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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:
20 Jul 2018 - 22 Aug 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to other study

Data source

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

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:
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 powder
Storage conditions: at room temperature

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: All test concentrations and control
- Sampling method: 1.9 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 substance to test medium.
Since the test item consisted of a mixture of two enantiomers, and because the test item was not completely soluble in test medium, WAFs were prepared individually at loading rates ranging between 1.0 and 100 mg/L. A three-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in test medium. The test item was not completely solube in test medium at the loading rates initially prepared. After the stirring period, the aqueous WAFs were collected by filtration through a 0.45 μm membrane filter (PES450, Supor®) and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure.
- Controls: Test medium without test item or other additives.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus 1820
- Source: In-house laboratory culture with a known history
- Method of breeding: cyclical parthenogenesis under specified breeding conditions
- Age of parental stock: > 2 weeks
- Age at test initiation: < 24 hours
- 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.

CULTIVATION
- Start of each batch: Approximately 250 newborn daphnids, i.e. less than 3 days old, were placed into 5 litres of M7 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.

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:
19.5 - 20.2°C
pH:
At start (t=0 h): 7.7-8.4
At end (t=48 h): 8.1-8.2
Dissolved oxygen:
At start (t=0 h): 8.4-8.5 mg/L
At end (t=48 h): 8.8-8.9 mg/L
Nominal and measured concentrations:
Nominal: control and WAFs individually prepared at loading rates of 10, 18, 32, 56 and 100 mg/L
Measured: The measured concentrations were at the level of nominal (i.e. 93 – 107% relative to nominal) throughout the test. Effect parameters were therefore expressed in terms of analytically confirmed nominal concentrations.
See Table 1 in 'Any other information on results' for details on measured concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 60 mL, all-glass, containing 50 mL of test solution
- Aeration: no aeration
- Feeding: no feeding
- No. of organisms per vessel: 5 daphnids
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Introduction of daphnids: Within 45 minutes after preparation of the test solutions.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Medium prepared with tap water purified by Reverse Osmosis and the following salts: CaCl2.2H2O: 211.5 mg/L, MgSO4.7H2O: 88.8 mg/L, NaHCO3: 46.7 mg/L, KCl: 4.2 mg/L
- Culture medium different from test medium: yes (M7)
- Intervals of water quality measurement: pH and dissolved oxygen: at the beginning and at the end of the test, for all concentrations and the control. Temperature of medium: continuously in a temperature control vessel, beginning at the start of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h daily.

EFFECT PARAMETERS MEASURED
- Immobility (including mortality) at 24 h and at 48 h.

RANGE-FINDING STUDY
- Combined Limit/Range-Finding Test concentrations: control and WAFs individually prepared at loading rates of 1.0, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: yes, expected EC50 between WAFs prepared at 10 and 100 mg/L.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (performed Jul 2018)

Results and discussion

Effect concentrations
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
78 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Remarks on result:
other: 95% confidence interval: 68-88 mg/L
Details on results:
- Behavioural abnormalities: none observed in the control group.
- Mortality of control: none
- Analytical measurements: Measured concentrations were at the level of nominal throughout the test, therefore the undissolved material removed during preparation of test solutions was most likely not test item related.
- No immobility was observed in the control and at the three lowest test concentrations throughout the exposure period. At the end of the test, respectively 5 and 90% immobility was observed at nominal concentrations of 56 and 100 mg/L. It should be noted that ≤10% immobility is allowed in the control, and therefore effects <10% were considered biologically not relevant. See Table 2 in 'Any other information on results' for full details on immobility.
- All water quality parameters remained within the requirements as laid down in the study plan throughout the test.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- The 48h-EC50 was 0.59 mg/L with a 95% confidence interval between 0.53 and 0.72 mg/L.
- The responses in this reference test with K2Cr2O7 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.
Reported statistics and error estimates:
The 48h-EC50-value was calculated from the probits of the percentages of affected daphnids and the logarithms of the corresponding test item concentrations using the maximum likelihood estimation method.
The 24h-EC50 could not be determined since the observed effects were below 50%.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Any other information on results incl. tables

Table 1: Final Test: Test Samples

Time of sampling

[hours]

Loading rate *

[mg/L]

Concentration analyzed
[mg/L]

Relative to initial

[%]

0

0

n.d.

 

 

10

10.7

 

 

18

16.8

 

 

32

32.0

 

 

56

54.9

 

 

100

100

 

48

0

n.d.

n.a.

 

10

10.6

99

 

18

16.7

99

 

32

32.0

100

 

56

54.2

99

 

100

98.5

99

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

n.d. Not detected.

n.a. Not applicable.

Table 2: Number of Introduced Daphnids and Incidence of Immobility in the Final Test 

Time (h)

Replicate


Nominal conc. (mg/L)

Control

10

18

32

56

100

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

1

4

B

0

0

0

0

0

5#

C

0

0

0

0

0

4

D

0

0

0

0

0

5

Total immobilised

0

0

0

0

1

18

Effect %

0

0

0

0

5

90

#Microscopic observation revealed no test item attached to the daphnids.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks' for details on validity criteria.
Conclusions:
The 48h-EC50 of the substance for Daphnia magna exposed to the substance was 78 mg/L based on analytically confirmed nominal concentrations (95% confidence interval between 68 and 88 mg/L).
Executive summary:

In a 48 -h acute toxicity test conducted according to OECD guideline 202, Daphnia magna were exposed to Water Accommodated Fractions of the substance, individually prepared at loading rates of 10, 18, 32, 56 and 100 mg/L and an untreated control (all with 4 replicates per concentration and 5 daphnids per replicate).

Measured concentrations were at the level of nominal (i.e. 93 - 97% relative to nominal) throughout the test. Therefore, nominal concentrations were used to express the effect parameters.

No immobility was observed in the control and the three lowest test concentrations throughout the test. At the end of the test, respectively 5 and 90% immobility was observed at nominal concentrations of 56 and 100 mg/L.

The 48h-EC50 of the substance for Daphnia magna (based on immobility) was 78 mg/L based on analytically confirmed nominal concentrations (95% confidence interval between 68 - 88 mg/L). The study met all validity criteria and is considered to be reliable without restrictions.