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:
28 April - 26 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to other study
Reason / purpose:
reference to other study
Reference
Endpoint:
water solubility
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 March 2017 - 26 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Reference:
Composition 0
Qualifier:
according to
Guideline:
EU Method A.6 (Water Solubility)
Version / remarks:
2014
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 105 (Water Solubility)
Version / remarks:
1995
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 830.7840 (Water Solubility)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes
Type of method:
column elution method
Test material information:
Composition 1
Key result
Water solubility:
29.5 µg/L
Conc. based on:
test mat.
Temp.:
20 °C
pH:
>= 7.9 - <= 8.1
Remarks on result:
other: average of two runs (maximum difference=10%)
Details on results:
Mean concentration in the water samples (n=10) obtained at the flowrate of 24 mL/h was 31.0 μg/L. CV= 9.2%.
Mean concentration in the water samples (n=10) obtained at the flowrate of 12 mL/h was 28.0 μg/L. CV= 9.0%.

The coefficient of variation (CV) (n=10) was ≤ 30% at both flow rates. It demonstrated that the system was in equilibrium at all flow rates.

The maximum difference on the mean values at both flow rates was ≤ 30%.

No test substance was detected in the samples from the blank column.

Conclusions:
The water solubility at 20°C was determined to be 29.5 μg/L.
Executive summary:

The water solubility of the substance was determined using the column elution method in a GLP study according to EC A.6, OECD 105 and OPPTS 830.7840.

Quantification was performed by UPLC-MS/MS. The water solubility at 20°C was determined to be 29.5 μg/L. The pH of the aqueous samples was 7.9-8.1.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
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
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Appearance: off-white powder
Test item storage: at room temperature
Specific details on test material used for the study:
Solubility in water: not indicated in the report; please refer to section 4.8 of this dossier
Stability in water: yes

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: all test concentrations and control.
- Frequency: at t=0 h and t=48 h
- Sampling method: 2.0 mL from the approximate center of the test vessels. At the end of the exposure period, replicates were pooled at each concentration before sampling.
- Sample storage conditions before analysis: samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Direct addition of the test item, highest loading rate of 100 mg/L, applying a three-day period of magnetic stirring to ensure maximum dissolution of the test item in medium. The aqueous Saturated Solution (SS) was collected by filtration through a 0.45 μm membrane filter (RC55, Whatman) and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium.
- Controls: Test medium without test item or other additives.
- All test solutions were clear and colorless at the end of the preparation procedure.

Test organisms

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

- Strain/clone: Daphnia magna (Straus, 1820)
- Source: In-house laboratory culture with a known history.
- Age of parental stock: >2 weeks old
- Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
- Age of test organisms at start of test: <24 h


BREEDING:
- Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel.
- Maximum age of the cultures: 4 weeks
- Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
- Temperature of the medium: 18-22 °C
- Feeding: Daily, a suspension of fresh water algae
- Medium: M7 medium

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h

Test conditions

Hardness:
180 mg/L expressed as CaCO3
Test temperature:
During test period: 20-21°C
pH:
Start: 7.8-7.9
End: 8.0-8.1
Dissolved oxygen:
Start: 8.5-8.9
End: 8.4-8.5
Nominal and measured concentrations:
Nominal loading rate: 100 mg/L (saturated solution)
Nominal loading rate during Final Test: Control, 10, 18, 32, 56, 100% SS.
Measured concentrations during Final Test: 100% SS: 0.081 mg/L at start, 0.051 mg/L at 48 h.
Based on the observed decrease in concentration during the exposure period, the geometric mean measured concentration was calculated to be 0.064 mg/L. The effect parameters are expressed based on the geometric mean of the measured concentration.
Details on test conditions:
TEST SYSTEM
- Test vessel: 60 mL, all-glass, containing 50 mL of test solution
- Aeration: no aeration
- No. of organisms per vessel: 5 daphnids per vessel
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO Medium, made by dissolving in Reverse-Osmosis purified tap water the following Macro salts: CaCl2.2H2O (211.5 mg/L), MgSO4.7H2O (88.8 mg/L), NaHCO3 (46.7 mg/L) and KCL (4.2 mg/L).
- Culture medium different from test medium: Yes.
- Intervals of water quality measurement: pH and dissolved oxygen were measured at the beginning and at the end of the Final Test, for all concentrations and the control. Temperature of the medium was continuously monitored in a temperature control vessel, beginning at the start of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light : 8 h dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility (including mortality) at 24 h and at 48 h.


