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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:
from 1991-02-14 to 1991-09-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP without deviations
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
from 1984
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPP 72-2 (Aquatic Invertebrate Acute Toxicity Test)
Version / remarks:
from 1982
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control), 0 (DMSO control), 250 and 500 µg/L (nominal)
- Sampling method: not reported
- Sample storage conditions before analysis: stored cool and dark
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 50 mg 2-ethylanthraquinone was dissolved in 10 mL DMSO to obtain a concentration of 5000 mg/L. This stock solution was diluted with DMSO to obtain a second stock solution of 2500 mg/L. To erlenmeyers of 1 liter, wrapped in aluminiumfoil and containing 800 mL ISO+-water, 80 µL of these stock solutions were added. These solutions were stirred for 2 hours to obtain homogeneous nominal test concentrations of 500 and 250 µg/L. The ISO+-water was prepared and aerated at least 24 hours prior to the test.
- Eluate: not applicable
- Differential loading: not reported
- Controls: A control with DMSO in ISO+-water (0.1 mL/L) and a control with ISO+-water were included in the test.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): DMSO
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain: laboratory culture of the Ecotoxicology Group, DUPHAR B.V
- Source: culture is in house since August 1986 and originates from TNO, the Netherland
- Age at study initiation (mean and range, SD): 0-24 hours
- Feeding during test: The water fleas were not fed during the test.
- Method of breeding: cultured in vessels with 2 liter reconstituted water ( ISO+) which was prepared and aerated at least 24 hours prior to use
- Type and amount of food during breeding: algal suspension of Chlorella pyrenoidosa ( 1 * 10^9 cells per day per vessel) and yeast/Mikromin suspension (1 mL per day per vessel)
- Feeding frequency: daily
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
Hardness of reconstituted dilution water: about 250 mg/L
Test temperature:
Control: 23.5 °C at 0 hours, 22.1 °C at 48 hours
DMSO-control: 22.4 °C at 0 hours, 22.4 °C at 48 hours
210 µg/L: 24.8 °C at 0 hours, 22.3 °C at 48 hours
374 µ/L: 24.5 °C at 0 hours, 22.3 °C at 48 hours

The temperature in the test vessels was measured one hour after removal of the test vessels from the climate chamber to the laboratory. Moreover, the thermometer used in this test appeared to overestimate the temperature by about 2°C. Therefore, the actual temperature was about 20-23°C.
pH:
Control: 7.9 at 0 hours, 7.9 at 48 hours
DMSO-control: 8.0 at 0 hours, 8.0 at 48 hours
210 µg/L: 7.8 at 0 hours, 8.0 at 48 hours
374 µ/L: 7.8 at 0 hours, 8.0 at 48 hours
Dissolved oxygen:
Control: 9.2 mg/L at 0 hours, 9.1 mg/L at 48 hours
DMSO-control: 9.2 mg/L at 0 hours, 9.3 mg/L at 48 hours
210 µg/L: 8.7 mg/L at 0 hours, 9.2 mg/L at 48 hours
374 µ/L: 8.7 mg/L at 0 hours, 9.2 at 48 hours
Nominal and measured concentrations:
Nominal test concentrations: 0 µg/L (control), 0 µg/L (DMSO-control), 250 µg/L, 500 µg/L
Mean measured test concentration during test period (controls measured at 0 and 48 hours, test concentrations measured at 0, 24 and 48 hours): < 27 µg/L (control),<27 µg/L (DMSO-control), 210 µg/L, 374 µg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml beaker glasses
- Type (delete if not applicable): not reported
- Material, fill volume: glass, 250 mL
- Aeration: not aerated
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): no renewal
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3
- Biomass loading rate: not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: not reported
- Composition of reconstituted dilution water:
ISO:
Deioised water
CaCl2 · 2H2O: 294 mg/L
MgSO4 · 7 H2O: 123 mg/L
NaHCO3: 64 mg/L
KCl: 5.8 mg/L
ISO+(same as ISO with the following elements in addition):
LiCl: 8.0 µg/L
RbCl: 0.4 µg/L
SrCl2 · 6H2O: 8.0 µg/L
NaBr: 50.0 µg/L
KI: 3.0 µg/L
MnCl2 · 4H2O: 3.0 µg/L
FeCl3: 1.7 µg/L
CuCl2 · 2H2O: 6.0 µg/L
CoCl2 · 6H2O: 1.1 µg/L
Na2MoO4 · 2H2O: 0.1 µg/L
ZnCl2: 1.0 µg/L
V2O5: 1.0 µg/L
NiSO4 · 6H2O: 4.0 µg/L
Al2O3: 1.0 µg/L
NaF: 5.0 µg/L
Na2CrO4 · 4H2O: 1.0 µg/L
PbCl2: 0.4 µg/L
SnCl2: 5.1 µg/L
SeO2: 1.0 µg/L
- Culture medium different from test medium: No
- Intervals of water quality measurement: not reported

