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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 January 2016 to 27 April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Analysis was conducted on 6.25, 12.5, 25, 50 and 100 % saturated solution test concentrations. Analysis of the test media samples was conducted at 0 hours (fresh media) and 48 hours (old media).
- Sampling method: At the start of the test (0 hours), ca 20 mL samples of freshly prepared test media were taken from the control, 6.25, 12.5, 25, 50 and 100 mg/L test media preparation flasks for chemical analysis.
At 48 hours, ca 20 mL samples were also taken for chemical analysis from the pooled old test media at control, 6.25, 12.5, 25, 50 and 100 mg/L test concentrations.
In each case duplicate samples were taken, one for chemical analysis and one as a ‘back-up’ should further analysis be required.
Analytical samples of the nominal 25 and 50 % saturated solutions at 0 hours were re-analysed due unexpected high concentrations, they were run in duplicate, therefore a mean of the two samples has been reported.
Due to problems seen during the test phase, injection volume had to be increased from 5 μL to 10 μL in order to give a better peak response, as sensitivity of the instrument was causing problems.
Vehicle:
no
Details on test solutions:
PRELIMINARY SOLUBILITY TRIAL AND MEDIA PREPARATION WORK
Initial visual solubility work was conducted at 100 mg/mL in acetone, however, after addition of acetone stock media to the test water the test substance was observed as white particulate in the test water. Solubility work was repeated to a lowest attempted concentration of 1 mg/L, which was not successfully in solution. Therefore, it was considered justifiable to carry out the test using a saturated solution preparation method, a media trial with analytical samples was conducted to analyse water solubility.

DEFINITIVE TEST
At the start of the test, the 100 % saturated solution stock media concentration was prepared by weighing ca 50 mg of test substance and adding to 500 mL of Elendt M4 medium. This was stirred for ca 20 hours. After stirring, the stock medium was then filtered through 0.45 μm filter giving the 100 % saturated solution top concentration. The remaining test concentrations were prepared by a serial dilution from the 100 % saturated solution top test concentration.
A control treatment was prepared by adding Elendt M4 medium only to the control vessels.
Media was prepared at 0 hours only.
The appearance, colour and behaviour of the test substance in the test media were recorded.
The dilution water used in the study was Elendt M4 medium.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain/clone: Daphnia magna (Straus)
- Age: Less than 24 hours old.
- Feeding during test: The Daphnia magna were not fed during the test.
- Parental Stock: The Daphnia magna are cultured in 1 000 mL glass beakers containing 1 000 mL of Elendt M4 medium. Each vessel and its contents are referred to as a 'culture'. New cultures are initiated with juvenile Daphnia magna (less than 24 hours old), at a density of approximately 10-20 daphnids per litre. The cultures are fed daily with a concentrated suspension of Chlorella vulgaris prepared in accordance with standard operating procedures.
The water in each culture was renewed or partially renewed at least once a week. Juveniles were removed when present in cultures using a sieve. Cultures were maintained up to a maximum of 4 to 5 weeks. Juveniles for use in acute toxicity tests were collected from the second brood onwards. Approximately 24 hours before a test was set up, juveniles present in the cultures were removed and discarded. Over the next 24 hours, juveniles for use in the test were removed from the culture using a wide bore pipette and transferred to fresh culture medium. The juveniles were then left for at least 1 hour before selecting actively swimming individuals for use. All juveniles used to initiate a test were less than 24 hours old.
All cultures, prior to and during toxicity tests, are maintained under fluorescent lighting on a 16-hour light:8-hour dark photoperiod.
Regular tests are conducted using a reference toxicant to ensure that Daphnia magna cultures are of the highest quality and sensitivity
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
19.1 – 20.1 °C
pH:
6.05 to 7.56
Dissolved oxygen:
7.01 to 9.56 mg/L
Nominal and measured concentrations:
Nominal concentrations: 6.25, 12.5, 25, 50 and 100 % saturated solutions.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers (100 mL) with clear petri dish lids.
- Material, size, headspace, fill volume: approximately 50 mL
- No. of organisms per vessel: Five organisms per vessel, added to each vessel with a wide bore glass pipette to avoid damaging the animals during transfer.
- No. of vessels per concentration/control: Four replicate test vessels were prepared for the control and each test concentration.

