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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:
30 November 2015 to 23 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
At the start of the test, the 100 % saturated solution top 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 23 hours. After stirring the test concentration was then filtered through 0.45 µm filter. A control treatment was prepared by adding Elendt M4 medium only to the control vessels.
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Nominal test concentration of 100 % saturated solution
- Sampling method: Concentration of test material in Elendt M4 medium was determined by dilution followed by MS/MS Detection TOF system.
- Sample analysis: At the start of the test (0 hours), ca 20 mL samples of freshly prepared test media were taken from the control and 100 % saturated solution (48.2 mg/L based on geometric mean measured concentration) 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 and 100 % saturated solution (48.2 mg/L based on geometric mean measured concentration).
In each case duplicate samples were taken, one for chemical analysis and one as a ‘back-up’ should further analysis be required.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The dilution water in the study was Elendt M4 medium. Initial visual solubility work was conducted at 100 mg/mL in Elendt M4 media, however it was observed as not in solution after 15 minute sonication and 48 hour stir. Therefore, it was considered justifiable to carry out a media trial to assess the water solubility of the test substance using a saturated solution preparation method, a media trial with analytical samples was conducted to analyse water solubility. However, the data for the media preparation work was poor and reproducibility was difficult, therefore, it was decided that the media preparation work would be DNU (data not used) and the media work would be for information purposes only. The media preparation work showed sample concentrations were consistent. Therefore, a saturated solution preparation method was used. The results observed in the media trial was consistent with that observed during the range-finder and definitive phases.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus
- Feeding during test: The daphnia were not fed during the test.

ACCLIMATION
- Acclimation period: Juvenile daphnia, less than 24 h old, were taken from healthy parental laboratory cultures. The Daphnia magna are cultured in 600 mL glass beakers containing 500 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 10-20 daphnids per vessel. 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 twice 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.
- Type and amount of food: A concentrated suspension of Chlorella vulgaris.
- Feeding frequency: The cultures are fed daily.
- Health during acclimation (any mortality observed):



Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Test temperature:
19.2 – 20.2
pH:
6.41 - 7.34
Dissolved oxygen:
8.17 - 9.32 mg/L
Nominal and measured concentrations:
Definitive test:
Nominal test concentration of 100 % saturated solution
Geometric mean measured concentration of 48.2 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers (100 mL) were used as test vessels
- Material, size, headspace, fill volume: The vessel was filled to approximately 50 mL
- Renewal rate of test solution (frequency/flow rate): No renewal. Media was prepared at 0 hours only.
- No. of organisms per vessel: 5 Daphnia (<24 hours old) per vessel
- No. of vessels per concentration: Four replicate test vessels
- No. of vessels per control: Four replicate test vessels

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water used in the study was Elendt M4 medium.

OTHER TEST CONDITIONS
- Adjustment of pH: No pH adjustment
- Photoperiod: The test was conducted with a 16-hour light: 8-hour dark photoperiod.

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 STUDY
- Test concentrations: The range-finding test was conducted at nominal test substance concentrations of 0.10, 1.0, 10 and 100 % saturated solution (0.0408, 0.206, 4.28, 44.7 mg/L based on geometric mean measured concentrations) under static test conditions. A control group was also included. Duplicate test vessels were prepared for the control and each test concentration. Based on geometric mean measured concentrations, the results of the range-finding test suggested that the 48-hour EC50 value would be greater than 44.7 mg/L.
- Results used to determine the conditions for the definitive study: Yes. Based on the results of a range-finding test a definitive limit test was conducted with a test concentration of 100 % saturated solution, a control group was also included.
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: The result is based on the loading rate of the test material
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Results derived empirically and based on the loading rate of the test material
Details on results:
- Other biological observations:
Daphnia were observed at 24 and 48 hours of exposure during this study and the primary endpoint indicating adverse effects was immobility. No effects were observed in the water control or 100 mg test material/L test vessels during this study.
After 48 hours, the concentration at which no significant (=10 %) dose related immobility occurred was 100 mg/L. The NOEC was therefore 100 mg/L. The 48-hour EC50 value was determined to be >100 mg/L.

CHEMICAL ANALYSIS RESULTS

The limit of quantification (LOQ) for the test material in Elendt M4 medium using this method was 0.1 mg/L.

