Registration Dossier

Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 01-29, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Batch No.: UGe-RS Kilo 1
Color: Yellow
Odor: Not specific
Storage conditions: room temperature
Solubility and stability: Very good solubility in water, stable in solution at least 2 days
Expiring date: 12.12.2018
Correction factor: 1.07
Specific details on test material used for the study:
Test Item name Yellow LF 6911
Batch No. UGe-RS Kilo 1
Appearance: Yellow powder
Purity: 93.3 % (organic part excluding water and salts not containing similar chromophore)
Solubility and stability: Very good solubility in water, stable in solution at least 2 days
Expiry date: 12 December 2018
Storage: Room temperature

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
For determination of the test item concentrations, four replicates samples (5 mL per replicate) were taken from the test vessels and from the control at the start and at the end of the test.

Test solutions

Vehicle:
no
Details on test solutions:
The test solution was prepared by mechanical dispersion. An amount of 0.03 g test item was dissolved in 300 mL test medium (ISO medium) resulting a nominal concentration of 100 mg/L. The test solution was freshly prepared in the test laboratory just before introduction of the test animals (start of the experiment).

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
Species and strain: Daphnia magna (Straus)
Origin: Laboratory of Hydrobiology (Central Agricultural Office, Directorate of Plant-, and Soil Protection) 2100 Gödöllő, Kotlán S. u. 3. Hungary
Breeding: The Daphnia are bred in the Laboratory of TOXI-COOP ZRT.
Number of animals: Twenty animals in both groups (concentration and untreated control), divided into 4 concurrent batches (5 animals per batch).
Age of animals: Less than 24 h old at the beginning of the test.
Sex: Female
Animal health: Apparently healthy animals were used in this test with a known history
Acclimatization: Test animals were bred under similar (or the same) conditions as that used during the exposure period (holding water, temperature, background colour etc.), therefore additional acclimatisation before the test was not necessary. Brood daphnids were maintained in dilution water at the test temperature for at least 48 hours prior to the start of the test.
Food and Feeding: The Daphnia were fed with concentrated algal suspension of Raphidocelis subcapitata during the holding. Test animals were not fed during the exposure.

Study design

Test type:
static
Water media type:
freshwater
Remarks:
Reconstituted water (ISO medium, according to OECD 202)
Limit test:
yes
Total exposure duration:
48 h

Test conditions

Hardness:
The reconstituted water (ISO medium) had an approximate theoretical total hardness of 249 mg/L (as CaCO3).
Test temperature:
The test temperature was in the range of 19.9 – 20.3°C measured in the test vessels during the test. The additionally measured ambient temperature within the climate chamber was between 19.6 – 20.5°C.
pH:
The pH of the test solution was not adjusted and not varied by more than 1.5 units in any one test. The pH was in the range of 7.91 – 8.04 during the test.
Dissolved oxygen:
The dissolved oxygen concentration was in the range of 7.70 – 8.08 mg/L during the test. pH
Salinity:
Substance Nominal concentration of the stock solutions (g/L)

CaCl2 x 2 H2O 11.76
MgSO4 x 7 H2O 4.93
NaHCO3 2.59
KCl 0.23
Conductivity:
N/A
Nominal and measured concentrations:
A concentration of 100 mg/L (limit concentration) and a concurrent control was included in the main test. The concentration of the test item was analytically determined at the start and at the end of the experiment. The test item was not detected in the untreated control group. In the treated group measured concentrations of test item were 98 % of the nominal at the start and 97 % at the end of the test. The measured test item concentration remained within ± 20 % of the nominal during the test period of 48 hours, therefore biological results are based on the nominal concentration.
Details on test conditions:
Performance of the Test
Twenty animals, divided into four groups (glass beaker; volume app. 50 mL) of five animals each (at least 4 mL test solution/animal) were exposed to 100 mg/L test item or included as control over a period of 48 hours. The test animals were not fed during the test. Each test vessel contained approximately 40 mL of test solution and was uniquely identified with study code, test group and replicate. Daphnia were observed for immobility or mortality by visual observation after 24 and 48 hours of exposure. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile.

Test Conditions
The test vessels were kept during the test in a climate chamber under controlled conditions. The dissolved oxygen concentration and pH were measured
in the test item treated and the control group at the start (before test solutions had been distributed into the test vessels) and in each test vessel at the end
of the test. The water temperature was measured in each test vessel at the start and at the end of the test. Additionally, the ambient temperature was measured continuously using a min/max thermometer in the climate chamber.