TEST MEDIUM / WATER PARAMETERS
See below for test water parameters.

OTHER TEST CONDITIONS
- Adjustment of pH: No adjustment
- Photoperiod: 16-hour light: 8-hour dark photoperiod.
- Water quality: The pH, dissolved oxygen concentration (% air saturation value (ASV) and mg/L) and temperatures were determined in freshly prepared test media at the start of the test and in the old media at 48 hours. Continuous temperatures were measured using a digital (min/max) thermometer in an additional vessel maintained in the study area. At 0 hours, water quality measurements were performed on media sampled directly from preparation flasks. At the end of the test, water quality measurements were conducted using pooled replicate samples of old test media.

EFFECT PARAMETERS MEASURED
After 24 and 48 hours, the Daphnia magna in each test vessel were observed for evidence of immobility.
The observations differentiated between mobile and immobile daphnids. An individual was considered immobile if, when the contents of the test vessel were briefly agitated, it did not swim during a 15-second period of observation. In addition, Daphnia magna submerged in the body of the test media and those that were held at the surface of the test media were also recorded.

RANGE-FINDING TEST
- Test concentrations: The range-finding test was conducted at nominal test substance concentrations of 0.10, 1.0, 10 and 100 % saturated solution under static test conditions. A control group was also included. Duplicate test vessels were prepared for the control and each test concentration.
- Results used to determine the conditions for the definitive study: 90 % immobility was reported at the highest dose at 48 hours. Based on nominal concentrations, the results of the range-finding test suggested that the 48-hour EC50 value would be between 10 and 100 mg/L.
Analysis of the test concentrations at 0 and 48 hours ranged from 0.0790 to 84.6 mg/L at 0 hours and 0.0783 to 99.9 mg/L at 48 hours.
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
84.91 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Results derived empirically
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Results derived empirically
Details on results:
After 48 hours, the concentration at which no significant (≤10 %) dose related immobility occurred was 50 mg/L. The NOEC was therefore 50 mg/L.
The 48-hour EC50 value was determined to be 84.91 mg/L.
All analytical results were within 80-120 % of nominal concentrations. Therefore, the results were based on nominal concentrations in terms of mg/L.
- Any observations that might cause a difference between measured and nominal values: The test preparations were observed to be colourless solutions throughout the duration of the test.
Reported statistics and error estimates:
Data Presentation and Analysis
Statistical analysis was performed using the CETIS program v 1.8.6.8. The EC50 values and corresponding 95 % confidence limits at 24 and 48 hours were calculated using linear interpolation. The toxicity results are expressed in terms of measured concentrations.
The NOEC was established by observation of the data.
The highest measured test concentration where no significant immobilisation (≤10 % immobile Daphnia magna) i.e. the no observed effect concentration (NOEC), and the lowest measured test concentration causing 100 % immobilisation based on observation of the data are also reported, where applicable.

Toxicity to Daphnia magna

The percentage immobility data are summarised below.

Nominal concentration (% saturated solution)

Cumulative immobilised Daphnia magna

(initial population: 5 Daphnia per replicate)

24 hours

48 hours

R1

R2

R3

R4

% Immobility

R1

R2

R3

R4

% Immobility

Control

0

0

0

0

0

0

0

0

0

0

6.25

0

0

0

0

0

0

0

0

0

0

12.5

0

0

0

0

0

0

0

0

0

0

25

0

0

0

0

0

0

0

0

0

0

50

0

0

0

0

0

0

0

0

0

0

100

0

2

0

0

10

2

2

3

4

55

Toxicity values and the corresponding NOEC and LOEC values are presented in the following table. Values are presented in terms nominal concentrations.

 

Nominal concentration (mg/L)

Statistical Test

 

24-hour

48-hour

 

EC50

>100 mg/L

84.91 mg/L

Linear Interpolation (ICPIN)

NOEC*

50 mg/L

50 mg/L

NA

Validity Criteria

The validity criteria for control immobility (≤10 %) and dissolved oxygen (≥3 mg/L) were both satisfied. The test is therefore considered valid.

