Registration Dossier

Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
Replicate D of the control group contained 4 instead of 5 daphnids, whereas replicate C of the WAF prepared at 18 mg/L received no daphnids. Evaluation: Because no significant effects were observed in this test there was no impact reliability of study.
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
yes
Remarks:
see above
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23 (2000)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: September 2015; signature: November 2015
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Definitive test: nominal concentrations: Solutions containing 0 (control), 10, 18, 32, 56 and 100 mg/L prepared as WAF: controls; Test medium without test substance or other additives.
- Sampling method: UPLC-analyses was conducted. See 'Details on analytical methods for more information'
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test item loadings were prepared as Water Accommodated Fraction (WAF). Before the preparation of test solutions, the test item was heated to 50 ⁰C in order to facilitate weighing. All test solutions were prepared separately at loading rates ranging from 1.0 to 100 mg/L (after correcting for the purity of the test item). After a 17-19 hour period of magnetic stirring to reach maximum solubility of the test item in test medium, the resulting mixtures were left to stabilize for one 60 – 92 minutes. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were siphoned off. The final test solutions were clear and ranged from colourless to slightly yellow. The highest concentration in the combined limit/range-finding test was slightly hazy.
- Controls: Twenty daphnids were exposed to each loading level and a control. Which consisted of: dilution water without test item tested under the same conditions as the test groups. Further a reference test was also conducted (non-concurrently) under static conditions to demonstrate the validity of the test system.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution): five Water Accommodated Fractions (WAF) of the test item with loading levels in the range of 0 (control), 10, 18, 32, 56 and 100 mg/L prepared in a series for each test item loading. All water accommodated fractions were analytically verified via analysis.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None reported.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnids
- Strain: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.
- Source: in-house laboratory cultures
- Age at study initiation (mean and range, SD): < 24 hours.
- Method of breeding: Parthenogenesis
- Feeding during test: No.
- Food type: During breeding: Daily, a suspension of fresh water algae. During test: Not applicable
- Amount: Not applicable.
- Frequency: During breeding: Daily ; During the test: Not applicable.

ACCLIMATION
- Acclimation period: None.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
The adjusted-ISO medium M7 hardness: 180 mg/L expressed as CaCO3 and the pH: 7.7 ± 0.3
Test temperature:
The temperature continuously measured in a temperature control vessel varied between 19.0 and 21.0°C during the test, and complied with the requirements as laid down in the protocol (18-22°C, constant within 2°C).
pH:
Test conditions remained within the limits prescribed by the guideline; pH: 7.7-8.2 not varying by 1.5 units the end of the test.
Dissolved oxygen:
Test conditions remained within the limits prescribed by the guideline; oxygen: > 3 mg/L at the end of the test. Actual oxygen: start: 8.7 ± 0.1 mg/L (control: 9.0 mg/L) and end: 9.7 ± 0.1 mg/L (control: 9.8 mg/L).
Nominal and measured concentrations:
Combined range-finding / limit test: nominal concentrations: 1.0, 10.0 and 100.0 mg/L (prepared as WAF); plus Control containing no test substance or other additives. Measured concentrations remained stable until the end of the exposure (98% of initial)
Definitive test: nominal concentrations: Solutions containing 0 (control), 10, 18, 32, 56 and 100 mg/L prepared as WAF.
Concentrations were measured to demonstrate the presence and stability of the test item in test medium. Samples taken from the highest concentration were analysed. The actual concentration was 45 mg/L at the start of the exposure and remained stable until the end of the test (97% of initial).
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 ml
- Type (delete if not applicable): closed airtight with reduced headspace; static.
- Material, size, headspace, fill volume: Glass
- Aeration: No aeration of the test solutions.
- No. of organisms per vessel: 20 per concentration; 5 per vessel containing 80 ml of test solution
- No. of vessels per concentration (replicates): 4 (four replicates, 5 daphnia per vessel).
- No. of vessels per control (replicates): 4 (four replicates, 5 daphnia per vessel).
- No. of vessels per vehicle control (replicates): Not applicable.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse Osmosis (RO-water, GEON Waterbehandeling); used to prepare adjusted-ISO Medium M7. The following macro salts were diluted in RO water and added to ISO Medium M7: Macro 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: No, adjusted-ISO Medium M7, renewed by 50% twice per week.

OTHER TEST CONDITIONS
- Adjustment of pH: None.
- Photoperiod:16 hours photoperiod daily.
- Light intensity: Not reported.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility (including mortality), 24 hours and at 48 hours.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: loading rates based on WAF
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: loading rates based on WAF; No EL50 could be estimated because the test substance proved to be non-toxic (EL50 > maximum loading rate)
Details on results:
- Behavioural abnormalities: None
- Observations on body length and weight: Not applicable.
- Other biological observations:
- Mortality of control: No mortalities.
- Other adverse effects control: None.
- Abnormal responses: None.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None.
- Effect concentrations exceeding solubility of substance in test medium: Not applicable. Test item was loaded at various water accomadated fraction (WAF) loadings.
Results with reference substance (positive control):
- Results with reference substance valid?: Yes.
- Mortality: None. Acute immobilisation observed only.
- EC50/LC50: 24h-EC50 was 0.69 mg/L. 48h-EC50 was 0.38 mg/L with a 95% confidence interval between 0.33 and 0.43 mg/L
- Other: The actual 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 test organism was in agreement with the historical data.

