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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
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:
adopted April 13, 2004
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Remarks:
The quantification of the test item was performed by HPLC analysis and UV/VIS-detection.
Details on sampling:
Stability of test concentration/s during exposure : Examined by chemical analysis (HPLC) at 0 and 48 hours.
Storage: Routinely, the samples were analysed immediately. Only in exceptional cases, they were stored overnight deep frozen and protected from light.
Vehicle:
no
Details on test solutions:
PRE-TREATMENTOF TEST ITEM AND PREPARATION OF TEST ITEM CONCENTRATIONS
To produce the only test item concentration, 100.8 mg of the test item were added to 1 litre of dilution water, treated for 1 h in an ultrasonic bath and stirred for 1 h on a magnetic stirrer. The pH was measured to be 7.9.
50 mL of the solution were taken and diluted with 0.5 mL of dilution water containing 5 daphnids resulting in a final concentration of 100 mg/L. For each test item concentration and the control 4 replicates were prepared.

STANDARD SOLUTIONS USED FOR CALIBRATION OF THE TEST ITEM
20.9 mg of the test item were dissolved and made up with Millipore water/Acetonitrile (1:1) in a 100 mL volumetric flask to prepare a stock solution of 209 mg/L. Defined volumes of this stock solution were diluted with Millipore water to obtain standard solutions in the range of 0.0523 to 2.09 mg/L.
The concentration of the lowest used calibration solution was employed as the limit of quantification.
These solutions were used to calibrate the HPLC-system.

STANDARD SOLUTIONS USED FOR VERIFICATION OF THE CALIBRATION OF THE TEST ITEM AND BLANK TEST
22.8 mg of the test item were dissolved and made up with Millipore water/Acetonitrile (1:1) in a 100 mL volumetric flask to prepare a verification solution with a nominal concentration of 228 mg/L. This solution was diluted with Millipore water to obtain a concentration of 0.912 mg/L. The daily verification of the calibration was performed with this diluted Standard Solution of Verification.
The mean recovery rate was calculated.
Millipore water without the test item was analysed (analytical blank).
Test organisms (species):
Daphnia magna
Details on test organisms:
-Name: Daphnia magna STRAUS, parthenogenetic females
-Source: Strain of Bundesgesundheitsamt Berlin
-Maintenance and Acclimatisation: A population of parthenogenetic females of synchronized age structure has been maintained for more than 20 years in the test facility under constant temperature conditions (20 +/- 2 °C) at a 16 : 8 hour light-dark photoperiod (light intensity: < 20 μE x m-2 x s-1). The culture water (so-called 'M4 medium') was partly renewed once a week. The Daphnia were exclusively fed unicellular green algae (Desmodesmus subspicatus) 'ad libitum'. Mortalities of parent Daphnia during the culture period were recorded daily. The neonates were separated from their parent Daphnia by filtration prior to the acute test.
Test type:
static
Water media type:
freshwater
Remarks:
Reconstituted water (so-called 'M4 medium' according to OECD 202 and EC Method C.2, annex 1) was used for the maintenance of the test animals and the preparation of stock and test solutions of the test item.
Limit test:
yes
Total exposure duration:
48 h
Remarks on exposure duration:
Immobilisation rates were recorded at 24-hour intervals. Additionally any abnormal behaviour or appearance of the Daphnia was reported every 24 hours.
Hardness:
14.5 °dH (= 259 mg/L CaCO3)
Test temperature:
18 - 22 °C +/- 1 °C;
Temperature of incubation unit : 19.5 to 19.7 °C
pH:
7.8 - 7.9
Dissolved oxygen:
9.0 - 9.3 mg/L (102 - 105%)
Nominal and measured concentrations:
100 mg/L (nominal)
Details on test conditions:
The temperature, the pH and the oxygen values were measured at the beginning and end of the test.

