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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:
18 Mar - 16 Sep 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD series on testing and assessment no. 23, ENV/JM/MONO (2000)6
GLP compliance:
yes (incl. certificate)
Remarks:
Staatliches Gewerbeaufsichtsamt Hildesheim, Germany
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control, 3.91, 6.25, 10, 16, 25.6% of a saturated solution were measured after 0 and 48 h (additionally the test item concentration in a saturated solution was measured at 0 h)
- Sampling method: At the start of the exposure (0 h), sampling was carried out after preparation of the test concentrations. At the end of the exposure (48 h), samples for the analyses of the old media were taken from additional replicates prepared with test media, but without daphnids, because the volume in the test vessels was not sufficient for the analyses. These additional replicates were incubated under test conditions until analysis. All sampling vials were completely filled leaving no headspace and were tightly closed.
- Sample storage conditions before analysis: If necessary, all samples were stored at 6 ± 2 °C until analysis. Prepared samples were stored in an autosampler at room temperature until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A saturated solution with a nominal concentration of 1 mL/L of the test item (corresponding to 1.04 g/L) was prepared with dilution water (ISO test water) four days prior to the start of the exposure. A glass flask was filled with 5000 mL of the dilution and the test item (5 mL) was placed by pipette onto the water surface, then the bottle was closed with a screw cap. A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out for three days with a magnetic stirrer at 30 ± 2 °C. After pre-incubation, the temperature of the solution was reduced to 21 -24 °C for 24 h. After completion of stirring, the saturated solution was removed by siphon from the bottom, since any undissolved material was expected to float on the surface due to the density of the test item. Five concentrations were prepared by dilution of the saturated solution with a separation factor of 2.
- Eluate: no
- Differential loading: no
- Controls: yes, test medium control
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No; The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). No Tyndall effect was observed. The saturated solution was used as the stock solution for the preparation of further dilution levels by diluting with ISO test water.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: Clone 5
- Method of breeding: Bred in Elendt M4 medium modified to a total hardness of 160 - 180 mg CaCO3/L. Daphnids were cultured in glass vessels (2-3 L capacity) filled with 1.8 L culture medium and incubated at 20 ± 2 °C in an incubator. The light/dark cycle was 16 h light/8 h darkness. Daphnids were fed at least 5 times per week ad libitum with a mix of unicellular green algae. Algae were cultured at the testing facility.
- Feeding during test: none
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
263 mg CaCO3/L
Test temperature:
19 - 20 °C
pH:
7.00 - 8.06
Dissolved oxygen:
5.68 - 7.90 mg O2/L
Conductivity:
640 µS/cm
Nominal and measured concentrations:
nominal: control, 3.91, 6.25, 10.0, 16.0, 25.6% of a saturated solution
measured: < LOQ, 0.717, 1.01, 1.54, 2.70, 4.42 mg/L (geometric mean measured concentration)
Details on test conditions:
TEST SYSTEM
- Test vessel: Sealed glass flasks (4.5 (ID) x 9.5 (H) cm) with screw caps (made from polypropylene) were used. A test volume of approximately 130 mL was provided in each test vessel.
- Type (delete if not applicable): closed
- 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: according to guideline
- Culture medium different from test medium: same as test
- Intervals of water quality measurement: The incubator temperature was recorded throughout the period of the test. Prior to the start of the exposure (0 h), pH-value, dissolved oxygen concentration, temperature, conductivity and total hardness of the dilution water were measured. At the start of the exposure (0 h), the water quality parameters of the fresh media were measured in one additional replicate (without daphnids) per concentration level and control. At the end of the exposure (48 h), the water quality parameters of the old media were measured in all replicates (containing daphnids) per concentration level and control.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light, 8 h dark
- Light intensity: diffuse light, light intensity of max. 20 µE*m-2*2-1 (max. 1340 lux)

EFFECT PARAMETERS MEASURED
- Immobilization after 24 and 48 h

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: control, 1, 10 and 100% of a saturated solution
- Results used to determine the conditions for the definitive study: 0% immobilization in the control, 10% immobilizazion at 1% solution, 85% immobilization at 10% solution, 100% immobilization at 100% solution
Reference substance (positive control):
yes
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
2.73 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CI
Remarks:
1.91 - > 4.42 mg/L
Details on results:
- Mortality of control: 0%
- Effect concentrations exceeding solubility of substance in test medium: No Tyndall effect was observed in the saturated solution of the test item, which was used as a stock solution, indicating that no emulsion was formed. The tested concentration levels were visually clear throughout the exposure period.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- ECx: EC50 (48 h) 2.03 mg/L (CI: 1.17 - 3.63 mg/L)
Reported statistics and error estimates:
The EC10- and the EC50-value after 48 h was calculated by sigmoidal dose-response regression with the software GraphPad Prism 5. The respective 95% confidence limits were calculated from the standard error and the t-distribution. All calculations were carried out from the best-fit values with the software GraphPad PrismS. The concentration-effect relationship after 48 hours is shown graphically. Since only a partial effect was observed, the calculation of the EC10- values (after 24 h) was not applicable and therefore estimated directly from the observation data. The EC50-value for the reference item and its 95% confidence limits were calculated accordingly.

Table 1: Immobilization of D. magna after 24 and 48 h of exposure in the definitive test

Geometric mean measured concentration [mg/L]

Immobilization [%]

24 h

48 h

Replicates

Replicates

1

2

3

4

Mean

1

2

3

4

Mean

4.42

20

0

20

60

25

40

60

80

100

70

2.70

40

20

20

20

25

40

20

60

60

45

1.54

40

20

0

20

20

60

20

40

20

35

1.01

0

0

0

0

0

0

0

0

0

0

0.717

0

0

0

0

0

0

0

0

20

5

Control

0

0

0

0

0

0

0

0

0

0

Table 2: Measured concentrations of the test item during the definitive test

Dilution level of the saturated solution [%]

0 h

48 h

Geometric mean measured concentration [mg/L]

Measured concentration [mg/L]

Measured concentration [mg/L]

%

100

16.1

Not determined since it was used as stock solution

25.6

4.37

4.48 (2)

103

4.42

16

2.63

2.78

106

2.70

10

1.58

1.50

95

1.54

6.25

0.948

1.07

113

1.01

3.91

0.682

0.753

110

0.717

Control

< LOQ

< LOQ

 

(2): Mean value of two replicates with different dilution factor

Validity criteria fulfilled:
yes
Conclusions:
The study resulted in an EC50 (48 h) of 2.73 mg/L based on the geometric mean of measured concentrations (OECD 202).

Description of key information

EC50 (48 h): 2.73 mg/L based on mobility of Daphnia magna (OECD 202)

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
2.73 mg/L

Additional information

One experimental study is available investigating the short-term effects of the substance to aquatic invertebrates. The study was performed according to the OECD 202 (GLP) with the water flea Daphnia magna. Dilutions of 3.91, 6.25, 10.0, 16.0, 25.6% of a saturated solution were tested under static conditions. It was ensured by an appropriate test design that the substance does not volatilize from the test vessels (closed vessels; no headspace). Immobilisation of D. magna was observed after 48 h resulting in an EC50 (48 h) of 2.73 mg/L based on geometric mean measured concentrations.