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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:
18-01-2002 to 07-04-2002
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:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: February 2000; signature: April 2000
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control), 0.025, 0.045, 0.080, 0.14, 0.25, 0.45, 0.80, 1.40, 2.50 mg/L (nominal ; based on the results of the pre-study media preparation trial).
Time Weighted Average equivalent concentrations: 0 (control), of 0.024, 0.037, 0.064, 0.13, 0.19, 0.37, 0.67, 1.2 and 2.0 mg/L (measured)
- Sampling method: Where possible all samples were prepared for analysis on the day of sampling. Duplicate samples were taken and stored frozen (approximately -20°C) for further analysis if necessary.
- Sample storage conditions before analysis: See above.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Direct dissolution of test item in culture medium to prepare stock; serial dilution of stock to prepare test media. During validation: Recoveries were in the region of 72 to 64% at 0.025 and 2.5 mg/L concentrations. A second media preparation trial was performed based on these results. Which indicated an inconsistent media preparation. The second trial was as follows: Duplicate test item dispersions at concentrations of 100 mg/L were prepared by dispersing known amounts of test item in known volumes of ASTM algal culture medium, reconstituted water or dechlorinated tap water and stirring each dispersion at a temperature of 25°C for 48 hours using a propeller stirrer at approximately 2000 rpm. After the 48-Hour stirring period the dispersions were cooled to 21°C or 14°C for 24 hours and the undissolved test item removed by filtration (0.2 µm) prior to samples being taken for analysis. In order to ensure that adsorption of the test item to the filter matrix was reduced to an insignificant level, the filter used was preconditioned by passing a suitable volume (1 litre) of the dispersion through the filter prior to collecting the saturated solution for testing. Analysis performed on the pretest trial preparation showed measured test concentrations of 2.381 and 2.637 mg/L for each of the two prepared saturated solutions. Given the consistency of the results it was considered that the method was suitable for use in the definitive test.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No precipitate reported using the second media preparation trial.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain: not reported
- Source: in-house laboratory cultures
- Age at study initiation (mean and range, SD): < 24 hours
- Method of breeding: Parthenogenesis
- Feeding during test: No. The daphnids were not fed during the study. During culture: The culture daphnids are fed daily ad libitum with algae suspension
- Food type: Not applicable.
- Amount: Not applicable.
- Frequency: Not applicable.

ACCLIMATION
- Acclimation period: None reported. Can be presumed to be < 2 hours in dilution water.
- Acclimation conditions (same as test or not): Yes.
- Type and amount of food: Not applicable.
- Feeding frequency: Not applicable.
- Health during acclimation (any mortality observed): Not applicable.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
In accordance with OECD TG 202 guideline.
Hardness:
Dilution water quality parameters: 0 hours: 250 mg CaCO3/L
Test temperature:
Actual: 21ºC
pH:
Control: 0 hours: pH 8.0 and 48 hours: 8.0 ; test groups: 0 hours: pH 8.0 and 48 hours: 8.0
Dissolved oxygen:
Control: 0 hours: 8.3 - 8.4 mg O2/L (93% ASV) and 48 hours: 8.1 mg O2/L (91% ASV); test groups: 0 hours: 8.3 - 8.4 mg O2/L (93-94% ASV) and 48 hours: 8.1-8.2 mg O2/L (91-92% ASV)
Salinity:
Not applicable.
Nominal and measured concentrations:
Definitive test: nominal test item concentrations: 0 (control), 0.025, 0.045, 0.080, 0.14, 0.25, 0.45, 0.80, 1.40, 2.50 mg/L (nominal ; based on the results of the pre-study media preparation trial).
Time Weighted Average equivalent concentrations: 0 (control), of 0.024, 0.037, 0.064, 0.13, 0.19, 0.37, 0.67, 1.2 and 2.0 mg/L (measured)
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass jars
- Type (delete if not applicable): open, but covered to reduce test item losses
- Material, size, headspace, fill volume: glass; 250 mL fill volume, headspace ca. 0 mL (minimal)
- Aeration: No aeration of the test solutions.
- No. of organisms per vessel: Control and test item: 20 per concentration, 10 per vessel (divided into 2 replicates)
- No. of vessels per concentration (replicates): 2 (two replicates, 10 daphnia per vessel).
- No. of vessels per control (replicates): 2 (two replicates, 10 daphnia per vessel).
- No. of vessels per vehicle control (replicates): Not applicable.
- Biomass loading rate: equivalent to 25 mL test media per animal

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water - analytical grade salts dissolved in purified water equivalent or similar to ISO Test water, according to OECD 202, Annex 3: composition (mg/L): KCl 5.8; NaHCO3: 64.8; CaCl2.2H2O: 294 ; MgSO4.7H2O: 123. pH 7.8 (adjusted if necessary).
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Metals: Not reported
- Pesticides: Not reported
- Chlorine: Not reported
- Alkalinity: Not reported
- Ca/mg ratio: Not reported
- Conductivity: <5 µS/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: 0 hours and 48 hours, (dissolved oxygen, pH and temperature)

OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Photoperiod: 16 h light / 8 hours dark, 20 minute dawn and dusk transition periods.
- Light intensity: Not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Immobility (or adverse effects including mortality), 24 hours and at 48 hours. An organism was considered to be immobile, if it was not able to swim in the water phase within 15 seconds after gentle agitation of the test vessel.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study: Two were conducted. The below relates to media preparation trial two and second RF test which was accepted.
- Test concentrations: 0 (control), 0.025, 0.25, 2.5 mg/L ; nominal test item concentration.
- Results used to determine the conditions for the definitive study: Yes. No immobilisation observed at 0.025 and 0.25 mg/L. However, immobilisation was observed at 2.5 mg/L.
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.5 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: (95% CL. 1.3-1.7 mg/L)
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
1.1 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: (95% CL. 0.97-1.2 mg/L)
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.67 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: None reported.
- Observations on body length and weight: Not applicable.
- Other biological observations:
- Mortality of control: No mortalities in control.
- Other adverse effects control: None reported.
- Abnormal responses: None reported.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None. It was speculated the decline in measured test concentrations was therefore considered to be due to possible accumulation of the test item in the test organisms or to any organic waste matter present in the test system.
- Effect concentrations exceeding solubility of substance in test medium: No.
Reported statistics and error estimates:
The EC50 value and associated confidence limits at 48 hours were calculated by the trimmed Spearman-Karber method (Hamilton et al. 1977)

Table 1.0: Cumulative immobilisation data in the definitive test

Nominal Concentration (mg/L)

Time Weighted Average Concentration (mg/L)

Cumulative population immobilised (n=20)

 

 

24 hours

48 hours

 

 

R1

R2

Total

%

R1

R2

Total

%

0 (control)

0

0

0

0

0

0

0

0

0

0.025

0.024

 

0

0

0

0

0

0

0

0.045

0.037

0

0

0

0

0

0

0

0

0.080

0.064

0

0

0

0

0

0

0

0

0.14

0.13

0

0

0

0

0

0

0

0

0.25

0.19

0

0

0

0

0

0

0

0

0.45

0.37

0

0

0

0

0

0

0

0

0.80

0.67

0

0

0

0

0

0

0

0

1.40

1.2

2

2

4

20

8

5

13

65

2.50

2.0

8

9

17

85

10

10

20

100

 

 

 

 

 

 

 

 

 

 

Validity criteria fulfilled:
yes
Conclusions:
The test item 48h-EC50 was 1.1 mg/L (C.I: 0.97 – 1.2 mg/L) based on time weighted average concentrations.
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 under GLP. Following preliminary range finding test in the range of 0.025, 0.25 and 2.5 mg/L, twenty daphnids in two replicates of 10 were exposed to an aqueous solution of the test item at time-weighted average concentrations of: 0.024, 0.037, 0.064, 0.13, 0.19, 0.37, 0.67, 1.2 and 2.0 mg/L for 48 hours under static test conditions. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in reconstituted water and then removing any undissolved test item by filtration. This saturated solution was then further diluted, as necessary, to provide the remaining test groups. The number of immobilised Daphnia were recorded after 24 and 48 hours. Physicochemical measurements on water quality were made throughout the test including: temperature, pH and oxygen concentration. The temperature was maintained at 21°C and the pH was 8.0 to 8.4 and the dissolved oxygen concentrations were 91 to 94%ASV. The validity criteria of the test guideline were fulfilled. Analysis of the fresh test solutions at 0 hours showed measured test concentrations to range from 0.027 to 2.2 mg/L. Analysis of the old or expired test media at 48 hours showed a marked decline in measured values which ranged from 0.020 to 1.9 mg/L. On this basis the test item was not maintained at ±20% of the initial concentrations therefore the effect levels were estimated based on time weighted average concentrations. Under the conditions of this study, the 48h-EC50 was 1.1 (C.I: 0.97 – 1.2) mg/L based on TWA concentrations.

Description of key information

EC50 (invertebrates) = 1.1 mg/L (C.I: 0.97 – 1.2 mg/L) mg/L based time weighted average concentrations, 48hour-freshwater, OECD TG 202, 2002

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
1.1 mg/L

Additional information

Key study : OECD TG 202, 2002: 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 under GLP. Following preliminary range finding test in the range of 0.025, 0.25 and 2.5 mg/L, twenty daphnids in two replicates of 10 were exposed to an aqueous solution of the test item at time-weighted average concentrations of: 0.024, 0.037, 0.064, 0.13, 0.19, 0.37, 0.67, 1.2 and 2.0 mg/L for 48 hours under static test conditions. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in reconstituted water and then removing any undissolved test item by filtration. This saturated solution was then further diluted, as necessary, to provide the remaining test groups. The number of immobilised Daphnia were recorded after 24 and 48 hours. Physicochemical measurements on water quality were made throughout the test including: temperature, pH and oxygen concentration. The temperature was maintained at 21°C and the pH was 8.0 to 8.4 and the dissolved oxygen concentrations were 91 to 94%ASV. The validity criteria of the test guideline were fulfilled. Analysis of the fresh test solutions at 0 hours showed measured test concentrations to range from 0.027 to 2.2 mg/L. Analysis of the old or expired test media at 48 hours showed a marked decline in measured values which ranged from 0.020 to 1.9 mg/L. On this basis the test item was not maintained at ±20% of the initial concentrations therefore the effect levels were estimated based on time weighted average concentrations. Under the conditions of this study, the 48h-EC50 was 1.1 (C.I: 0.97 – 1.2) mg/L based on TWA concentrations.