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Ecotoxicological information

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:
From 17 September 2018 to 19 March 2019
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
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Inspection dates 29-30-31 May 2018 / Signature date 15 November 2018
Specific details on test material used for the study:
- Storage conditions: To be stored refrigerated (2-8°C) protected from light, stored under nitrogen after first opening.
- Vapour pressure: 1.3 Pa at 20°C and 2.4 Pa at 25°C (OECD Guideline 104, effusion method, LPL 2018)
- Water solubility: 39.7 mg/L (iSafeRat v1.7 prediction). Experimental study on-going at the date of the present study
- Log Kow: 3.93 (iSafeRat v1.7 prediction). Experimental study on-going at the date of the present study
- Stability: Stable (less than 10% loss) from pH 2-12 (Firmenich internal data)
Analytical monitoring:
yes
Details on sampling:
Single samples for analysis were taken from the control and all test concentrations at the start and at the end of the test (t=48h).
Test samples (specimen) were injected directly after a dilution by a factor of two with acetonitrile (500μL sample + 500 μL acetonitrile). If the sample concentrations were too high and not included in the concentration range of the calibration, the samples were diluted appropriately with a test water/acetonitrile (50/50 v/v) mixture.
Samples were injected as soon as possible after their preparation.
A blank sample of test water/acetonitrile (50/50) was injected every analytical sequence in order to check that no instrument or glassware contamination had occurred. Blank samples were considered as valid if their concentrations were below the limit of detection.
Vehicle:
no
Details on test solutions:
PREPARATION OF STOCK AND TEST SOLUTIONS
The mixing vessel was a cylindrical glass bottle sealed with screw cap and fitted with a drain port near the bottom for drawing off the saturated stock solution. The volume of the mixing vessel was
approximately 1 L. A magnetic stirring bar was placed in the vessel and approx. 1 L test water was added. Then an excess of the test item (approx. 1000 mg) was carefully added directly to the surface of the test water. The mixing vessel was thereafter closed immediately. The mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 23 hours of gentle stirring in the dark at room temperature, the contents of the vessel was allowed to stand undisturbed for at least 1 hour before use. The first 100 mL were discarded via the drain port. Samples were taken from the remaining stock solution and chemically analysed. Then the stock solution was diluted with test water as necessary to obtain the required test concentrations into 500-mL volumetric flasks based on the measured concentration of the stock solution (39.5 mg.L-1). Each flask was inverted several times before filling the test vessels (without headspace) to ensure adequate mixing and homogeneity of test solutions. After filling and introduction of daphnids the vessels were sealed immediately. The test solutions in test vessels were observed to be clear and colourless. The test was carried out without adjustment of the pH.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Species: Daphnia magna (Straus), clone 5
- Sex: Female
- Origin: LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine Campus Bridoux - Bât. IBISE, 8, rue du Général Delestraint - 57070 METZ, bred in the Laboratoires des Pyrénées et des Landes.
- Reason for selection: Characteristic and common representative of freshwater zooplankton which has been selected as an internationally accepted invertebrate species.
- Validity of batch: Daphnids originated from a healthy stock, showing no signs of stress such as mortality, presence of males, ephippia or discoloured Daphnids.
- Age at test start: < 24 hours old
- Test water: Reconstituted water (Elendt M4 medium), as prescribed in OECD Guideline 202. The pH of this solution was in the range of 6 to 9 and the total water hardness was approximately 250 mg.L-1 (as CaCO3).
- Breeding conditions: Daphnids were cultured in the Laboratoires des Pyrénées et des Landes under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in all-glass vessel containing test water. Cultures were maintained at a density of 1 adult daphnid per 25 mL of culture medium. Daphnids were fed at least three
times a week with a suspension of algal cells (Pseudokirchneriella subcapitata) up to 0.1-0.2 mg C.Daphnia.-1day.-1. The water was changed three times a week. These culture conditions maintained the daphnids in the parthenogenetic reproductive stage.

Daphnids were introduced into the test vessels each completely filled with test solution and stoppered without headspace immediately after filling the test vessels with test solutions.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
The total water hardness was approximately 250 mg/L (as CaCO3).
Test temperature:
The temperature of the test medium was situated between 20.8 and 21.0°C throughout the test (average value: 20.9°C)
pH:
From 8.04 to 8.45
Dissolved oxygen:
From 8.27 mg/L to 8.43 mg/L
Salinity:
Not applicable
Conductivity:
No data
Nominal and measured concentrations:
- Nominal concentrations (spaced by a factor of 2.00): 0.75, 1.50, 3.00, 6.00 and 12.00 mg test item.L-1.
- Geometric means of the measured concentrations: 0.70, 1.58, 3.08, 6.12 and 12.61 mg.L-1.
Details on test conditions:
TEST SYSTEM
- Test vessel: 60-mL glass flasks sealed with assembled screw cap with hole and PTFE/silicone septum. The test vessels were filled with test water without headspace
- Type (delete if not applicable): closed
- Aeration: No aeration of the test solutions occurred throughout the test.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable. A static test was performed (stability of the test item was demonstrated in closed and static conditions during the range-finding test).
- Renewal rate of test solution (frequency/flow rate): Not applicable
- No. of organisms per vessel: 5 daphnids
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4 replicates. Test water without test substance but treated in the same way as the test substance solutions.

