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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:
17 December 2012 to 12 February 2013
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)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Water samples were taken from the control and each test group (replicates R1 – R4 pooled).
- Sampling method: Samples were taken at 0 and 48 hours for quantitative analysis. Samples at the No Observed Effect Concentration and above only were analysed.
- Sample storage conditions before analysis: Samples were stored at approximately -20 °C prior to analysis. Duplicate samples were taken and stored at approximately -20 °C for further analysis if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
An amount of test material (2200 mg) was dispersed in 11 litres of reconstituted water with the aid of a propeller stirrer at a rate of approximately 1500 rpm for a period of 24 hours. After stirring, any undissolved test material was removed using a 0.2 μm Gelman Acrocap filter (initial approximate 100 mL discarded to pre-condition the filter) to give the 100 % v/v saturated solution. Serial dilutions were then performed in reconstituted water to give the required saturated solution test concentrations. Aliquots of the saturated solution test concentrations were then each separately added to a final volume of 1 litre of reconstituted water to give the required test concentrations.
Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Source: The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
- Age at study initiation (mean and range, SD): 1st instar, less than 24 hours old
- Method of breeding: Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
- Feeding during test: Daphnia were not fed during the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
250 mg/L (as CaCO3)
Test temperature:
20 - 21 °C
pH:
8.0 - 8.2
Dissolved oxygen:
8.7 - 9.0 mg O2/L
Nominal and measured concentrations:
0.010, 0.032, 0.10, 0.32, 1.0 % v/v saturated solution (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass jars
- Type : closed
- Fill volume: 200 mL
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water
- Preparation of Reconstituted water: 25 mL of CaCl2.2H2O (11.76 g/L), MgSO4.7H2O (4.93 g/L), NaHCO3 (2.59 g/L) and KCl (0.23 g/L) was added per litre deioinsed water.
- pH: 7.8 ± 0.2 adjusted (if necessary with NaOH or HCl
- Conductivity: < 5 µS/cm
- Culture medium different from test medium: yes. Culture medium was Elendt M7 medium
- Intervals of water quality measurement: Water temperature and light intensity were recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration were measured using a Hach HQ30d Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light / 8 hours dark with 20 minute dawn/dusk transition
- Light intensity: 664 - 687 lux

EFFECT PARAMETERS MEASURED
Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

TEST CONCENTRATIONS
- Range finding study (Test 1)
- Test concentrations: 0.10, 1.0, 10, 100 % v/v saturated solution (nominal)
- Results: Due to complete immobilisation in all concentrations used in the initial range-finding test it was necessary to conduct a second range-finding test.

- Range finding study (Test 2)
- Test concentrations :0.0010, 0.010, 0.10, 1.0 % v/v saturated solution (nominal)
- Results used to determine the conditions for the definitive study: no immobilisation was observed at the nominal test concentrations of 0.0010 and 0.10 % v/v saturated solution. However, immobilisation was observed at 0.10 and 1.0% v/v saturated solution.
Based on this information nominal test concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0% v/v saturated solution were selected for the definitive test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.18 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 0.098 - 0.33 mg/L (95 % CL)
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.099 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
0.33 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Immobilisation data: None of the Daphnia in the control, 0.010, 0.032 and 0.10 % v/v saturated solutions were immobilised during the 48 hour exposure period. However, all of the Daphnia in the 0.32 and 1.0 % v/v saturated solutions were immobilised within 24 hours of exposure (see Table 3)
- Observations: The test preparations were observed to be clear colourless solutions for the duration of the test.
Results with reference substance (positive control):
- Results with reference substance valid? Yes. A positive control used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
Exposure conditions for the positive control were similar to those in the definitive test.
The No Observed Effect Concentrations after 24 and 48 hours were 0.56 and 0.32 mg/L respectively. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.
The results from the positive control with potassium dichromate were within the normal range for this reference item.
- EC50/LC50: 48 hour EC50 0.45 mg/L (95 % C.I. 0.42-0.48 mg/L)
Reported statistics and error estimates:
The EC50 value and associated confidence limits at 24 and 48 hours were calculated using the geometric mean method using the ToxCalc software package.

Table 1: Cumulative Immobilisation Data in the Initial Range-finding Test

Nominal conc.

