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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:
Experimental start date: . Experimental completion date:
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control and test item concentration at 0 and 48 hours.
- Sampling method: For the determination of the actual test item concentration, duplicates samples were taken from the test medium and the control at the start and at the end of the test after 48 hours (stability samples).
For the 48-hour stability samples, additional flasks with adequate volumes of the freshly prepared test medium and the control were incubated over the test period under the same conditions as in the actual test (but without daphnids). Sampling from the test beakers with daphnids was not possible because the volume of test medium in the actual test (4 x 50 mL) was too small for the analytical requirements (500 mL per sample). The concentration of the test item was analytically determined in the duplicate test medium samples taken at the start and the end of the test. From the control samples, only one of the duplicate samples was analysed from each sampling time.

- Sample storage conditions before analysis:
The samples were analysed immediately after sampling without prior storage.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: In order to assess the toxicity of the test substance containing to daphnids, a water accommodated fraction (WAF) with a loading rate of 100 mg/L was tested. Additionally, a control (test water without test item) was tested in parallel. Thus, a limit test was performed in accordance with the EU Commission Directive 92/69/EEC to demonstrate that WAFs of the test item have no toxic effects on the daphnids up to the loading rate of 100 mg/L. The test method was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures.
The WAF was prepared as follows: A dispersion with a loading rate of 100 mg/L was prepared by dispersing 249.9 mg of the test item in 2500 mL of test water using intense stirring on a magnetic stirrer. The dispersion was stirred for 3 hours at room temperature in the dark to dissolve a maximum amount of the test item in the dispersion. Ultrasonic treatment was not applied to avoid accelerating hydrolysis.
The short stirring period of 3 hours was chosen because the components of the test item are not stable in water (results of a pre-experiment to determine the solubility and stability of the test item in test water, without GLP). Then, the dispersion of the test item was filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 pm) after the 3 hour stirring period. The test medium was prepared just before introduction of the daphnids (= start of the test).
The actual concentration of the test item in the test medium was analytically determined.
The limit test was based on the results of a range-finding test and the results of a pre-experiment to determine the solubility of the test item (without GLP). However, loading rates above 100 mg/L were not tested according to the EU Commission Directive 92/69/EEC.
- Controls: a control (test water without test item) was tested in parallel.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): n/a
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): n/a
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia
- Strain/clone: Straus, Clone 5
- Justification for species other than prescribed by test guideline: n/a
- Source: originally supplied by the University of Sheffield/UK in 1992, defined by the supplier as clone 5. Since that time, the clone is bred in the laboratories of RCC in reconstituted water of the quality identical to the water quality used in the tests (in respect to pH, main ions, and total hardness) and under temperature and light conditions identical to those of the tests.
- Age of parental stock (mean and range, SD): 6—24 hours old and were not first brood progeny.
- Feeding during test: None
- Food type:
- Amount:
- Frequency:

ACCLIMATION
- Acclimation period: Not required: daphnia are held in water identical to the water quality used in the tests (in respect to pH, main ions, and total hardness) and under temperature and light conditions identical to those of the tests.
- Acclimation conditions (same as test or not): n/a
- Type and amount of food: not reported
- Feeding frequency: not reported
- Health during acclimation (any mortality observed): not reported
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
2.5 mmol/l, 250 mg/l as CaCO3
Test temperature:
20 - 21 °C
pH:
7.7 - 7.8
Dissolved oxygen:
8.2 - 8.5 mg/l
Conductivity:
not reported
Nominal and measured concentrations:
Nominal: control and 100 mg/l loading rate WAF.
Measured:
Details on test conditions:
TEST SYSTEM
- Test vessel: Beaker
- Type (delete if not applicable): open / closed: covered with glass plates to reduce the loss of water and to avoid the entry of dust into the solutions.
- Material, size, headspace, fill volume: 100 mL glass beakers filled with 50 mL of test medium.
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): n/a
- Renewal rate of test solution (frequency/flow rate): n/a
- 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): n/a
- Biomass loading rate: The loading rate was less than one daphnia per 5 mL of test solution

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water: analytical grade salts were dissolved in purified water.
- Total organic carbon: not reported
- Alkalinity: 0.8 mmol/l
- Ca/mg ratio: 4:1 based on molarity
- Culture medium different from test medium: no
- Intervals of water quality measurement: At the start and at the end of the test, the pH values, the dissolved oxygen concentrations, and the water temperature were determined in the single test concentration and the control. The appearance of the test medium was recorded at the start of the test and after 24 and 48 hours.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16-hour light to 8-hour dark photoperiod with a 30 minute transition period.
- Light intensity: between 520 and 710 Lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The daphnids were observed for immobility after 24 and 48 hours of exposure.

VEHICLE CONTROL PERFORMED: yes/no: n/a

RANGE-FINDING STUDY
- Test concentrations: The limit test was based on the results of a range-finding test and the results of a pre-experiment to determine the solubility of the test item (without GLP). However, loading rates
above 100 mg/L were not tested according to the EU Commission Directive 92/69/EEC. Results of the range-finding test are not reported in the study report.
- Results used to determine the conditions for the definitive study: Results of the range-finding test are not reported in the study report.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
mortality
Remarks on result:
other: highest concentration tested
Key result
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
mortality
Remarks on result:
other: highest concentration tested
Details on results:
- Behavioural abnormalities: None reported
- Observations on body length and weight: Not reported
- Other biological observations: None reported
- Mortality of control: No
- Other adverse effects control: None reported
- Abnormal responses: None 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: n/a
Results with reference substance (positive control):
48 h EC50: 0.83 mg/l

Table: Effect of the test substance on mortality of Daphnia magna

 Loading rate (mg/l)  Mean measured concentration (µg/l)  Number of daphnids tested  Immobilised daphnids after 24 hours     Immobilised daphnids after 48 hours  
       Number  %  Number  %
 Control  -  20  0  0  0  0
 100  <0.7  20  0  0  0  0
Validity criteria fulfilled:
yes
Conclusions:
A 48 hour EL50 value of >100 mg/l (nominal, WAF loading rate) (highest concentration tested) has been determined for the effects of the submission substance (CAS 352230-22-9) on mortality of Daphnia magna, in accordance with OECD Guideline 202 (Daphnia sp., Acute immobilisation Test).

Description of key information

48-hour EL50: >100 mg/L (nominal, WAF loading rate) (highest concentration tested); effects of the submission substance (CAS 352230-22-9) on mortality of Daphnia magna.

Key value for chemical safety assessment

Additional information

A 48-hour EL50 value of >100 mg/L (nominal, WAF loading rate) (highest concentration tested) has been determined for the effects of the submission substance (CAS 352230-22-9) on mortality of Daphnia magna, in accordance with OECD Guideline 202 (Daphnia sp., Acute immobilisation Test).

The study reports an analytically determined test substance concentration of <0.7 µg/L (<LOQ, mean measured). This value reflects the low water solubility of the test substance, but losses may also have occurred as a result of filtering of the test solution prior to addition of test organisms, and the potential for adsorption of the test substance onto the filter matrix.

Each constituent of the test substance has a slow hydrolysis half-life at pH 7 (predicted to be >200 to >630 hours). However, the hydrolysis rate greatly increases with higher pH; 2 to 5.3 hours at pH 9. This test was conducted at pH 7.7 -7.8. Some hydrolysis is likely to have occurred in the timescale of the test. In view of the test media preparation method and the static exposure regime it is likely that the test organisms were exposed predominantly to the parent substance at the beginning of the test, and to a mixture of the parent substance and the hydrolysis products of each constituent of the tested substance towards the end of the test.