TEST MEDIUM / WATER PARAMETERS
Reconstituted water (Elendt M4 medium), as prescribed by OECD Guideline 202

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16h light : 8h dark
- Light intensity: no data

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility and abnormal behaviour were determined by visual observation after 24 and 48 hours. Daphnids were considered to be immobile if they were not able to swim within 15 seconds after gentle agitation of test vessels.

RANGE-FINDING STUDY
Ten daphnids per concentration (5 per vessel, in duplicate) were exposed to the nominal concentrations of 0.5, 1.0, 10.0, 32.0 mg/L and to a concentration close to the solubility limit of the test item in test water (40.0 mg/L), and to a control. After 48 hours of exposure, immobilisation rates were 0% at 0.5 and 1.0 mg/L, and 100% at 10.0, 32.0 and 40.0 mg/L.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (Batch n°16H244108; purity Min. 99.8 %; expiry date 16 January 2019)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
2.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: 2.06 - 2.56 mg/L
Duration:
48 h
Dose descriptor:
other: The highest test concentration without observed effect
Effect conc.:
1.58 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
WATER QUALITY PARAMETERS AND ENVIRONMENTAL CONDITIONS THROUGHOUT THE TEST
The results of measurement of pH and oxygen concentrations remained within the limits prescribed by the study plan (pH: 6.0-9.0, not varying by more than 1.5 units; oxygen: ≥ 60% of the air-saturation value at the end of the test), suggesting that pH and oxygen concentrations were not influenced by the test item.
Turbidity measurements confirmed the absence of undissolved material in the stock solution (represented by turbidity analysis of the stock solution, in comparison with the turbidity value of the
control).
The temperature of the test medium was situated between 20.8 and 21.0°C throughout the test (average value: 20.9°C), and complied with the requirements as laid down in the study plan (20°C ± 2°C, constant within 1°C).

ANALYTICAL RESULTS
The analytical results of this test showed that concentrations of test solutions prepared following the outlined procedures were reproducible. Indeed, test item concentrations were satisfactorily
maintained within ± 20% of the nominal concentrations throughout the test. Besides, analyses of samples taken from all test concentrations at the start and the end of the test revealed that test item
levels were stable, with losses of test item < 20%, except for the nominal concentration of 0.75 mg/L where losses of 35% were observed. For this reason, the evaluation of the effects on Daphnia magna was based on the geometric means of the measured concentrations (as recommended by OECD Guideline 202).
See analytical results in the table 6.1.3/2 in "Any other information on results incl. tables".

BIOLOGICAL RESULTS
After 48 hours of exposure, immobilisation rates were 0% at 0.70 and 1.58 mg/L, 95% at 3.08 mg/L, and 100% at 6.12 and 12.61 mg/L. Therefore, the highest concentration without effect was 1.58 mg/L and the lowest concentration with 100% immobilisation was 6.12 mg/L.
See biological results in the table 6.1.3/1 in "Any other information on results incl. tables".
Results with reference substance (positive control):
On September 4, 2018 (KD18-002; most recent test), the 24h-EC50 was 1.105 mg/L. Hence, the sensitivity of the clone of Daphnia magna was in agreement with OECD 202 (expected 24h-EC50: 0.6 mg/L to 2.1 mg/L) at this time.
Reported statistics and error estimates:
Statistical analyses were performed by the computer program ToxRat.

Table 6.1.3/1: Acute immobilisation of daphnids after 24 and 48 hours in the final test

Nominal concentration(mg test item.L-1)

Measured concentration (geometric mean)

(mg test item.L-1)

Replicate

Number of daphnids exposed

Response at 24h

Response at 48h

Number

Total %

Number

Total %

Control

0

1

2

3

4

5

5

5

5

0

0

0

0

0

0

0

0

0

0

0.75

0.70

1

2

3

4

5

5

5

5

0

0

0

0

0

0

0

0

0

0

1.50

1.58

1

2

3

4

5

5

5

5

0

0

0

0

0

0

0

0

0

0

3.00

3.08

1

2

3

4

5

5

5

5

1

0

0

1

10

5

4

5

5

95

6.00

6.12

1

2

3

4

5

5

5

5

0

1

1

4

30

5

5

5

5

100

12.00

12.61

1

2

3

4

5

5

5

5

4

4

5

1

70

5

5

5

5

100

Table 6.1.3/2: Concentrations of the test item (mg/L) in test water - final test

Nominal

concentration

(mg.L-1)

Measured

concentration

(mg.L-1)

Geometric mean measured concentrations

Start

(t=0h)

End (t=48h)

mg.L-1

%

nominal

Control

Abs.