Validity criteria fulfilled:
yes
Conclusions:
Based on nominal concentrations, the 48-hour EC50 value was determined to be 84.91 mg/L. The corresponding No Observed Effect Concentration (NOEC) was considered to be 50 mg/L under the conditions of the study.
Executive summary:

The potential of the test material to cause acute toxicity to Daphnia magna was investigated in a study conducted accordance with the standardised guideline OECD 202 under GLP conditions.

A study was conducted to determine the 48-hour acute toxicity of the test material to the freshwater planktonic crustacean, Daphnia magna. Based on the results of a range-finding test, for which the key results only have been reported, a definitive test was conducted with test concentrations of 6.25, 12.5, 25, 50 and 100 % saturated solutions, a control group was also included. The test was conducted under static test conditions with media preparation at 0 hours only.

At the start of the test, five juvenile Daphnia magna were added to each test vessel. The Daphnia magna in each test vessel were observed at 24 and 48 hours. The Daphnia magna were not fed during the test.

For the 6.25, 12.5, 25, 50 and 100 % saturated solution test concentrations, analysis of the freshly prepared media at 0 hours showed measured concentrations to be 5.39, 11.3, 27.2, 48.4 and 97.4 mg/L, respectively (86 – 109 % of nominal). At 48 hours, analysis of the old pooled media showed measured concentrations of 6.19, 11.2, 25.7, 54.1, 110.2 mg/L, respectively (99 – 110 % of nominal).

The validity criteria for control immobility (≤10 %) and dissolved oxygen (≥3 mg/L) were both satisfied. The test is therefore considered valid.

All analytical results were within 80-120 % of nominal concentrations. Therefore, the results were based on nominal concentrations in terms of mg/L.

The 24 h EC50 was > 100 mg/L. The 48 h EC50 was 84.9 mg/L. The NOEC was 50 mg/L (derived empirically). The validity criteria for control immobility (10 %) and dissolved oxygen ( 3 mg/L) were both satisfied. The test is therefore considered positive.

Based on nominal concentrations, the 48-hour EC50 value was determined to be 84.91 mg/L. The corresponding No Observed Effect Concentration (NOEC) was considered to be 50 mg/L under the conditions of the study.

Description of key information

Based on nominal concentrations, the 48-hour EC50 value was determined to be 84.91 mg/L. The corresponding No Observed Effect Concentration (NOEC) was considered to be 50 mg/L under the conditions of the study.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
84.91 mg/L

Additional information

The potential of the test material to cause acute toxicity to Daphnia magnawas investigated in a study conducted accordance with the standardised guideline OECD 202 under GLP conditions.

A study was conducted to determine the 48-hour acute toxicity of the test material to the freshwater planktonic crustacean, Daphnia magna.Based on the results of a range-finding test, for which the key results only have been reported, a definitive test was conducted with test concentrations of 6.25, 12.5, 25, 50 and 100 % saturated solutions, a control group was also included. The test was conducted under static test conditions with media preparation at 0 hours only.

At the start of the test, five juvenile Daphnia magna were added to each test vessel. The Daphnia magna in each test vessel were observed at 24 and 48 hours. The Daphnia magnawere not fed during the test.

For the 6.25, 12.5, 25, 50 and 100 % saturated solution test concentrations, analysis of the freshly prepared media at 0 hours showed measured concentrations to be 5.39, 11.3, 27.2, 48.4 and 97.4 mg/L, respectively (86 – 109 % of nominal). At 48 hours, analysis of the old pooled media showed measured concentrations of 6.19, 11.2, 25.7, 54.1, 110.2 mg/L, respectively (99 – 110 % of nominal).

The validity criteria for control immobility (≤10 %) and dissolved oxygen (≥3 mg/L) were both satisfied. The test is therefore considered valid.

All analytical results were within 80-120 % of nominal concentrations. Therefore, the results were based on nominal concentrations in terms of mg/L.

The 24 h EC50 was > 100 mg/L. The 48 h EC50 was 84.9 mg/L. The NOEC was 50 mg/L (derived empirically). The validity criteria for control immobility (10 %) and dissolved oxygen (3 mg/L) were both satisfied. The test is therefore considered positive.

Based on nominal concentrations, the 48-hour EC50 value was determined to be 84.91 mg/L. The corresponding No Observed Effect Concentration (NOEC) was considered to be 50 mg/L under the conditions of the study.

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