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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

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Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Remarks:
Conducted according to guideline in effect at time of study conduct
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 22.82% solids in water

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
One sample plus a back-up sample from each test concentration, including the dilution water control, on day 0 before the test solutions were poured into the replicate test chambers, and from 2 of the 4 replicate chambers (replicates B and C) at test end (day 2). In addition to the neat test solution samples, one sample plus a back-up sample of test solutions diluted 1:3 test solution:methanol (5 mL test solution + 15 mL methanol) from each test concentration, including the dilution water control, on day 0 of the test before the test solutions were poured into the replicate test chambers and from the 2 replicate chambers (replicate B and C) at test end. All samples and back-up samples were stored refrigerated when not in use.

Test solutions

Vehicle:
no
Details on test solutions:
Test substance solutions were prepared by direct addition of test substance to dilution water, testing laboratory well water. The dilution water control and test solutions were prepared by adding the appropriate amount of test substance to dilution water in 1-L glass beakers (final volume 1 L) and stirring for approximately 2 hours. Based on visual observations, the dilution water control was clear with no colour and lacked precipitate at test start and through the duration of the test. The nominal 32.9 and 65.7 mg/L test concentrations were clear with no colour; the nominal 131 and 263 mg/L test concentrations were slightly cloudy; and the nominal 526 mg/L test solution was cloudy at test start. All test concentration solutions had undissolved test substance present and a surface film at test start.
Dilution water originated from the testing laboratory well, which is 480-feet deep and is cased and sealed to bedrock. The hardness of the well water is adjusted to approximately 100-140 mg/L as CaCO3 by the flow-proportioned addition of CaCl2. The well water is then aerated, passed through a green sand filter to remove iron, and filtered through 50-, 10-, and 3.0-μm cartridge filters to remove particulates. The water is heated or chilled as appropriate and distributed through aged polyvinyl chloride piping. Dilution water quality was acceptable based on OECD 202 guideline specifications, and no quantifiable concentrations of pollutants and pesticides were present in the most recent semiannual dilution water analysis.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
Daphnia magna were reared at the testing laboratory in 1000-mL glass beakers (10 per beaker at start of culture) and 250-mL glass beakers (1 per beaker at start of cultures) which contained 1000 mL and 200 mL, respectively, of dilution water, testing laboratory well water, held at approximately 20°C. Daphnids were fed daily, Monday to Friday, with 3 mL/L of a yeast, cereal leaves, and trout chow (YCT) mixture and the green alga, Pseudokirchneriella subcapitata, at a rate of approximately 62,500 cells/mL of culture media. The YCT mixture was standardized to 1700-2100 mg/L solids. The combination of YCT and alga is equivalent to approximately 0.1-0.2 mg total organic carbon per adult daphnid per feeding day. YCT is analysed periodically to assure that contaminants are not present at levels that would be expected to affect the scientific integrity of the study. Green algae are not routinely analysed for contaminants because they are cultured in a defined laboratory medium prepared using reagent grade chemicals and deionized water. Neonates used in this test were less than 24 hours old, were collected from 1000-mL glass beakers, and were collected from the 4th brood of 14-day old parent daphnids. Sickness, injury, and abnormalities were not seen and ephippia were not being produced by the parent daphnids. No adult immobility was seen in the culture used for testing during the 48-hour pretest period. Daphnia magna were identified by labels on the culture beakers and test chambers.