Abs.

N.A.

N.A.

0.75

0.87

0.57

0.70

93

1.50

1.65

1.52

1.58

105

3.00

3.07

3.09

3.08

103

6.00

6.30

5.95

6.12

102

12.00

12.81

12.41

12.61

105

N.A.: not applicable

% = Percent of expected nominal concentration in test item.

Abs.= Absence: concentrations below the LOQ (0.45 mg.L-1) and the LOD (0.13 mg.L-1).

Validity criteria fulfilled:
yes
Remarks:
In the control, no daphnids became immobilised nor trapped at the surface of the water nor showed signs of stress. Dissolved oxygen concentration at the end of the test was ≥ 60% of the air-saturation value in controls and test vessels.
Conclusions:
The toxic effect of test item to the freshwater invertebrate Daphnia magna was investigated in a closed static test. Under the experimental conditions and based on the geometric means of measured
exposure concentrations, the 48-hour EC50 value was determined at 2.30 mg/L (95% CL: 2.06 - 2.56 mg/L).
Executive summary:

A study was performed to assess the acute toxicity of test substance to Daphnia magna, according to OECD Guideline 202, EU Method C2 with GLP compliance. The criterion measured was the EC50 (Median Effective Concentration), a statistically derived concentration which is expected to cause immobility in 50% of the daphnids within a period of 48 hours. Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed to an aqueous solution of the test item over 48 hours at the required nominal test concentrations of 0.75, 1.50, 3.00, 6.00 and 12.00 mg/L and to a control. The immobilisation of the daphnids was determined in a closed static 48-hour test by visual observation after 24 and 48 hours. Samples taken from the control and all test concentrations were analysed at the start and the end of the test in order to determine if concentrations of the test item were maintained.

The test item levels were found to be relatively stable throughout the test, with test item losses < 20% of the initial concentrations, except for the nominal concentration of 0.75 mg/L where losses of 35% were observed compared to the initial concentration. Therefore, the evaluation of the effects on Daphnia magna was based on the geometric means of the measured concentrations: 0.70, 1.58, 3.08, 6.12 and 12.61 mg/L. After 48 hours of exposure, immobilisation rates were 0% at 0.70 and 1.58 mg/L, 95% at 3.08 mg/L, and 100% at 6.12 and 12.61 mg/L.

Under the experimental conditions and based on the geometric means of measured exposure concentrations, the 48-hour EC50 value was determined at 2.30 mg/L (95% CL: 2.06 - 2.56 mg/L). No effect was observed at 1.58 mg/L measured.

Description of key information

OECD Guideline 202, EU Method C2, GLP, key study, validity 1:

48h-EC50 (Daphnia magna) = 2.30 mg/L (95% CL: 2.06 - 2.56 mg/L), based on geometric mean measured concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
2.3 mg/L

Additional information

One experimental study is available to assess the acute toxicity of the registered substance to Daphnia magna, according to OECD Guideline 202, EU Method C2 with GLP compliance. Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed to an aqueous solution of the registered substance over 48 hours at the required nominal test concentrations of 0.75, 1.50, 3.00, 6.00 and 12.00 mg/L and to a control. The immobilisation of the daphnids was determined in a closed static 48-hour test by visual observation after 24 and 48 hours. Samples taken from the control and all test concentrations were analysed at the start and the end of the test in order to determine if concentrations of the test item were maintained. The test item levels were found to be relatively stable throughout the test, with test item losses < 20% of the initial concentrations, except for the nominal concentration of 0.75 mg/L where losses of 35% were observed compared to the initial concentration. Therefore, the evaluation of the effects on Daphnia magna was based on the geometric means of the measured concentrations: 0.70, 1.58, 3.08, 6.12 and 12.61 mg/L. After 48 hours of exposure, immobilisation rates were 0% at 0.70 and 1.58 mg/L, 95% at 3.08 mg/L, and 100% at 6.12 and 12.61 mg/L.

Under the experimental conditions and based on the geometric means of measured exposure concentrations, the 48-hour EC50 value was determined at 2.30 mg/L (95% CL: 2.06 - 2.56 mg/L).