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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:
21 March 2017 to 27 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: YBF1002/16
- Expiration date of the lot/batch: 17 October 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature over silica gel, in the dark

Analytical monitoring:
yes
Details on sampling:
- The concentration of the test material in the test preparations was verified by chemical analysis at 0 and 48 hours.
- The samples were stored frozen prior to analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
-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.
-Based on this information the test material was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document and so a media preparation trial was conducted in order to determine the solubility of the test material under test conditions.
-The variable nature of the results from the centrifuged samples in the trial suggested that this method of preparation was not suitable at effectively removing all of the undissolved material from the media. In addition, as there was no increase in dissolved test material when extending the stirring period beyond 24 hours, the test material was prepared as a saturated solution at an initial loading rate of 100 mg/L, stirred via propeller stirrer for 24 hours prior to the removal of any undissolved test material by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 1 litre discarded in order to pre-condition the filter) to give a nominal concentration of approximately 0.52 mg/L as test material.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Source: derived from in-house laboratory cultures.
- Feeding during test: no

ACCLIMATION
- Type and amount of food: A mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension.
- Feeding frequency: daily
- 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.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
250 mg/L as CaCO3
Test temperature:
21 to 22 °C
pH:
7.8 to 8.2
Dissolved oxygen:
8.2 to 9.1 mg O2/L
Nominal and measured concentrations:
RANGE-FINDING TEST
Nominal: 0.10, 1.0, 10 and 100 % v/v saturated solution.
Measured: 0.31 mg/L as Ytterbium, equivalent to 0.52 mg/L as test material (100 % v/v solution tested only)

DEFINITIVE TEST
Nominal: 100 % v/v saturated solution.
Measured: 0.39 mg/L as Ytterbium (equivalent to 0.52 mg/L as test material)
Details on test conditions:
TEST SYSTEM
- Test vessel: 150 mL glass jars
- Type: closed
- Material, size, headspace, fill volume: 100 mL
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Reconstituted water- Elendt M7 Medium (for culture)
- Ingredients (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.
-Reconstituted Water – ISO Medium (for tests)
-Ingredients (mg/L): CaCl2.2H2O 294, MgSO4.7H2O 123, NaHCO3 65 and KCl 5.8

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.
- Light intensity: between 200 and 1200 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.
-Water Quality Criteria: Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. The light intensity during the light period was measured using an ATP Instrumentation Lux meter. The appearance of the test media was recorded daily.
Verification of Test Concentrations: Samples were taken from the control and the 100 % v/v saturated solution test group from the bulk test preparation at 0 hours and from the pooled replicates at 48 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at and stored frozen for further analysis if necessary.

RANGE-FINDING STUDY
- Test concentrations: 0.10, 1.0, 10 and 100 % v/v saturated solution
- Results used to determine the conditions for the definitive study: yes.
- In the range-finding test, two replicate vessels were prepared per concentration and 5 Daphnids were placed in each test and control vessel. Vessels were maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized Daphnids were recorded.

DATA EVALUATION
- Statistical Analysis: An estimate of the EC50 values was given by inspection of the immobilisation data.
- Validation Criteria
The results of the test are considered valid if the following performance criteria are met:
- No more than 10% of the control daphnids show immobilisation or other signs of disease or stress (e.g. discolouration or unusual behaviour such as trapping at the surface water).
- 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.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 0.52 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: equivalent to > 0.39 mg/L Ytterbium
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.52 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: equivalent to 0.39 mg/L Ytterbium
Details on results:
RANGE-FINDING TEST
- No immobilisation was observed at the test concentrations of 0.10 and 100 % v/v saturated solution. A single daphnid was observed to be immobilised in the 1.0 and 10 % v/v saturated solution test groups, however, this was considered to be due to natural causes and as such considered not to have had an impact on the outcome of the test. Sub lethal effects were observed in the control group and the 0.10, and 100 % v/v saturated solution test groups, however, these effects were considered not to have had an impact on the outcome of the test.
- Based on this information, a single test concentration of four replicates, of 100 % v/v saturated solution was selected for the definitive test.
- Chemical analysis of the 100 % v/v saturated solution test preparation at 0 hours showed that a measured concentration of 0.31 mg/L as ytterbium (equivalent to 0.42 mg/L as test material) was obtained.

