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Short-term toxicity to aquatic invertebrates

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Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 January -21 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
(No chemical analysis was conducted. The test substance is an UVCB, and no chemical analysis is available for the whole compound. Therefore, in agreement with the sponsor, no chemical analysis was performed.)
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
yes
Remarks:
(No chemical analysis was conducted. The test substance is an UVCB, and no chemical analysis is available for the whole compound. Therefore, in agreement with the sponsor, no chemical analysis was performed.)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Green liquor 2
- Chemical name: Sulphite liquors and Cooking liquors, green.
- Molecular formula (if other than submission substance): UVCB (a substance from sulphate/Kraft process)
- Appearance: Yellowish liquid
- pH: over 11
- Solubility: In water: The substance is a water solution, The dry solids content of the tested sample is 18.7% dry solids/81.3% water by weight (at 20 deg. C))
- Impurities (identity and concentrations): UVCB
- Lot/batch No.: Not stated
- Expiry date: Not available.
- Stability under test conditions: No data available
- Storage condition of test material: Room temperature. Storage in the dark but may be used under light.

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:Nominal test substance concentrations of 3.1, 6.2, 12.4, 24.7, 49.4, 98.8 and 196.5 mg/L (wet weigth basis, 75% water included) were diluted from two stock solutions of 98.8 mg/L and 393.0 mg/L. The stock solutions were prepared by addition of 24.7 mg and 39.3 mg test substance to respectively 250 mL and 100 mL reconstituted water according to ISO 6341.
- Controls: Reconstituted water only
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable
- Concentration of vehicle in test medium (stock solution and final test solution): Not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No.

Test organisms

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).
- Strain: not applicable.
- 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: 1.32 mg/L for the batch used (DM281009). 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 88 hours after initiation of hatching.
- Feeding during test: none.
- Food type: not applicable.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None.

Test conditions

Hardness:
250 mg CaCO3/L
Test temperature:
19-20 °C
pH:
The pH was between 7.6 and 9.0 in the test substance media and 7.7 in the control media.
Dissolved oxygen:
at least 8.7 mg/L
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal: 3.1, 6.2, 12.4, 24.7, 49.4, 98.8 and 196.5 mg/L (wet weight basis, 81.3 % water included)
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
- No. of vessels per vehicle control (replicates): not applicable.
- Biomass loading rate: Not applicable.

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. ISOD070509). The water was aerated and the pH, the dissolved oxygen concentration and the water hardness were checked before using.
- 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.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: <2.2
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study: No.
Reference substance (positive control):
yes
Remarks:
24 h EC50 for potassiumdichromate: 1.1 mg/L

Results and discussion

Effect concentrationsopen allclose all
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
43 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
wet weight
Basis for effect:
mobility
Remarks on result:
other: 95% CL: lower: 35 mg/L, upper: 52 mg/L, slope: 2.3
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
40 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
wet weight
Basis for effect:
mobility
Remarks on result:
other: 95% CL: lower: 32 mg/L, upper: 50 mg/L, slope: 2.4
Duration:
24 h
Dose descriptor:
EC100
Effect conc.:
ca. 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
wet weight
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
ca. 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
wet weight
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC0
Effect conc.:
ca. 25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
wet weight
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
ca. 6.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
wet weight
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
6.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
wet weight
Basis for effect:
mobility
Remarks:
based on immobilised and trapped daphnia
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
6.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
wet weight
Basis for effect:
mobility
Remarks:
based on immobilised daphnia only
Details on results:
- Behavioural abnormalities: none.
- Observations on body length and weight: not applicable
- Other biological observations: Two daphnia were trapped at the water surface of the test medium C6 after 24 hours of exposure. After 48 hours one daphnia each was trapped at the water surface of the test media C1 and C2 and two in C6.
- Mortality of control: One immobile daphnia in control after 48 hours of exposure
- Other adverse effects control: No.
- Abnormal responses: No.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No.
Results with reference substance (positive control):
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 24 h EC50 of 1.1 mg/L which is wihtin 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.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
EC50, 48 h: 40 mg/L (wwt.) 7.5 mg/L (dwt.)
EC0, 48 h: ca. 6.2 mg/L (wwt.) 1.2 mg/L (dwt.)
NOEC, 48 h: 6.2 mg/L (wwt.) 1.2 mg/L (dwt.) (based on immobilised and trapped daphia)
NOEC, 48 h: 6.2 mg/L (wwt.) 1.2 mg/L (dwt.) (based on immobilised daphnia only)
Executive summary:

The acute toxicity of"Green liquor 2" to Daphnia magna was determined for regulatory purposes.

Regulation (EC) 440/2008: C.2.: Daphnia sp. Acute Immobilisation Test andOECD-Guideline 202: "Daphnia sp., Acute Immobilisation Test", (13thApril 2004) were applied.

Nominal test substance concentrations of 3.1, 6.2, 12.4, 24.7, 49.4, 98.8 and 196.5 mg/L (wet weight, 75% water included) were diluted from two stock solutions of 98.8 mg/L and 393.0 mg/L. The stock solutions were prepared by addition of 24.7 mg and 39.3 mg test substance to respectively 250 mL and 100 mL reconstituted water according to ISO 6341.

Reconstituted water only was used for the negative control group.

20 neonates ofDaphnia magna, hatched from ephippia and less than 24 hours old were exposed in each of the 7 test substance groups and in the control group. Exposure was for 48 h in a static test. The dry solids content of the tested sample was 18.7% dry solids/81.3% water by weight (at 20 deg. C)).

Results (GL2 sample):

EC50, 48 h: 40 mg/L (wet weight), 7.5 mg/L (dry weight)

EC0, 48 h: ca. 6.2 mg/L (wet weight), 1.2 mg/L (dry weight)

NOEC, 48 h: 6.2 mg/L (wet weight), 1.2 mg/L (dry weight) (based on immobilised and trapped daphia).

NOEC, 48 h: 6.2 mg/L (wet weight), 1.2 mg/L (dry weight) (based on immobilised daphnia only)