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Light intensity: reduced light conditions (less than 100 lux) because additional studies showed that 2-ethylanthraquinone is highly unstable in water under influence of light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobility (examined at 24 and 48 hours), slower movement, uncontrolled movement, laying down on bottom of test vessel, abnormal shape (examined at 48 hours)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: In a range-finding study, the presence of 2-ethylanthraquinone did not clearly cause mortality at the nominal test concentrations up to 250 µg/L. Therefore, the final test was carried out at 2 concentrations, one slightly above and one slightly below the water solubility level.
- Range finding study: yes
- Test concentrations: up to 250 µg/L
- Results used to determine the conditions for the definitive study: The water fleas were exposed during 48 hours to various concentrations of 2-ethylanthraquinone in test vessels in a range-finding test. In this study the presence of 2-ethylanthraquinone did not clearly cause mortality at the nominal test concentrations up to 250 µg/L. Therefore, the final test was carried out at 2 concentrations, one slightly above and one slightly below the water solubility level.
Reference substance (positive control):
yes
Remarks:
potassium-bichromate (performed every year, last test: February 1990)
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
270 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CL: [220, 320]
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
< 210 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: immobility
- Observations on body length and weight: not reported
- Other biological observations: At 24 and 48 hours after test initiation some of the water fleas were floating on the surface of the test solution. These effects are not considered to be compound related as they occurred in nearly all test vessels.
- Mortality of control: 4/31 (control), 2/29 (DMSO-control)
- Other adverse effects control: not reported
- Abnormal responses: not reported
- 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: The EC50.48h is above the solubility level of 2-ethylanthraquinone (245 µ g/L).
Results with reference substance (positive control):
EC50.48 h: 0.42 mg/L
95% confidence interval: 0.34 - 0.48 mg/L

Immobility of Daphnia magna during the acute toxicity test with 2-ethylanthraquinone

Mean measured

concentration

(µg/L)

Number of immobile

water fleas

Total number

of immobile

water fleas

Number of

water fleas

tested

Percentage (%)

immobility

after 48 hours

 

0-24h

24-48h

 

 

 

0 (control)

2

2

4

31

10

0 (DMSO-control)

0

2

2

29

210

2

7

9

30

30

374

27*

-2*

25

30

82

*Because of low light intensity a good measurement at 24 hours was not possible, and the number of immobile daphnids was overestimated.

Validity criteria fulfilled:
yes
Conclusions:
Based on the mean measured test concentrations at the initiation of the test the EC50.48 h was 270 µg/L with 95% confidence interval of 220-320 µg/L. The EC50.48h is above the solubility level of 2-ethylanthraquinone (245 µg/L). The NOEC (no observed effect concentration) was <210 µg/L.
Executive summary:

The acute toxicity of 2-ethylanthraquinone to water fleas (Daphnia magna, Crustaceae) was tested according to OECD TG 202 at nominal test concentrations of 250 and 500 µg/L in ISO-water. As 2-ethylanthraquinone is highly unstable in water under influence of light, the test was conducted under reduced light conditions (400 lux) to reduce the decrease in test concentration. Samples of the test concentrations were taken 0, 24 and 48 hours after test initiation and analysed with HPLC. During the test, the concentration of

2-ethylanthraquinone remained above 80% of the initial test concentration. Based on the mean measured test concentrations at the initiation of the test the EC50.48 h was 270 µg/L with 95% confidence interval of 220-320 µg/L. The EC50.48h is above the solubility level of 2-ethylanthraquinone (245 µg/L). The NOEC (no observed effect concentration) was <210 µg/L.

Description of key information

In a short-term toxicity test with daphnia, the EC50 after 48 hours was 270 µg/L and the NOEC was <210 µg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
270 µg/L

Additional information

The acute toxicity of 2-ethylanthraquinone to water fleas (Daphnia magna, Crustaceae) was tested according to OECD TG 202 at nominal test concentrations of 250 and 500 µg/L in ISO-water. The test substance was dissolved in DMSO prior to the preparation of test solutions due to its low water solubility. As 2-ethylanthraquinone is highly unstable in water under influence of light, the test was conducted under reduced light conditions (400 lux) to reduce the decrease in test concentration. Samples of the test solutions were taken 0, 24 and 48 hours after test initiation and analysed with HPLC. During the test, the concentration of

2-ethylanthraquinone remained above 80% of the initial test concentration. Based on the mean measured test concentrations at the initiation of the test the EC50 after 48 h was 270 µg/L with a 95% confidence interval of 220-320 µg/L. The EC50 after 48h was above the solubility level of 2-ethylanthraquinone (245 µg/L). The NOEC (no observed effect concentration) was <210 µg/L.

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