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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:
2017-12-12 to 2018-08-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

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:
other: solid
Details on test material:
-
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Japan/ Batch No.: 7J13AA
- Expiration date of the lot/batch: 15.10.2018
- Purity test date: 2018


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in dark
- Stability under test conditions: Stable under normal conditions
Hazardous reactions : Reactions with strong acids.
Hazardous decomposition products: None with proper storage and handling.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
Prior to start of the test sufficent master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10exp3 cells/mL.
The flasks were plugged with polyurethane foam stoppers and kept under constant illumination at 24+/-1 °C until the algal cell density was approximately 10 Exp4 to 10DExp cells/mL.



Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Reconstituted water (Elendt M7 medium)
- Concentrations: in the range of 0.010 to 0.10 mg/L and 0.025 to 0.15 mg/L
- Sampling method: pretreatment
- Sample storage conditions before analysis: The samples were analyzed on the day of receipt.

Test solutions

Vehicle:
yes

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: N/A
- Justification for species other than prescribed by test guideline: N/A
- Source: In house laboratory cultures
- Age of parental stock (mean and range, SD):
- Feeding during test
- Food type: algal suspension and a GM300 suspension

Adult daphnids were maintained in 150 mL glass beakers containing 100 mL Elendt M7 medium (see Annex 2) in a temperature controlled room maintaining the water temperature at 18 to 22 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and GM300 suspension. Culture conditions ensured that reproduction was by parthenogenesis. 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. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Study design

Test type:
semi-static
Water media type:
other: Reconstituted water (Elendt M7 medium)
Limit test:
no
Total exposure duration:
48 h

Test conditions

Test temperature:
18-22°C ±1 °C
pH:
7.9-8.3
Dissolved oxygen:
≥3 mg/L
Nominal and measured concentrations:
nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L.
Measured concentrations: range from 70% to 102% of nominal concentrations
Details on test conditions:
TEST SYSTEM
- Test vessel: Flasks
- Type: open
- Aeration: No
- Semi static
- No. of organisms per vessel: 20 daphnids (4 replicates of 5 animals)
- No. of vessels per concentration (replicates): 4

TEST MEDIUM / WATER PARAMETERS
Ingredient Final Concentration
(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
D(+) biotin (vitamin H) 0.00075


- Source/preparation of dilution water:
Envigo La./ Elendt M7 Medium

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the prepared media (reconstituted water) was 7.9 ±0.3 and stored at approximately 21 ºC.
- Photoperiod: 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods
- Light intensity: 721-912 Lux: The light intensity during the light period was measured using an ATP Instrumentation Lux meter.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L. Prior to use the test item was heated to 80 ºC in order to homogenize.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Sub-Lethal Effects:
Sub-lethal effects of exposure were observed in the 0.10, 0.32 and 1.0 mg/L test preparations.
These responses were reduced mobility, pale, and trapping at surface.












Results with reference substance (positive control):
Positive Control

A positive control (Envigo study number LK67NP) used potassium dichromate as the reference item 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.

Analysis of the immobilization data was carried out using the Trimmed Spearman-Karber method at 24 hours and the Binomial Distribution method at 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package with results based on the nominal test concentrations and gave the following results:

Time Point EC50 95% Confidence Limits NOEC LOEC
(Hours) (mg/L) (mg/L) (mg/L) (mg/L)

24 0.79 0.73 - 0.86 0.56 1.0

48 0.75 0.56 - 1.0 0.56 1.0



The NOEC is based upon equal to or less than 10% immobilization at this concentration.

The results from the positive control with potassium dichromate were within the normal range for this reference item*.
Reported statistics and error estimates:
None

Any other information on results incl. tables

Results for Range -Findings Samples:

 Time point (Hours)

 Nominal Concentration of Test Item in

Range-Finding Sample

 Sample Preparation Factor

F

 Determined Concentration of Test Item

in Range-Finding Sample c (mg/L)

 Percentage of

Nominal Concentration (%)

 0  0.10  4  0.102  102
 0  1.0  8  0.784  78
 0  10  80  7.02  70
 48  0.10  4  0.0781  78
 48  1.0  8  0.649  65
 48  10  80  6.99  70

Results for Test Samples:

Time point (Hours)

 Nominal Concentration of Test Item in

Range-Finding Sample

 Sample Preparation Factor

F

 Determined Concentration of Test Item

in Range-Finding Sample c (mg/L)

 Percentage of

Nominal Concentration (%)

0 (fresh)  Control  4 <LOQ -
0  (fresh) 0.10  4  0.110 110
0  (fresh) 0.32  4  0.325 102
0  (fresh) 1.0  8  1.01 101
0  (fresh) 3.2 40  3.23 101
0  (fresh)  10 80  9.57 96
24 (old) Control 4  <LOQ  -
24 (old)  0.10 4 0.0937  94
24 (old)  0.32 4 0.299  93
24 (old)  1.0 8 0.928  93
24 (old)  3.2 40 3.16  99
24 (old)  10 80 9.50  95
24 (fresh)  Control 4 <LOQ  -
24 (fresh)  0.10 4 0.0987  99
24 (fresh)  0.32 4 0.313  98
24 (fresh)  1.0 8 0.969  97
 48 (old)  Control 4 <LOQ  -
48 (old)  0.10 4 0.0876  88
48 (old)  0.32 4 0.296  93
48 (old)  1.0 4 0.968  97

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.
Analysis of the freshly prepared test preparations at 0 and 24 hours showed measured test concentrations to range from 96% to 110% of nominal
. There was no significant decline in the corresponding old or expired test preparations at 24 and 48 hours (88% to 99% of nominal) and so results are based on nominal test concentrations only.
Executive summary:

Exposure of Daphnia magna to the test item gave the following results:

 Time point (hours)  EC50 (mg/L)  95% Confidence Limits (mg/L)  No Observed Effect Concentration (NOEC) (mg/L)  Lowest Observed Effect Concentration (LOEC) (mg/L)
 48  0.52  Not determined due to nature of data  0.32  1.0