Table 1. pH and oxygen (mg/L) concentrations during the final test

Test item loading rate / mg/L

Start (t=0 h)

End (t=48 h)

 

pH

O2

pH

O2

Control

8.2

9.0

8.3

9.8

10

8.1

8.7

8.2

9.7

18

8.0

8.7

8.2

9.7

32

8.0

8.7

8.2

9.7

56

7.9

8.7

8.2

9.7

100 (45)

7.9

8.6

8.1

9.7

( ) between brackets the measured concentration is given (mg/L)

 

During the combined limit test/range finder: the responses recorded during the combined limit/range-finding test. No immobility was observed at any of the test concentrations after 24 hours of exposure. At the end of the test, 95% of daphnids were immobilised in the highest concentration. A number of daphnids were trapped at the surface in the control treatment. Based on the observations made in this test, the 48h-EL50 was between WAFs prepared at 10 and 100 mg/L. Therefore, samples taken from these two WAFs were analysed. The actual concentrations were 6.1 and 59 mg/L at the start of the test, respectively. Measured concentrations remained stable until the end of the exposure (98% of initial).

 

Table 2. Number of introduced daphnids and incidence of immobility in the definitive test

Time

/ h

Replicate

Test item loading rate / mg/L

 

 

Control

10

18

32

56

100 (45)

0

A

5

5

5

5

5

5

 

B

5

5

5

5

5

5

 

C

5

5

0

5

5

5

 

D

4

5

5

5

5

5

 

Total introduced

19

20

15

20

20

20

24

A

0

0

0

0

0

0

 

B

0

0

0

0

0

0

 

C

0

0

na

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

1

 

C

0

0

na

0

0

1

 

D

0

0

0

0

0

0

 

Total

immobilised

0

0

0

0

0

2

 

Effect %

0

0

0

0

0

10

( ) between brackets the measured concentration is given (mg/L)

n.a. – not applicable, there were no daphnids introduced to this vessel

 

No significant immobilisation or other effects were observed in any of the group tested. It should be noted that the guideline allows 10% effect in the control, which is considered biologically insignificant. Concentrations were measured to demonstrate the presence and stability of the test item in test medium. Samples taken from the highest concentration were analysed. The actual concentration was 45 mg/L at the start of the exposure and remained stable until the end of the test (97% of initial).

Validity criteria fulfilled:
yes
Conclusions:
The test substance 48h-EL50 was > 100 mg/L based on nominal loadings into the WAF test system
Executive summary:

The acute toxicity to Daphnia magna was carried out according to OECD TG 202 Daphnia sp., Acute Immobilisation Test and EU Method C.2 guidelines. Twenty Daphnia magna per test group (4 replicates, 5 per vessel) were exposed to an untreated control group and to a WAF prepared at a test item loading rates up to 100 mg/L. In the combined range-finding test ten daphnia per test group (2 replicates, 5 per vessel) were exposed to WAFs prepared at loading rates of 1.0, 10.0 and 100.0 mg/L (after correcting for the purity of the test item). All final test solutions were clear and colourless. Concentrations were measured to demonstrate the presence and stability of the test item in test medium. A final test was performed based on the results of a combined limit/range-finding test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to WAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test. Concentrations were measured to demonstrate the presence and stability of the test item in test medium. Samples taken from the highest concentration were analysed. The actual concentration was 45 mg/L at the start of the exposure and remained stable until the end of the test (97% of initial). The study met the acceptability criteria prescribed by the study plan and was considered valid. Under the conditions of the present study, the 48h-EL50 was beyond the range tested, i.e. the maximum loading rate of 100 mg/L. Therefore, the 48h-EL50 was > 100 mg/L.

Description of key information

EL50 (invertebrates) = > 100 mg/L ; WAF loading 48hour-freshwater, OECD TG 202, 2016

Key value for chemical safety assessment

Additional information

The acute toxicity to Daphnia magna was carried out according to OECD TG 202 Daphnia sp., Acute Immobilisation Test and EU Method C.2 guidelines. Twenty Daphnia magna per test group (4 replicates, 5 per vessel) were exposed to an untreated control group and to a WAF prepared at a test item loading rates up to 100 mg/L. In the combined range-finding test ten daphnia per test group (2 replicates, 5 per vessel) were exposed to WAFs prepared at loading rates of 1.0, 10.0 and 100.0 mg/L (after correcting for the purity of the test item). All final test solutions were clear and colourless. Concentrations were measured to demonstrate the presence and stability of the test item in test medium. A final test was performed based on the results of a combined limit/range-finding test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to WAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test. Concentrations were measured to demonstrate the presence and stability of the test item in test medium. Samples taken from the highest concentration were analysed. The actual concentration was 45 mg/L at the start of the exposure and remained stable until the end of the test (97% of initial). The study met the acceptability criteria prescribed by the study plan and was considered valid. Under the conditions of the present study, the 48h-EL50 was beyond the range tested, i.e. the maximum loading rate of 100 mg/L. Therefore, the 48h-EL50 was > 100 mg/L.