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 - 30 May 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 - 25 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:
22.8 µg/L
Conc. based on:
test mat.
Temp.:
19.9 °C
pH:
>= 8 - <= 8.1
Remarks on result:
other: average of two runs (maximum difference= 27%)
Details on results:
Mean concentration in the water samples (n=10) obtained at the flowrate of 25 mL/h was 25.8 μg/L. CV= 5.9%.
Mean concentration in the water samples (n=10) obtained at the flowrate of 12 mL/h was 19.7 μg/L. CV= 6.0%.

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 19.9°C was determined to be 22.8 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 19.9°C was determined to be 22.8 μg/L. The pH of the aqueous samples was 8.0 -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:
Physical appearance: white crystalline powder
Test item storage: In refrigerator (2-8°C)
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:
- Nominal concentrations: Control, WAFs prepared at 1.0, 10, and 100 mg/L
- Sampling method: 2.0 mL from the approximate centre of the test vessels, at t=0 h and t=48 h.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
- At the end of the exposure period, the replicates were pooled at each concentration before sampling.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: direct addition of test item to test medium - loading rates were individually prepared at 1.0, 10 and 100 mg/L. A two-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by filtration through a 0.45 µm membrane filter (RC55, Whatman) and used as test concentrations.
- Controls: Test medium without test item or other additives
- 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 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:
yes
Total exposure duration:
48 h

Test conditions

Hardness:
180 mg/L expressed as CaCO3
Test temperature:
20°C
pH:
Start: 7.9
End: 7.8
Dissolved oxygen:
Start: 8.4-8.8
End: 8.7-9.0
Nominal and measured concentrations:
- Nominal loading: Control and WAF prepared at a nominal loading rate of 100 mg/L
- Measured concentrations: WAF at 100 mg/L at start: 0.0368 mg/L; at 48 h: 0.00708 mg/L
Based on the observed decrease in concentration during the exposure period, the geometric mean measured concentration was calculated as 0.016 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 80 mL of test solution
- Aeration: No aeration
- No. of organisms per vessel: 5 per vessel
- No. of vessels per concentration (replicates): 4 for WAF at 100 mg/L, 2 for WAFs at 1.0 and 10 mg/L
- 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 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, M7
- Intervals of water quality measurement: pH and dissolved oxygen were measured at the beginning and at the end of the test, for the control and the WAF at 100 mg/L. Temperature of medium was continuously measured 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 24h and at 48 h.


RANGE-FINDING STUDY
- Test concentrations: Control, WAFs prepared at 1.0, 10, and 100 mg/L
- Results used to determine the conditions for the definitive study: not applicable; the range-finding study was combined with a limit test and the results are used to draw final conclusions.
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate (performed February 2017)

Results and discussion

Effect concentrations
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 0.016 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Details on results:
- No immobility of daphnids was observed at any of the test concentrations and the control during the test period.
- Behavioural abnormalities: At 24 h, two daphnids in two separate replicates of Control were observed trapped at the surface of the test solution. These organisms were reimmersed into the respective solutions before recording of mobility.
- Mortality of control: None observed.
- Other adverse effects control: No signs of disease or stress, for example discoloration, were observed.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: After 24 and 48 hours of exposure unknown brown particles were observed on the bottom of the control vessels. These particles, however, did not affect the daphnids and therefore the test was accepted.
- Effect concentration exceeding solubility limit in test medium: yes. The sample of WAF at 100 mg/L at the start of the test showed a measured concentration of 0.037 mg/L, which decreased during the exposure period to 0.0071 mg/L. Based on this result, the average exposure concentration was calculated to be 0.016 mg/L, which was considered the solubility limit of the test item in test medium.
Results with reference substance (positive control):
- Results with reference substance valid? Yes.
Actual responses are within the ranges of the expected responses at the different concentrations (i.e., the 48h-EC50 was between 0.3 and 1.0 mg/L). Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at the Test Facility.
- Relevant effect levels:
The 24h-EC50 was 0.64 mg/L with a 95% confidence interval between 0.56 and 0.73 mg/L.
The 48h-EC50 was 0.48 mg/L with a 95% confidence interval between 0.43 and 0.53 mg/L.
Reported statistics and error estimates:
No statistics were reported.

Any other information on results incl. tables

Number of Introduced Daphnids and Incidence of Immobility

Time (h)

Replicate

Diona-R, WAF prepared at x mg/L

Control

1.0

 

10

 

100
(0.016)

0

A

5

5

5

5

B

5

5

5

5

C

5

5

D

5

5

Total introduced

20

10

10

20

24

A

0

0

0

0

B

0

0

0

0

C

0 [1]

 

 

0

D

0 [1]

 

 

0

Total immobilised

0

0

0

0

Effect %

0

0

0

0

 

 

 

 

48

A

0

0

0

0

B

0

0

0

0

C

0

 

 

0

D

0

 

 

0

Total immobilised

0

0

0

0

Effect %

0

0

0

0

[ ]: number of daphnids observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

( ): Average exposure concentration

Analysis results Combined Limit/Range-finding Test: Reserve test Samples

Time of sampling
[hours]

Date of sampling

Date of
analysis1

Loading rate2

[mg/L]

Concentration
analyzed
[mg/L]

Relative to
initial
[%]

0

02 May 2017

30 May 2017

0

n.d.

 

 

 

 

100

0.0368

 

48

04 May 2017

30 May 2017

0

n.d.

n.a.

 

 

 

100

0.00708

19

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

2      A water accommodated fraction (WAF) prepared at the loading rate.

n.d.Not detected.

n.a.Not applicable.

In the initial analysis of the samples from this Daphnia magna test the low accuracy samples fel ouside the acceptability criterion range (70 -110%) and therefore the analysis was repeated with reseve samples.

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. >0.016 mg/L based on the geometric mean measured concentration in the highest treatment group tested.
Executive summary:

In a 48 h acute toxicity test conducted according to OECD guideline 202, Daphnia magna were exposed to Water Accomodated Fractions (WAF) in a combined limit/range-finding study at the following nominal loading rates: 0 (Control), 1.0, 10, and 100 mg/L. The geometric mean measured concentration in the WAF prepared at 100 mg/L was calculated to be 0.016 mg/L, which was considered the solubility limit of the test item in test medium. No immobility was observed at any of the test concentrations and the control during the exposure period. The 48h-EC50 for Daphnia magna exposed to the test subtance was above the solubility limit, i.e. >0.016 mg/L. The study is considered to be reliable without restrictions.