Study design

Test type:
static
Water media type:
freshwater
Total exposure duration:
48 h

Test conditions

Hardness:
EDTA hardness: 151 to 162 mg/L as CaCO3
Test temperature:
20.9°C (mean) with a range of 20.7 to 21.0°C
pH:
8.2 to 8.4
Dissolved oxygen:
8.6 to 8.8 mg/L
Nominal and measured concentrations:
Nominal concentrations: 0, 7.5, 15, 30, 60, and 120 mg total solids/L (equivalent to 0, 35.7, 65.9, 131, 263, and 526 mg/L test substance)
Mean, measured concentrations: 0, 4.92, 9.85, 16.4, 42.6, and 102 mg total solids/L
Mean, measured concentrations of total solids ranged from 56 to 85% of nominal total solids concentrations based on 22.82% content by analysis.
Concentrations were chosen based on the results from an initial screening test.
One major active component was used to determine concentrations of the test substance because it eluted as a well-resolved peak with a retention time of approximately 5.15 minutes and is considered representative of the total solids content.
Details on test conditions:
Test chambers were 250-mL glass beakers containing 200 mL of test solution (approximately 6.5-cm test solution depth). Four replicate test chambers were used per test concentration with 5 test organisms in each chamber (20 test organisms per concentration). The test chambers were covered with a glass plate during the test. Random numbers were used to assign the position of test concentrations in the waterbath. A sign was placed above the waterbath identifying the definitive test by its unique study description, which included the work request and service code. The individual test chambers were labelled with total solids concentration and replicate. A recirculating waterbath was used to maintain mean temperature in the test chambers during the test at approximately 20°C. In addition, a continuously-recording thermometer was used to check for temperature variation in the waterbath. A photoperiod of 16 hours light and 8 hours darkness was employed, which included 30 minutes of transitional light preceding and following the 16-hour light interval. Dissolved oxygen concentration, pH, and temperature were measured in 2 replicates of the dilution water control and test substance concentrations. These measurements were taken at test start, at total immobility in a test concentration (if necessary), and at test end. Total alkalinity, EDTA hardness, and conductivity were measured in the dilution water control and highest test substance concentration at test start. Test solutions were not aerated during the test. Test solutions were disposed of in an appropriate manner at test end. Total solids concentrations of the dilution water control and test substance concentrations were verified by chemical analysis in a sample collected from the mixing vessel at test start and in 2 replicates at test end.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 102 other: mg total solids /L
Nominal / measured:
meas. (arithm. mean)
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 526 mg/L
Nominal / measured:
nominal
Basis for effect:
mobility
Details on results:
Exposure of test organisms to nominal test substance concentrations of 32.9, 65.7, 131, 263, and 526 mg/L, equivalent to mean, measured total solids concentrations of 4.92, 9.85, 16.4, 42.6, and 102 mg total solids/L resulted in 5, 15, 20, 10, and 5% immobility, respectively, at the end of 48 hours. Sublethal effects included lethargy, observed in the mean, measured 16.4 (1 of 16) and 42.6 (3 of 18) test solutions at 48 hours. No immobility or sublethal effects were seen in the dilution water control. The 48-hour EC50 values were not calculated because mobility was greater than 50% in all test solutions, and are reported as greater than the high test concentration.
During the test, the dilution water control was clear with no colour and lacked precipitate. Based on visual observations, the nominal 32.9 and 65.7 mg/L test concentrations were clear with no colour; the nominal 131 and 263 mg/L test concentrations were slightly cloudy; and the nominal 526 mg/L test solution was cloudy at 24 and 48 hours. Undissolved test substance was observed settled on the bottom of the test chambers in the nominal 131, 263, and 526 mg/L test solutions at 24 hours and additionally in the nominal 65.7 mg/L test solution at 48 hours.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
48-hour EC50, based on mean, measured concentrations and immobility > 102 mg total solids /L.
48-hour EC50, based on nominal concentrations > 526 mg/L .
Executive summary:

The acute toxicity of the test substance to unfed Daphnia magna neonates, less than 24 hours old at test start, was determined in an unaerated, 48-hour, static test. The study was conducted with 5 concentrations of test substance and a dilution water control at a mean temperature of 20.9°C (range of 20.7-21.0°C). Four replicates with 5 daphnids per replicate were used per test substance concentration or dilution water control. Mean, measured concentrations of total solids ranged from 56 to 85% of nominal total solids concentrations based on 22.82% content by analysis. During the test, the dilution water control was clear with no colour and lacked precipitate. Based on visual observations, the nominal 32.9 and 65.7 mg/L test concentrations were clear with no colour; the nominal 131 and 263 mg/L test concentrations were slightly cloudy; and the nominal 526 mg/L test solution was cloudy at 24 and 48 hours. Undissolved test substance was observed settled on the bottom of the test chambers in the nominal 131, 263, and 526 mg/L test solutions at 24 hours and additionally in the nominal 65.7 mg/L test solution at 48 hours. All water quality parameters were within acceptable limits during the exposure.

Exposure of test organisms to nominal test substance concentrations of 32.9, 65.7, 131, 263, and 526 mg/L, equivalent to mean, measured total solids concentrations of 4.92, 9.85, 16.4, 42.6, and 102 mg total solids/L resulted in 5, 15, 20, 10, and 5% immobility, respectively, at the end of 48 hours. Sublethal effects included lethargy, observed in the mean, measured 16.4 (1 of 16) and 42.6 (3 of 18) test solutions at 48 hours. No immobility or sublethal effects were seen in the dilution water control. The 48-hour EC50 values, based on immobility and nominal test substance concentrations and mean, measured concentrations of total solids, were greater than 526 mg/L and greater than 102 mg total solids/L, respectively.