DEFINITIVE TEST
- Verification of Test Concentrations: Analysis of the 100 % v/v saturated solution test preparation at 0 hours showed that a measured concentration of 0.39 mg/L as ytterbium (equivalent to 0.52 mg/L as test material) was obtained. There was no significant change in the measured concentrations at 48 hours and so the results are based on 0-hour measured test concentrations only.
- Immobilisation Data: There was no immobilisation in 20 daphnids exposed to a test concentration of 100 % v/v saturated solution for a period of 48 hours. Exposure of Daphnia magna to the test material gave EC50 values based on the 0-hour measured concentration of greater than 0.39 mg/L as ytterbium (equivalent to 0.52 mg/L as test material). The No Observed Effect Concentration 0.39 mg/L as ytterbium (equivalent to 0.52 mg/L as test material).
- Sub-Lethal Effects: In the control group after 24 hours exposure, a single daphnid was observed to be trapped at the surface. As this effect did not last to the end of the test, and affected only a single daphnid, this was considered not to have had an impact on the outcome of the test.
- Validation Criteria: The test was considered to be valid given that none of the 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.
- Water Quality Criteria: Temperature was maintained at 21 to 22 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH. Throughout the test the light intensity was observed to be in the range 426 to 563 Lux.
- Observations on Test Material Solubility: At the start and throughout the test the control and test solutions were observed to be clear colourless solutions.
Results with reference substance (positive control):
- 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. Exposure conditions for the positive control were similar to those in the definitive test.
- 48h-EC50: 0.64 mg/L, NOEC: 0.56 mg/L
- The No Observed Effect Concentration is based upon equal to or less than 10% immobilisation at this concentration.
- The results from the positive control with potassium dichromate were within the normal range for this reference material.
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study the acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and based on the 0-hour measured test concentration gave a 48-hour EC50 value of greater than 0.39 mg/L as ytterbium (equivalent to 0.52 mg/L as test material). The No Observed Effect Concentration was 0.39 mg/L as ytterbium (equivalent to 0.52 mg/L as test material). This study showed that there were no toxic effects at saturation.
Executive summary:

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

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test material using traditional methods of preparation e.g. ultrasonication and high shear mixing. A preliminary media preparation trial indicated that a dissolved test material concentration of approximately 0.52 mg/L as test material was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this material under test conditions.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test material at a concentration of 100 % v/v saturated solution for 48 hours at a temperature of 21 to 22 °C under static test conditions. The test material solution was prepared by stirring an excess (100 mg/L) of test material in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test material was removed by filtration (0.2 μm Sartorius Sartopore filter, first approximate 1 litre discarded in order to pre-condition the filter) to produce a 100 % v/v saturated solution of the test material. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Chemical analysis of the test preparation at 0 hours showed that a measured ytterbium concentration of 0.39 mg/L (equivalent to 0.52 mg/L as test material). There was no significant change in the measured concentration at 48 hours and so the results are based on 0 -hour measured test concentrations only.

Under the conditions of the study the exposure of Daphnia magna to the test material gave EC50 values based on the 0 -hour measured concentration of greater than 0.39 mg/L (equivalent to 0.52 mg/L as test material). The No Observed Effect Concentration was 0.39 mg/L (equivalent to 0.52 mg/L as test material). This study showed that there were no toxic effects at saturation.

Description of key information

Under the conditions of the study the 48 hour exposure of Daphnia magna to the test material gave EC50 values based on the 0-hour measured concentration of greater than 0.39 mg/L (equivalent to 0.52 mg/L as test material). The No Observed Effect Concentration was 0.39 mg/L (equivalent to 0.52 mg/L as test material). This study showed that there were no toxic effects at saturation.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
0.52 mg/L

Additional information

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

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test material using traditional methods of preparation e.g. ultrasonication and high shear mixing. A preliminary media preparation trial indicated that a dissolved test material concentration of approximately 0.52 mg/L as test material was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this material under test conditions.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test material at a concentration of 100 % v/v saturated solution for 48 hours at a temperature of 21 to 22 °C under static test conditions. The test material solution was prepared by stirring an excess (100 mg/L) of test material in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test material was removed by filtration (0.2 μm Sartorius Sartopore filter, first approximate 1 litre discarded in order to pre-condition the filter) to produce a 100 % v/v saturated solution of the test material. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Chemical analysis of the test preparation at 0 hours showed that a measured ytterbium concentration of 0.39 mg/L (equivalent to 0.52 mg/L as test material). There was no significant change in the measured concentration at 48 hours and so the results are based on 0-hour measured test concentrations only.

Under the conditions of the study the 48 hour exposure of Daphnia magna to the test material gave EC50 values based on the 0-hour measured concentration of greater than 0.39 mg/L (equivalent to 0.52 mg/L as test material). The No Observed Effect Concentration was 0.39 mg/L (equivalent to 0.52 mg/L as test material). This study showed that there were no toxic effects at saturation.