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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:
10 January 2017 to 12 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: The control, solvent control and each test group
- Sampling method: Samples were taken from the control, solvent control and each test group from the freshly prepared bulk test preparation at 0 and 24 hours and from the old or expired pooled replicates at 24 and 48 hours for quantitative analysis.
- Sample storage conditions before analysis: All samples were stored frozen prior to analysis. Duplicate samples were taken at and stored frozen for further analysis if necessary.
Vehicle:
yes
Remarks:
dimethylformamide
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
A nominal amount of test material (100 mg) was dissolved in dimethylformamide and the volume adjusted to 25 mL to give the 4.0 mg/mL stock solution from which a series of dilutions was made to give further solvent stock solutions of 0.102, 0.256, 0.64 and 1.6 mg/mL. Each stock solution was inverted several times to ensure adequate mixing and homogeneity. Aliquots (200 µL) of the solvent stock solutions were each separately dispersed into 2 litres of test water with the aid of vigorous magnetic stirring for 30 minutes. Following the stirring period any undissolved test material was removed by centrifugation at 40 000 g for 30 minutes to give the test concentrations.

Preliminary solubility work conducted indicated that the test material was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. A test concentration of 0.40 mg/L (by visual inspection) was obtained using a preliminary solution in dimethylformamide. Based on this information the test material was categorised as being a ‘difficult substance’ as defined by OECD. Therefore a media preparation trial was conducted in order to determine the solubility of the test material under test conditions.
The use of a saturated solution method of preparation was considered inappropriate for this test material as centrifugation was not able to remove all of the undissolved test material present and filtration resulted in measured concentrations of less than the LOQ, possibly due to adsorption to the filter matrices.
Observations made on the untreated test sample taken from the solvent spike preparation showed some undissolved test material remained. It was therefore considered that centrifugation was required in order to ensure that all undissolved test material was removed. As there was a slight decline in the measured concentration obtained between the samples centrifuged at 10 000 and 40 000 g it was considered that centrifugation at 10 000 g was insufficient to ensure all undissolved test material was removed.
Based on the trial the test material was prepared using a solvent spike solution method of preparation at an initial loading rate of 0.40 mg/L, stirred for a period of 30 minutes prior to the removal of any undissolved test material by centrifugation at 40 000 g for 30 minutes to give a nominal test concentration of approximately 0.077 mg/L.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Source: In-house laboratory cultures
- Age: First instar. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.
- Feeding during test: No

ACCLIMATION
- Acclimation conditions: Adult daphnids were maintained in 150 mL glass beakers containing Elendt M7 medium as used for the range-finding and definitive tests in a temperature controlled room maintaining the water temperature at 18 to 22 °C. The lighting cycle was controlled to give a cycle with 16 hours of light and 8 hours of darkness with 20 minute dawn and dusk transition periods. Culture conditions ensured that reproduction was by parthenogenesis.
- Type of food: A mixture of algal suspension (Desmodesmus subspicatus) and flake food suspension
- Feeding frequency: Each culture was fed daily
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
21 to 22 °C
pH:
7.8 to 8.1
Dissolved oxygen:
8.3 to 9.4 mg O2/L
Nominal and measured concentrations:
- Nominal concentrations: 0.0102, 0.0256, 0.064, 0.16 and 0.40 mg/L
- Geometric mean measured concentration: 0.031 mg/L (0.40 mg/L nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: 40 mL glass test vessels
- Type: Sealed
- Material, size, headspace, fill volume: Completely filled
- Aeration: No
- Renewal rate of test solution: Fresh solution after 24 hours. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24-hour old test media into the fresh test media. Concentrations at which 100 % immobilisation was observed after 24 hours exposure were not renewed.
- 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): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water, Elendt M7 Medium. The medium contained the following ingredients (final concentration in mg/L): H3BO3 0.715, MnCl2.4H2O 0.090, LiCl 0.077, RbCl 0.018, SrCl2.6H2O 0.038, NaBr 0.004, Na2MoO4.2H2O 0.016, CuCl2.2H2O 0.004, ZnCl2 0.013, CoCl2.6H2O 0.010, KI 0.0033, Na2SeO3 0.0022, NH4VO3 0.00058, Na2EDTA.2H2O 0.625, FeSO4.7H2O 0.249, CaCl2.2H2O 293.8, NaHCO3 64.8, MgSO4.7H2O 123.3, Na2SiO3.9H2O 10, KCl 5.8, NaNO3 0.274, K2HPO4 0.184, KH2PO4 0.143,Thiamine hydrochloride 0.075, Cyanocobalamine (vitamin B12) 0.0010 and D(+) biotin (vitamin H) 0.00075. The pH of the prepared media was 7.9 ± 0.3 and it was stored at approximately 21 °C.
- Culture medium different from test medium: No
- Intervals of water quality measurement: Water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours of light and 8 hours of darkness with 20 minute dawn and dusk transition periods
- Light intensity: between 200 and 1200 Lux (measured range 463 to 796 Lux)

EFFECT PARAMETERS MEASURED:
Test Organism Observations: Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that daphnia were considered to be immobilised if they were unable to swim within 15 seconds after gentle agitation.