Analysis of the test media samples was conducted at 0 hours (fresh media) and 48 hours (old media).

For the 100 % saturated solution, analysis of the freshly prepared test media at 0 hour showed achieved concentrations of 60.0 mg/L, respectively. For old test media at 48 hours showed measured concentrations of 38.8 mg/L.

The reduction in measured concentration observed over the 48-hour test period was contrary to that observed during the range-finding test which indicated that the test substance was stable over 48 hours. A review of the data could not give a definitive reason for this. Given that the 48-hour old media result was comparable to the results obtained during the range-finding test, it was considered possible that the 0 hour results were possibly due to a small amount of test substance passing through the filter during media preparation although this could not be confirmed. The geometric mean measured concentration was calculated to be 48.2 mg/L. This value was again comparable to that observed during the range-finding test.

Concentration of test material solutions

The measured value of the solutions represents just the soluble fraction. Having consulted the guidance documents OECD 23 Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures and OECD 2000 Use of the Harmonized system for the Classification of Chemicals which are Hazardous for the Aquatic Environment, both confirm that use of the loading rate is advocated for expressing exposure/reporting data concerning WAFs. It is therefore considered to be scientifically sound to report the concentration of the limit dose solution as 100 mg/L based on the loading rate.

Test Media Description

The test preparations were observed to be colourless solutions throughout the duration of the test.

Validity Criteria

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

Numbers of immobilised Daphnia magna at 24 and 48 hours

Concentration
(mg/L based on loading rate)

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

100

0

0

0

0

0

0

1

0

0

5

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study the 48-hour EC50 was determined to be >100 mg/L and the corresponding NOEC was determined to be 100 mg/L based on the loading rate.
Executive summary:

The toxicity of the test material to the freshwater invertebrate, Daphnia magna, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 202.

Based on the results of a range-finding test a definitive limit test was conducted with a test concentration of 100 % saturated solution, a control group was also included. The test was conducted under static test conditions. 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 and the primary endpoint indicating adverse effects was immobility. The Daphnia magna were not fed during the test.

The measured value of the solutions represents just the soluble fraction (geometric mean measured concentration was calculated to be 48.2 mg/L). Having consulted the guidance documents OECD 23 Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures and OECD 2000 Use of the Harmonized system for the Classification of Chemicals which are Hazardous for the Aquatic Environment, both confirm that use of the loading rate is advocated for expressing exposure/reporting data concerning WAFs. It is therefore considered to be scientifically sound to report the concentration of the limit dose solution as 100 mg/L based on the loading rate.

No effects were observed in the water control or 100 mg test material/L test vessels during this study. After 48 hours, the concentration at which no significant (=10 %) dose related immobility occurred was 100 mg/L.

Under the conditions of the study the 48-hour EC50 was determined to be >100 mg/L and the corresponding NOEC was determined to be 100 mg/L based on the loading rate.

Description of key information

Under the conditions of the study the 48-hour EC50 was determined to be >100 mg/L and the corresponding NOEC was determined to be 100 mg/L based on the loading rate.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
100 mg/L

Additional information

The toxicity of the test material to the freshwater invertebrate, Daphnia magna, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 202. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Based on the results of a range-finding test a definitive limit test was conducted with a test concentration of 100 % saturated solution, a control group was also included. The test was conducted under static test conditions. 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 and the primary endpoint indicating adverse effects was immobility. The Daphnia magna were not fed during the test.

The measured value of the solutions represents just the soluble fraction (geometric mean measured concentration was calculated to be 48.2 mg/L). Having consulted the guidance documents OECD 23 Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures and OECD 2000 Use of the Harmonized system for the Classification of Chemicals which are Hazardous for the Aquatic Environment, both confirm that use of the loading rate is advocated for expressing exposure/reporting data concerning WAFs. It is therefore considered to be scientifically sound to report the concentration of the limit dose solution as 100 mg/L based on the loading rate.

No effects were observed in the water control or 100 mg test material/L test vessels during this study. After 48 hours, the concentration at which no significant (=10 %) dose related immobility occurred was 100 mg/L.

Under the conditions of the study the 48-hour EC50 was determined to be >100 mg/L and the corresponding NOEC was determined to be 100 mg/L based on the loading rate.