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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:
25 - 27 Oct 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Staatliches Gewerbeaufsichtsamt Hildesheim, Germany
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control, and 100 mg/L (limit concentration)
- Sampling method: At the start of the exposure (0 h), sampling was carried out after preparation of the limit concentration. At the end of the exposure (48 h), samples were taken from additional replicates, which were prepared with test media, but without daphnia. These additional replicates were incubated under test conditions until sampling.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until the start of the analysis, if necessary. Prepared samples were stored in the autosampler at room temperature until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The limit concentration (100 mg/L test item weighed out) was freshly prepared with dilution water prior to the start of the exposure (at 0 h)
- Controls: Culture medium without test item incubated under the same conditions as the test group.
- Evidence of undissolved material: The limit concentration was visually clear during the exposure period
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: Clone 5
- Source: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), Berlin, Germany
- Age of daphnia at test start: Less than 24 h old.
- Culture conditions: In 2 – 3 L glass vessels with approximately 1.8 L Elendt M4 culture medium, at 20 ± 2 °C, in an incubator, 16 h illumination, light intensity max. 1500 lux.
- Food type: A mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus (origin of the food algae: Sammlung von Algenkulturen (SAG), Göttingen, Germany, cultured at the test facility).
- Amount: 1E+06 cells/mL
- Frequency: The culture daphnia were fed at least 5 times per week ad libitum.
- Feeding during test: no


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Juvenile daphnia were removed from the culture vessels at the latest 24 h before the start of the exposure and discarded. The juveniles born within the following period of max. 24 h preceding the exposure were used for the test. No first brood progeny was used for the test.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
168 mg CaCO3/L
Test temperature:
19 - 20 °C
pH:
0 h: 7.82 (control), 7.12 (limit concentration)
48 h: 7.53 - 7.58 (control), 7.51 - 7.53 (limit concentration)
Dissolved oxygen:
0 h: 8.98 mg/L (control), 8.88 mg/L (limit concentration)
48 h: 7.35 - 7.83 mg/L (control), 6.94 - 7.83 mg/L (limit concentration)
Conductivity:
464 µS/cm (at 0 h)
Nominal and measured concentrations:
control, 100 mg/L (nominal)
< LOQ, 93.5 mg/L (mean of 0 h and 48 h values measured)
Details on test conditions:
TEST SYSTEM
- Test vessel: beakers
- Type : Loosely covered with watch glasses.
- Material, size, headspace, fill volume: Material: glass; Size: 4 (ID) x 7 (H) cm, 50 mL; Fill volume: 20 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: Dilution water was same as culture medium.
- Culture medium different from test medium: Same as test medium.
- Intervals of water quality measurement: Prior to the start of the exposure (0 h), the water quality parameters (i.e. pH-value, dissolved oxygen concentration, temperature, conductivity and total hardness) of the dilution water wree measured. At the start of the exposure (0 h), the water quality parameters of the fresh media (i.e. pH-value, dissolved oxygen concentration) were measured in one additional replicate (without daphnia) of the limit concentration and the control. At the end of the exposure (48 h), the water quality parameters of the old media were measured in all replicates (containing daphnia) of the limit concentration and the control.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light/ 8 h dark cycle
- Light intensity: Diffuse light, max. 1500 lux.
- Other: 20 g (= 20 mL) test solution per replicate was weighed out into each test vessel. The daphnia were inserted with a small amount of dilution water by a pipette.

EFFECT PARAMETERS MEASURED:
Immobilisation: after 24 and 48 h

RANGE-FINDING STUDY
- Test concentrations: Control, 1, 10, and 100 mg/L nominal concentration
- Results used to determine the conditions for the definitive study: Yes, no effects on the mobility of the daphnia were observed.
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
Details on results:
- Other biological observations: No.
- Mortality of control: 0%
Results with reference substance (positive control):
- Results with reference substance valid? Yes, within the valid range 0.6 - 2.1 mg/L, according to OECD 202 (2004).
- Relevant effect levels: EC50 (24 h) = 1.26 mg/L (potassium dichromate), 95% confidence limits = 1.04 - 1.45 mg/L
Reported statistics and error estimates:
An EC50-value was calculated for the reference item by sigmoidal dose-response regression. The respective 95% confidence limits wer calculated from the standard error and the t-distributin,. All calculations were carried out from the best-fit values with the software GraphPad Prism5.

ANALYTICAL RESULTS

The measured concentration in the limit concentration was 94% of the nominal concentation at the start of the exposure (0 h) and 93% of the nominal concentration at the end of the exposure (48 h).

The measured test item concentrations were within ± 20% of the nominal concentration. This indicates that the test item concentration was successfully maintained for the duration of the test.

Therefore, the effect concentrations were based on the nominal concentration of the test item.

BIOLGICAL RESULTS

5% immobility (1 of 20 daphnia) was observed in the nominal limit concentration 100 mg/L of the test item after 48 h. Since the test guidelines accept 10% immobility in the control group and since no other adverse effects were observed in the definitive test and in the range-finding test at the highest tested concentration level, the 5% are considered to be not test item-related.

Description of key information

EC50 (48 h) > 100 mg/L (nominal, limit concentration, D. magna, OECD 202)

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
100 mg/L

Additional information

There is one GLP study available investigating the acute toxicity of the substance to aquatic invertebrates according to the OECD guideline 202.

In a static test, Daphnia magna was exposed to a nominal limit concentration of 100 mg/L test item, which was previously established in a preliminary range-finding test, for 48 h. Negative controls were run in parallel. The concentration of the test item was analytically verified by HPLC-DAD at the start and at the end of the exposure (0 and 48 h).

The limit concentration was visually clear throughout the exposure period. The measured concentration of the test item in the limit concentration was 94% of the nominal concentration at the start of the exposure (0 h) and 93% of the nominal concentration at the end of the exposure (48 h). This indicates that the test item concentration was successfully maintained for the duration of the test. Since the measured test item concentrations were within ± 20% of the nominal concentration, the effect concentration is based on the nominal concentration.

After 48 h, 5% immobilization (1 of 20 daphnia) was observed in the limit concentration. However, since the testing guideline accepts 10% immobility in the control group and no other adverse effects were otherwise observed, this finding was not considered test item-related.