VEHICLE CONTROL PERFORMED: Yes, exposed to 100 µL/L Dimethylformamide

RANGE-FINDING STUDY
- Test concentrations: 0.010, 0.10 and 0.40 mg/L.
- Results used to determine the conditions for the definitive study: Yes; no immobilisation was observed at the test concentrations of 0.010 and 0.10 mg/L, however, immobilisation was observed at 0.40 mg/L.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 0.031 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.031 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
At the start and throughout the test all control and test solutions were observed to be clear colourless solutions. No immobilisation was seen throughout the test and all daphnids were observed to be normal throughout the test.
Exposure of Daphnia magna to the test material gave EC50 values of greater than 0.031 mg/L. The No Observed Effect Concentration was 0.031 mg/L.
The test concentration of 0.031 mg/L was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and having due regard to the amount of auxiliary solvent permitted in the study under the OECD Guidelines. No sub-lethal effects of exposure were observed throughout the test.

VALIDATION CRITERIA
The results of the test are considered valid if the following performance criteria are met: No more than 10 % of the control and solvent control daphnids show immobilisation or other signs of disease or stress (e.g. discoloration or unusual behaviour such as trapping at the surface water) and the dissolved oxygen concentration at the end of the test is equal to or greater than 3 mg/L in the control and test vessels.
The test was considered to be valid given that none of the control or solvent control daphnids showed immobilisation or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.
Results with reference substance (positive control):
- Results with reference substance valid: Yes
A positive control study used potassium dichromate as the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L. The 48 h-EC50 was determined to be 1.2 mg/L with 95 % confidence limits of 1.1 to 1.3 mg/L and the 48 h-NOEC was determined to be 0.56 mg/L.
The results from the positive control with potassium dichromate were within the normal range for this reference material at the testing facility.
Reported statistics and error estimates:
An estimate of the EC50 values was given by inspection of the immobilisation data.
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, the 48-hour EC50 value was greater than 0.031 mg/L. The No Observed Effect Concentration was 0.031 mg/L.
Executive summary:

The acute toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guidelines OECD 202 and EU Method C.2 under GLP conditions in an acute immobilisation test using Daphnia magna.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test material at nominal concentrations of 0.0102, 0.0256, 0.064, 0.16 and 0.40 mg/L for 48 hours at a temperature of 21 to 22 °C under semi-static test conditions. The number of immobilised daphnia was recorded after 24 and 48 hours.

No immobilisation was seen throughout the test and all daphnids were observed to be normal throughout the test.

Analysis of the freshly prepared 0.40 mg/L test preparations at 0 and 24 hours showed measured test concentrations of 0.049 and 0.036 mg/L, respectively. Analysis of the old test media at 48 hours showed no significant change, however, analysis of the old test media at 24 hours showed measured test concentrations had declined to less than the limit of quantification (LOQ) of the analytical method employed which was determined to be 0.025 mg/L. Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentration in order to give a "worst case" analysis of the data. The geometric mean measured test concentration of the 0.40 mg/L nominal concentration was determined to be 0.031 mg/L. The test concentration of 0.031 mg/L was the highest attainable test concentration due to the limited solubility of the test material in water and auxiliary solvent.

Under the conditions of the study, the 48-hour EC50 value was greater than 0.031 mg/L. The No Observed Effect Concentration was 0.031 mg/L.

Description of key information

The acute toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guidelines OECD 202 and EU Method C.2 under GLP conditions in an acute immobilisation test using Daphnia magna. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).


No immobilisation was seen throughout the test and all daphnids were observed to be normal throughout the test.


The test concentration of 0.031 mg/L (geometric means) was the highest attainable test concentration due to the limited solubility of the test material in water and auxiliary solvent.


Under the conditions of the study, the 48-hour EC50 value was greater than 0.031 mg/L. The No Observed Effect Concentration was 0.031 mg/L.

Key value for chemical safety assessment

Additional information