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Short-term toxicity to aquatic invertebrates

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short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 September 2003 to 1 October 2003
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
according to guideline
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
Analytical monitoring:
Details on sampling:
Samples of the control and test substance exposure solutions were analysed at the start and end of the test to verify the test substance concentration. The samples were collected from approximately mid-depth of the test vessel using a spigot-valve assembly. Aliquots of the solutions were stored in a 4 °C refrigerator after sample preparation. These solutions were clear and colourless in appearance.
Details on test solutions:
Exposure solutions containing the test substance at nominal concentrations of 1 mg/L were tested. The exposure solutions were prepared by direct addition of a test substance stock solution to the test media. The stock solution was prepared to contain the test substance at a concentration of 10 mg/mL be weighing approximately 0.25 g of test substance into a 25 mL volumetric flask that was brought to volume with a carrier solvent, N,N-dimethyl formamide (DMF). The stock solution was sonicated for approximately 2-3 minutes and observed. Undissolved test substance was not observed. The 1 mg/L exposure solutions were prepared by pipetting 2 mL of stock solution into separate test vessels containing 20 litres of laboratory dilution water. Dilution water and carrier solvent controls were also prepared. Carrier solvent controls were prepared by pipetting 2 mL of solvent into test vessels containing 20 litres of laboratory dilution water. The solutions were stirred using stir plates and Teflon-coated stir bars for 10 minutes.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Strain: Straus
- Source: in house cultures
- Age at study initiation: < 24 hours
- Collection: The brood stock was cultured under test conditions in the water used for preparing the test substance solutions. Approximately 24 hours before the test an ample number of gravid adult daphnids were transferred into 20 cm diameter bowls containing laboratory culture water and fed. The neonates produced in the following 24 hour period were used as the test organisms. The neonates were collected by pipette and transferred directly into the test vessels.

- Acclimation period: 24 hours
Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
Total hardness: 123.182 mg CaCO3/L
Test temperature:
20 ± 1 ºC
8.4 to 8.6
Dissolved oxygen:
8.5 to 8.8 mg/L
Nominal and measured concentrations:
1 mg/L (nominal)
0.92 mg/L (measured)
Details on test conditions:
- Test vessel: 250 mL Pyrex® glass beakers containing 200 mL of test solution with approximately 80 cm3 of headspace.
- Type : closed
- Aeration: none
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2

- Source/preparation of dilution water: Water is pumped from Lake Ontario and treated at a water treatment facility and stored in an underground reservoir. The water is subsequently pumped into the laboratory where it passes through a set of polypropylene filters, a series of activated carbon filters and through another set of polypropylene filters and is finally treated with sodium thiosulfate to eliminate trace levels of residual chlorine.
- Total organic carbon: 6.067 mg/L
- Particulate matter: 172.5 mg/L
- Metals: Aluminum 0.124 mg/L. Arsenic 0.004 mg/L, Barium 0.05 mg/L, Boron 0.022 mg/L, Cadmium 0.003 mg/L, Chromium 0.005 mg/L, Cobalt 0.007 mg/L, Copper 0.006 mg/L, Iron 0.023 mg/L, Lead 0.005 mg/L, Manganese 0.002 mg/L, Mercury 0.002 mg/L, Nickel 0.007 mg/L, Silver 0.003 mg/L and Zinc 0.011 mg/L
- Pesticides: 4,4'-DDT 0.013 µg/L, Aldrin 0.007 µg/L, Alpha-BHC 0.007 µg/L, Chlordane 0.04 µg/L, Diazinon 0.132 µg/L, Dieldrin 0.024 µg/L, Endrin 0.011 µg/L, Gamma-BHC/Lindane 0.009 µg/L, Malathion 0.094 µg/L, Mirex 0.04 µg/L, Parathion 0.099 µg/L
- Chlorine: 23.432 mg/L
- Alkalinity: 90.505 mg CaCO3/L
- Conductivity: 298.636 µmhos/cm
- Intervals of water quality measurement: Most recent sample collected: June 9, 2003
Reference substance (positive control):
Key result
48 h
Dose descriptor:
Effect conc.:
> 0.92 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: immobility and behavioural effects
Remarks on result:
other: Observed to be greater than the limit of solubility for the test substance
Details on results:
- Behavioural abnormalities: none observed
- Mortality of control: none

The highest tested concentration resulting in no immobility was determined to be 0.92 mg/L. The lowest tested concentration causing 100 % immobility was not determined.

Test validity
The following criteria for a valid test were met during the study:
1. The control mortality was not greater than 10 %
2. The dissolved oxygen level did not fall below 2 mg/L.
3. No abnormal occurrences that might have influenced the outcome of the test were noted.
4. Test daphnids in the control groups were not trapped at the surface of the water.
5. The pH did not vary by more than one unit.
Reported statistics and error estimates:
Statistical analysis of the data was not performed due to lack of observable effects. The 24 and 48 h EC50 values were estimated to be greater than the highest tested concentration of 0.92 mg/L.
Validity criteria fulfilled:
Under the conditions of the test, the 48 hour EC50 value of the testt substance to Daphnia is greater than the limit of solubility (0.92 mg/L).
Executive summary:

In a GLP compliant study conducted in line with OECD Guideline 202 and EU Method C.2, the acute toxicity of butanoic acid, 4-[[4-[7-chloro-6-(1,1-dimethylethyl)-3H-pyrazolo[1,5-b][1,2,4]triazol-2-yl]phenyl]amino]-4-oxo, tetradecyl ester to Daphnia was investigated. The 48 hour EC50 was determined to be greater than the limit of solubility of the substance (0.92 mg/L).

Description of key information

Key study:- Light (2003b) 'An acute aquatic effects limit test with the Daphnid, Daphnia magna' was conducted in line with OECD Guideline 202 and EU Method C.2. The 48 hour EC50 of the test substance was determined to be greater than the limit of solubility for the substance (0.92 mg/L).

Key value for chemical safety assessment

Additional information

The key study (Light, 2003b) was performed in compliance with GLP and to OECD Guideline 202 and EU Method C.2 with a sufficient level of detail to assess the quality of the presented data. The study was performed to a good standard in line with accepted, standardised guidelines and was assigned a reliability score of 1 using the principles for assessing data quality as set out in Klimisch et al. (1997). The 48 hour EC50 of the test substance was determined to be greater than the limit of solubility for the substance (0.92 mg/L).