EXPOSURE CONDITIONS
-Test vessels : 100 mL glass beakers covered with watch glasses holding 5 neonates in 50 mL of test medium.
-Experimental design : 1 test concentration plus 1 control
5 neonates per vessel, 4 replicates per concentration/control
no feeding during the exposure period
static system
-Method of initiation : neonates were placed in prepared media
-Photoperiod : 16 h light : 8 h dark
-Temperature of incubation unit : 19.5 to 19.7 °C
-Aeration : none
-Test item concentration/s : 100 mg/L (nominal)
-Method of administration : direct weighing
-Medium renewal : none
-Duration of exposure: 48 hours
-Criteria of effects : The criterion of adverse effects used in this study was the item-induced alteration of the normal mobility behaviour and the loss of locomotory actions of the neonates, observed at 24 and 48 hours.
Reference substance (positive control):
no
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
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC100
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC0
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
No toxic effects against Daphnia were observed at a limit test concentration of 100 mg/L under exposure conditions.
The results are expressed in terms of nominal concentrations. Effective concentrations correspond to 103.3% of nominal values at 0 hours and to 102.4% of nominal values at 48 hours.
Reported statistics and error estimates:
Daphnia were exposed to the test item added to dilution water at a limit test concentration of nominally 100 mg/L for a period of 48 hours. At this concentration no toxic effects against Daphnia were observed at the end of the 48 hour study period, thus no statistical analysis was required to determine the EC 50.

Abiotic parameters at test start and at the end of the test

       Nominal test concentration [mg/L]

          At test start (0 h)

          At the end of the test (48 h)
 Temperature  Oxygen  Oxygen  pH value  Temperature  Oxygen  Oxygen  pH value
 [°C]  [mg/L]  [%]    [°C]  [mg/L]  [%]  
 Control  20.1  9.2  102  7.8  19.7  9.3  103  7.9
 100  20.7  9.0  105  7.9  20.3  9.1  102  7.9

Analysis

 Nominal concentration     HPLC value [mg/L]
 [mg/L]  at 0 hours  at 48 hours
 control  --  < 0.052
 100  103.346  102.431
Validity criteria fulfilled:
yes
Remarks:
The immobilisation and other abnormalities in the controls did not exceed 10 % by the end of the test. The dissolved oxygen concentration remained above 3 mg/L throughout the exposure period.
Conclusions:
Daphnia exposed to 100 mg/L of Anthraquinone-2-sulfonic acid sodium salt monohydrate in a limit test according to OECD guideline No. 202 unter static conditions showed no toxic effects (EC50 > 100 mg/L after 48 hours).
Executive summary:

A study was performed to assess the acute toxicity of Anthraquinone-2-sulfonic acid sodium salt monohydrate to Daphnia magna STRAUS under static conditions

The study was conducted in accordance with the Council Regulation (EC) No 440/2008, Method C.2 ‘Acute toxicity for Daphnia’ (2008) which is equivalent to OECD Guideline for Testing of Chemicals No. 202 'Daphnia sp., Acute Immobilisation Test' (adopted April 13, 2004).

The Daphnia were exposed to a limit test concentration of nominally 100 mg/L of Anthraquinone-2-sulfonic acid sodium salt monohydrate dissolved in dilution water. Auxiliaries used to prepare the test media were an ultrasonic bath and a magnetic stirrer.

Observations were made on the swimming ability and the immobilisation rate, respectively, after 24 and 48 hours of exposure.

No toxic effects against Daphnia were observed at a limit test concentration of 100 mg/L under exposure conditions (48hr-EC50 > 100 mg/L).

The results are expressed in terms of nominal concentrations. Effective concentrations correspond to 103.3% of nominal values at 0 hours and to 102.4% of nominal values at 48 hours.

The hardness of the dilution water used was 14.5 °dH (= 259 mg/L CaCO3).

All validity criteria are met.This toxicity study is classified as acceptable and satisfies the guideline requirements for toxicity to aquatic invertebrates.

Description of key information

Daphnia exposed to 100 mg/L of Anthraquinone-2-sulfonic acid sodium salt monohydrate in a limit test according to OECD guideline No. 202 unter static conditions showed no toxic effects (EC50 > 100 mg/L after 48 hours).

Key value for chemical safety assessment

Additional information

The results are expressed in terms of nominal concentrations. Effective concentrations correspond to 103.3% of nominal values at 0 hours and to 102.4% of nominal values at 48 hours.

The hardness of the dilution water used was 14.5 °dH (= 259 mg/L CaCO3).