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Ecotoxicological information

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:
from 8 March to 12 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Nominal test substance concentrations of 30, 63, 125, 250, 500 and 1000 mg test substance per litre reconstituted water according to ISO 6341 were tested.
- Sampling method: Samples of the test media for determination of the actual test substance concentrations were taken immediately before introduction of the Daphnia and after 48 hours.
- Sample storage conditions before analysis: The samples were analysed at the day of sampling.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution of nominal 1000 mg/L was prepared by addition of 265.9 mg test substance (6 % water content) to 250 mL reconstituted water according to ISO and stirring for 5 minutes. The test media were prepared by diluting the stock solution of nominal 1000 mg/L with reconstituted water.
- Controls: For the negative control group only reconstituted water was used.
- Evidence of undissolved material: no.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Neonates of Daphnia magna (Crustacea, Cladocera), were obtained by hatching ephippia, supplied as "Daphtoxkit F TM magna" (Daphnia magna; Fa. Micro BioTests Inc; Kleimoer 15, B-9030 Mariakerke (Gent); BELGIUM).
- Source:Ephippia were supplied in the microbiotest "Daphtoxkit F TM " (Daphnia magna; Fa. Micro BioTests Inc; Kleimoer 15, B-9030 Mariakerke (Gent); BELGIUM). The batch of ephippia is provided together with a specification sheet with batch number and date of expiry. Additionally for confirmation of the sensitivity of the test animals of the used batch the results of a reference test with potassium dichromate are provided by Microbiotest. 24h EC50 of potassium dichromate: 1.13 mg/L for the batch used. The acceptable range of the EC50 24h according to the OECD guideline 202 is 0.6 mg/L to 2.1 mg/L.
- Age at study initiation (mean and range, SD): less than 24 hours old.
- Method of breeding: Neonates were obtained by hatching ephippia. The ephippia were hatched in the laboratory in a petri dish containing reconstituted water in a temperature controlled room at approx. 20-22 °C under continuous illumination of approximately 6 000 Lux. According to MicroBioTests Inc. the largest hatching occurs between 72 and 80 hours and the Daphnia must be collected at the latest 90 hours after initiation of hatching. The neonates were collected for this test at approximately 83 hours after initiation of hatching.
- Feeding during test: none.

ACCLIMATION
- Acclimation period: <24 h.
- Acclimation conditions (same as test or not): same.
- Type and amount of food: Approximately 2 hours before the start of the test the neonates were fed with a suspension of Spirulina powder, as also in traditional Daphnia cultures the neonates have the opportunity to take up some feed particles.
- Feeding frequency: once

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
none
Hardness:
230 mg CaCO3/L.
Test temperature:
20 °C.
pH:
The pH was between 7.6 and 7.7 in the test substance media and between 7.6 and 7.7 in the control media.
Dissolved oxygen:
At least 8.7 mg/L.
Salinity:
not applicable.
Nominal and measured concentrations:
Nominal: 0.0, 30.00, 63.00, 125.00, 250.00, 500.00 and 1000.00 mg/L
Actual, at time 0 h: 0.0, 27.70, 58.38, 117.81, 227.79, 463.82 and 940.43 mg/L
Actual, at time 48 h: 0.0, 15.65, 24.56, 77.76, 156.83, 329.33 and 543.36 mg/L.
Geometric mean of the actual concentrations: 0.0, 20.82, 37.86, 95.71, 189.00, 390.83 and 714.84 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: Disposable multiwell test plates with 30 test wells with a transparent lid, provided with the Daphtoxkit F. Each plate has four 15 mL wells (replicates A, B, C, D) for each of the control and the concentrations tested. Additionally, for each concentration the plates are provided with "rinsing wells" to prevent dilution of the test substance during the transfer of the daphnia from the hatching petri dish to the test wells.
- Material, size, headspace, fill volume: 15 mL policarbonate wells (according to Microbiotest), filled with 10 mL medium.
- Aeration: No extra aeration, but slight circulation of air is still possible when the test plate is closed.
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable.
- Renewal rate of test solution (frequency/flow rate): not applicable.
- 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: The stock solutions for preparation of the reconstituted water according to ISO 6341 were obtained by MicroBioTests Inc. (Batch No. ISOD191007). The water was aerated and the pH, the dissolved oxygen concentration and the water hardness were checked before using. (Table 1)
- Culture medium different from test medium: no.
- Intervals of water quality measurement: Temperature daily. Oxygen and pH at the start and the end of the test. Total hardness before the start.