(% v/v saturated solution)

Cumulative immobilised Daphnia (initial population: 10 per replicate)

24 hours

48 hours

Control

1

*

0.10

10

*

1.0

10

*

10

10

*

100

10

*

*The test was stopped at 24 hours after exposure due to 100 % immobilisation in all test concentrations.

Table 2: Cumulative Immobilisation Data in the Second Range-finding Test

Nominal conc.

(% v/v saturated solution)

Cumulative immobilised Daphnia (initial population: 10 per replicate)

24 hours

48 hours

Control

0

0

0.0010

0

0

0.010

0

0

0.10

3

5

1.0

10

10

Table 3: Cumulative Immobilisation Data in the Definitive Test

Nominal conc. (% v/v saturated solution)

Cumulative immobilised Daphnia (initial population: 5 per replicate)

24 hours

48 hours

R1

R2

R3

R4

Total

R1

R2

R3

R4

Total

Control

0

0

0

0

0

0

0

0

0

0

0.010

0

0

0

0

0

0

0

0

0

0

0.032

0

0

0

0

0

0

0

0

0

0

0.10

0

0

0

0

0

0

0

0

0

0

0.32

5

5

5

5

20

5

5

5

5

20

1.0

5

5

5

5

20

5

5

5

5

20

R1 - R4 = Replicates 1 to 4

Validity criteria fulfilled:
yes
Conclusions:
Based on the mean-measured test concentrations of the test media, the 48-hour EC50 was 0.18 mg/L with 95 % confidence limits of 0.098 – 0.33 mg/L and the No Observed Effect Concentration was 0.098 mg/L.
Executive summary:

The acute toxicity of the test material to Daphnia magna was investigated in a GLP study which was conducted in accordance with standardised guidelines OECD 202 and EU Method C.2 under static conditions.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 % v/v saturated solution for 48 hours. The number of immobilised Daphnia were recorded after 24 and 48 hours.

The No Observed Effect Concentration and above only were analysed. Analysis of the freshly prepared media at 0 hours for the 0.10, 0.32 and 1.0 % v/v saturated solution test concentrations showed measured concentrations of 0.098, 0.34 and 0.91 mg/L, respectively. Analysis of the old media at 48 hours for the 0.10, 0.32 and 1.0 % v/v saturated solution test concentrations showed measured concentrations of 0.099, 0.32 and 1.1 mg/L. It was considered appropriate to base the results on mean-measured test concentrations.

The 48-hour EC50 for the test item to Daphnia magna, based on mean-measured test concentrations, was 0.18 mg/L with 95 % confidence limits of 0.099 – 1.0 mg/L. The Lowest Observed Effect Concentration was considered to be 0.33 mg/L. The No Observed Effect Concentration was 0.099 mg/L.

Description of key information

The 48-hour EC50 for the test material to Daphnia magna based on mean-measured test concentrations was 0.18 mg/L with 95 % confidence limits of 0.099 – 1.0 mg/L.  

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.18 mg/L

Additional information

The acute toxicity of the test material to Daphnia magna was investigated in a GLP study which was conducted in accordance with standardised guidelines OECD 202 and EU Method C.2 under static conditions. The study was assigned a reliability score of 1 in accordance with the principles for assessing data quality as set forth by Klimisch et al (1997).

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 % v/v saturated solution for 48 hours. The number of immobilised Daphnia were recorded after 24 and 48 hours.

The No Observed Effect Concentration and above only were analysed. Analysis of the freshly prepared media at 0 hours for the 0.10, 0.32 and 1.0 % v/v saturated solution test concentrations showed measured concentrations of 0.098, 0.34 and 0.91 mg/L, respectively. Analysis of the old media at 48 hours for the 0.10, 0.32 and 1.0 % v/v saturated solution test concentrations showed measured concentrations of 0.099, 0.32 and 1.1 mg/L. It was considered appropriate to base the results on mean-measured test concentrations.

The 48-hour EC50 for the test item to Daphnia magna, based on mean-measured test concentrations, was 0.18 mg/L with 95 % confidence limits of 0.099 – 1.0 mg/L. The Lowest Observed Effect Concentration was considered to be 0.33 mg/L. The No Observed Effect Concentration was 0.099 mg/L.