Test type: Static test, based on the results obtained during analytical method validation (Study Number: 805-100-1545). The test item was shown to be stable for 48 hours. Temperature: The test temperature was in the range of 19.9 – 20.3°C measured in the test vessels during the test. The additionally
measured ambient temperature within the climate chamber was between 19.6 – 20.5°C.

Oxygen Concentration: The dissolved oxygen concentration was in the range of 7.70 – 8.08 mg/L during the test.
pH: The pH of the test solution was not adjusted and not varied by more than 1.5 units in any one test. The pH was in the range of 7.91 – 8.04 during the test. Light-Dark Cycle: The light-dark cycle during the test was 16 hours light (artificial illumination) and 8 hours darkness.
Hardness: The reconstituted water (ISO medium) had an approximate theoretical total hardness of 249 mg/L (as CaCO3).

Preliminary Range-Finding Test
In order to select appropriate test concentrations for use in the definitive test, a non-GLP preliminary range-finding test was conducted to determine the
approximate toxicity of the test item. Ten daphnids (divided into 2 replicates) in each test concentration and control were exposed for 48 hours.
The preliminary range-finding test was not performed in compliance with the GLPRegulations and are excluded from the Statement of Compliance (Statement of the Study Director), but the raw data of these tests will be archived under the study code of present study.

Nominal concentrations [mg/L] Untreated control 0.1 1 10 100
Number of treated animals 10 10 10 10 10
Number of immobilised animals 0 0 0 0 0


Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
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
Key result
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
There was no immobilisation observed in twenty daphnids exposed to each test item treated and control group. Further no abnormal behaviour
or appearance of test animals was noted

Immobilisation or any sub-lethal effects were not detected either in the control or in the test item treated group.
The dissolved oxygen concentration in was greater 3 mg/L in each test vessel at the end of the test.
All validity criteria were within acceptable limits and therefore the study considered valid.
Results with reference substance (positive control):
For the evaluation of the quality of the Daphnia clone and the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study was: 01 – 02 March 2016.
The 24-h EC50: 1.26 mg/L, (95 % confidence limits: 1.11 – 1.55 mg/L)
Reported statistics and error estimates:
A limit test was performed and toxic effects were not observed, therefore statistical analysis was not necessary. The NOEC, LOEC and ECx values of the test item were determined directly from the raw data.
Electronic

Any other information on results incl. tables

Preliminary Range-Finding

Test In order to select appropriate test concentrations for use in the definitive test, a non-GLP preliminary range-finding test was conducted to determine the approximate toxicity of the test item. Ten daphnids (divided into 2 replicates) in each test concentration and control were exposed for 48 hours. The preliminary range-finding test was not performed in compliance with the GLPRegulations and are excluded from the Statement of Compliance (Statement of the Study Director), but the raw data of these tests will be archived under the study code of present study

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The test item Yellow LF 6911 had no toxic effect at 100 mg/L concentration (i.e. limit test concentration) on Daphnia magna; the EC10, EC20, EC50 results and the LOEC are higher than 100 mg/L.
Executive summary:

Concentrations: Limit test; using a single concentration of 100 mg/L; concurrent control ran. The analytically measured test item concentrations remained within ± 20 % of the nominal over the test period of 48 hours, therefore the biological results are based on the nominal concentration.

Test design: Twenty animals, divided into four groups (glass beaker; volume app. 50 mL) of five animals each were exposed to the test concentration or run as control for 48 hours. The immobility or mortality of the Daphnia will be determined by visual observation after 24 and 48 hours of exposure.

Analytics: Concentrations were determined using reverse phase HPLC method with VIS detection from the test group and the control at the start and end of the test. Endpoints: EC10, EC20, EC50, NOEC and LOEC for 48h test period. Statistics: Statistical analysis was not necessary; endpoints were determined directly from the raw data.

Validity: All validity criteria were met and therefore the study can be considered as valid

In conclusion: the test item Yellow LF 6911 had no toxic effect at 100 mg/L concentration (i.e. limit test concentration) on Daphnia magna; the EC10, EC20, EC50 results and the LOEC are higher than 100 mg/L.