OTHER TEST CONDITIONS
- Adjustment of pH: no.
- Photoperiod: 4 a.m. to 8 p.m.
- Light intensity: not applicable.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
24 and 48 hours after the start of the exposure to the test substance the neonates were observed for their swimming behaviour. Animals not able to swim within approximately 15 s after gentle agitation of the test container were evaluated as immobilised. Animals trapped at the surface were counted, to evaluate a possible influence of the test substance to the water surface tension. Only the mobile daphnia trapped on the water surface were regarded as trapped but not the immobilised ones.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: < 2.2
- Justification for using less concentrations than requested by guideline: not applicable.
Reference substance (positive control):
yes
Remarks:
yes (24h EC50 (potassium dichromate): 1.1 mg/L)
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
ca. 562 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limit lower : 511 mg/L, upper: 617 mg/L, slope 3.44
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
424 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limit lower : 361 mg/L, upper: 497 mg/L, slope 2.72
Duration:
24 h
Dose descriptor:
EC100
Effect conc.:
> 715 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
ca. 715 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC0
Effect conc.:
ca. 391 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
ca. 189 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
189 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: none. No daphnia were trapped at the water surface.
- Observations on body length and weight: not applicable.
- Other biological observations: no
- Mortality of control: no immobilisation in the negative control group.
- Other adverse effects control: no.
- Abnormal responses: no.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no.
- Effect concentrations exceeding solubility of substance in test medium: not applicable.



Results with reference substance (positive control):
- EC50: 1.1 mg/L
- Other: To check the reliability of the test conditions for acute toxicity studies to Daphnia magna at Seibersdorf Labor GmbH a static reference test with K2Cr2O7 is conducted at least yearly. The last reference test was conducted in December 2009 and gave a 24h EC50 of 1.1 mg K2Cr2O7 per litre (95% confidence interval of 0.86 to 1.5 mg/L) which is within the acceptability range of 0.6 - 2.1 mg/L given by the OECD guideline 202.
Reported statistics and error estimates:
The EC50-values and their confidence limits were calculated with the moving average method of W. Thompson, modified by C.S. Weil, Drug and Chemical Toxicology, 6 (6), 595-603 (1983), if appropriate.

Results

·      The water hardness was 230 mg CaCO3/L.

·      pH and temperature were sufficiently stable throughout the test period.

·      Dissolved oxygen concentration was always higher than 3 mg/L.

·      All test media were clear and colourless throughout the test period.

Actual test substance concentrations:

Group

Nominal test substance concentration (mg/L)

Actual concentration
at 0 h
(mg/L)

Actual concentration at 48 h
(mg/L)

Difference
(0-48 h) between the actual concentrations (%)

Geometric mean 0 - 48 h (mg/L)

K (Control)

0.00

0.00

0.00

-

0.00

C1

30.00

27.70

15.65

-43.51

20.82

C2

63.00

58.38

24.56

-57.94

37.86

C3

125.00

117.81

77.76

-34.00

95.71

C4

250.00

227.79

156.83

-31.15

189.00

C5

500.00

463.82

329.33

-29.00

390.83

C6

1 000.00

940.43

543.36

-42.22

714.84

- Immobilisation:

Group

Mean actual test substance concentration (mg/L)

Immobilisation at 24 h

Immobilisation at48 h

K (Control)

0.00

0 / 20

0 / 20

C1

20.82

0 / 20

1 / 20

C2

37.86

0 / 20

0 / 20

C3

95.71

0 / 20

0 / 20

C4

189.00

1 / 20

2 / 20

C5

390.83

1 / 20

6 / 20

C6

714.84

16 / 20

20 / 20

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test substance to Daphnia magna was determined according to OECD 202 (2004). The 48h-EC50 was determined to be 424 mg/L.
Executive summary:

The acute toxicity of the test substance to Daphnia magna was determined according to OECD 202 (2004). Mean actual test substance concentrations of 20.82, 37.86, 95.71, 189.00, 390.83 and 714.84 mg perlitre reconstituted water according to ISO 6341 were obtained bydiluting the stock solution prepared at the start of the test. The stock solution was prepared by addition of nominal 265.9 mg test substance (6 % water content) per 250 mL reconstituted water and stirring for 5 minutes. Reconstituted water only was used for the control group. 20 neonates of Daphnia magna, hatched from ephippia and less than 24 hours old were exposed in eachof the 6 test substance groups and in the control group. Exposure was for 48 h in static test. The water hardness was 230 mg CaCO3/L, the pH and temperature were sufficiently stable throughout the test period. The Dissolved oxygen concentration was always higher than 3 mg/L. All test media were clear and colourless throughout the test period. The validity criteria were met.

The 48h-EC50 was determined to be 424 mg/L.

Description of key information

The acute toxicity of the test substance to Daphnia magna was determined according to OECD 202 (2004). The 48h-EC50 was determined to be 424 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

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

Additional information

The acute toxicity of the test substance to Daphnia magna was determined according to OECD 202 (2004). Mean actual test substance concentrations of 20.82, 37.86, 95.71, 189.00, 390.83 and 714.84 mg perlitre reconstituted water according to ISO 6341 were obtained bydiluting the stock solution prepared at the start of the test. The stock solution was prepared by addition of nominal 265.9 mg test substance (6 % water content) per 250 mL reconstituted water and stirring for 5 minutes. Reconstituted water only was used for the control group. 20 neonates of Daphnia magna, hatched from ephippia and less than 24 hours old were exposed in eachof the 6 test substance groups and in the control group. Exposure was for 48 h in static test. The water hardness was 230 mg CaCO3/L, the pH and temperature were sufficiently stable throughout the test period. The Dissolved oxygen concentration was always higher than 3 mg/L. All test media were clear and colourless throughout the test period. The validity criteria were met.

The 48h-EC50 was determined to be 424 mg/L.