RANGE-FINDING STUDY
- Test concentrations: Control, 1, 10, 100% SS.
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate (performed Feb 2017)

Results and discussion

Effect concentrations
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 0.064 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: None observed in Control; not reported for test substance concentrations.
- Other biological observations: No signs of disease or stress, for example discoloration, were observed in Control; not reported for test substance concentrations.
- Mortality of control: None
- Other adverse effects control: None
- Effect concentrations exceeding solubility of substance in test medium: Yes
- Analysis of the 100% SS sample at the start of the Final Test showed a measured concentration of 0.081 mg/L, decreasing to 0.051 mg/L at the end of the test. The average exposure concentration (geometric mean) was calculated to be 0.064 mg/L. No precipitation was observed at any of the replicates, therefore 0.064 mg/L was considered the solubility limit of Norethyl in test medium. No immobility was observed at any of the test concentrations and the control during the exposure period of the Final Test.
Results with reference substance (positive control):
- Results with reference substance valid? Yes.
- The actual response was within the ranges of the expected responses (48h-EC50 between 0.3 and 1.0 mg/L). The sensitivity of this batch of D. magna was in agreement with the historical data collected at the Test Facility.
- Relevant effect levels:
24h-EC50: 0.64 mg/L with a 95% confidence interval 0.56-0.73 mg/L
48h-EC50: 0.48 mg/L with a 95% confidence interval 0.43-0.53 mg/L
Reported statistics and error estimates:
No statistics reported.

Any other information on results incl. tables

Number of Introduced Daphnids and Incidence of Immobility in the Final Test

Time (h)

Replicate

Norethyl, %SS prepared at 100 mg/L

Control

10

 

18

 

32

 

56

 

100

(0.064)

0

A

5

5

5

5

5

5

B

5

5

5

5

5

5

C

5

5

5

5

5

5

D

5

5

5

5

5

5

Total introduced

20

20

20

20

20

20

24

A

0

0

0

0

0

0

B

0

0

0

0

0

0

C

0

0

0

0

0

0

D

0

0

0

0

0

0

Total immobilised

0

0

0

0

0

0

Effect %

0

0

0

0

0

0

 

 

 

 

 

 

48

A

0

0

0

0

0

0

B

0

0

0

0

0

0

C

0

0

0

0

0

0

D

0

0

0

0

0

0

Total immobilised

0

0

0

0

0

0

Effect %

0

0

0

0

0

0

( ): Average exposure concentration

Final Test: Test Samples

Time of sampling
[hours]

Date of sampling

Date of
analysis1

Percentage of ss2
[%]

Analyzed concentration
[mg/L]

Relative to
initial
[%]

0

20 June 2017

26 June 2017

0

n.d.

 

 

 

 

100

0.0810

 

48

22 June 2017

26 June 2017

0

n.d.

n.a.

 

 

 

100

0.0507

63

1     Samples were stored in the freezer (≤ -15°C) until the day of analysis.

2     Percentage of a saturated solution (ss) prepared at a loading rate of 100 mg/L.

n.d.      Not detected.

n.a.Not applicable.

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
For details on validity criteria see 'Overall remarks' section.
Conclusions:
The 48h-EC50 of the test substance for Daphnia magna was above the solubility limit in test medium, i.e. >0.064 mg/L based on the geometric mean measured concentration in the highest treatment group tested.
Executive summary:

In a 48h acute toxicity study conducted according to OECD guideline 202, Daphnia magna were exposed to Norethyl under static conditions at the following nominal concentrations: 0 (Control), 10, 18, 32, 56, and 100% of the Saturated Solution prepared at a loading rate of 100 mg/L. The geometric mean measured concentration in the SS was calculated to be 0.064 mg/L, which was considered the solubility limit in test medium. No immobility was observed at any of the test concentrations and the control during the exposure period. Therefore, the 48h-EC50 was above the solubility limit, i.e. >0.064 mg/L. The study is considered to be reliable without restrictions.