treatment)
Dissolved oxygen:
0 h: 8.35 (control), 8.50 (treatment) mg O2/L
48 h: 8.31 (control), 8.02 (treatment) mg O2/L
Nominal and measured concentrations:
Control and 100 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: test tubes
- Type: Open
- Material, size, headspace, fill volume: Glass test tubes filled with 20 mL test solution.
- Aeration: No
- 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: demineralized water
- Culture medium different from test medium: No, reconstituted water (Elendt M4, Elendt 1990) was used for breeding as well as for the study.
- Intervals of water quality measurement: The pH-values of the test preparations were measured in the test item group and in the control group at the beginning of the study and after 48 h. The water temperature was measured in a separate vessel containing fully demineralized water (without daphnia), kept under experimental conditions using MCPS. The highest and the lowest values measured were documented. The oxygen content in mg O2/L was measured in the test item group and in the control group at the beginning and at the end of exposure.
- Other: For the preparation of the Elendt M4 medium, the stock solutions were mixed and autoclaved immediately. After preparation the reconstituted water was aerated for 24 h.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light/8 h dark
- Light intensity: The mean light intensity measured before start of the study was 624 Lux and 644 Lux at the end of the study.

EFFECT PARAMETERS MEASURED:
Immobilization: observed at 24 and 48 h

RANGE-FINDING STUDY
- Test concentrations: 100 mg/L
- Results used to determine the conditions for the definitive study: In a pre-test no immobilization was observed at a concentration of 100 mg/L under open static conditions. Therefore, the solution of a nominal test item concentration of 100 mg/L was tested in the present study.
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:
act. ingr. (dissolved fraction)
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 0 mg/L
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Remarks on result:
other:
Remarks:
Effect concentration refers to max. calculated water solubility (EPI Suite)
Details on results:
- Mortality of control: 0%
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No remarkable observations were made concerning the appearance of the solution of the test item preparation. The test medium was a clear preparation and stayed unchanged throughout the study.
Results with reference substance (positive control):
- Results with reference substance valid? Yes, the EC50 (24 h) was within the range of the published data (0.6 - 1.7 mg/L, Council Regulation (EC) No. 440/2008 and 0.6 - 2.1 mg/L, OECD guideline 202). Reference substance was last tested on 20 - 22 Oct 2015.
- Relevant effect levels: EC50 (24 h) = 0.753 mg/L potassium dichromate (95% confidence interval: 0.646 - 0.883 mg/L).

Daphnia exposed to an aqueous preparation of a nominal concentration of 100 mg/L test substance revealed no aquatic toxicity of the test substance.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study, an aqueous solution of nominal 100 mg/L of the test item revealed no aquatic toxicity in the test item. The 48-h EC50 was > 4.457E-7 mg/L (nominal > 100 mg/L) and, thus, could not be determined in this study.
Executive summary:

The objective of this study was to evaluate the influence of the test item on the mobility of Daphnia magna accoridng to OECD Guideline 202. 

Juvenile daphnids were exposed to a nominal test item concentration of 100 mg/L (limit test) over 48 hours, in an open static system. The daohnids were observed for immobilization 24 and 48 hours after placing in the aqueous test item solution. The study comprised of one test item group with four test vessels containing five daphnids each, i.e. 20 daphnids in total. Additionally, one control group (20 daphnids) was used.

The test item concentration in the reconstituted water was not quantified at the start and the end of this study. Because of the low water solubility (4.457e-7 mg/L), the compound cannot be detected with standard analytical methods and the development of an analytical method with a sufficiently low detection and quantification limit is complex. Due to the absence of any adverse effects at saturation concentration, the study was performed without any analytical concentration verification. 

Daphnids exposed to an aqueous preparation of a nominal concentration of 100 mg/L of the test item were not affected. 

Description of key information

Under the conditions of the present study, an aqueous solution of nominal 100 mg/L of the test item revealed no aquatic toxicity in the test item. The 48-h EC50 was > 4.457E-7 mg/L (nominal > 100 mg/L) and, thus, could not be determined in this study.

Key value for chemical safety assessment

Fresh water invertebrates

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

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