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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
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)
according to guideline
ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))
GLP compliance:
Specific details on test material used for the study:
- Name of test material (as cited in study report): XTJ 568
- Physical state: Clear colourless liquid
- Analytical purity: 97.8%
- Lot/batch No.: 8191-34
- Expiration date of the lot/batch: 01 January 2004
- Stability under test conditions: stability in water: at least 4 hours (determined at NOTOX)
- Storage condition of test material: at room temperature in the dark
- Other: specific gravity: > 0.94 g/cm3
Analytical monitoring:
Details on sampling:
Range finding test: no sampling for determination of actual test concentrations
Pre-test: no sampling for determination of actual test concentrations
Final test:
- Concentrations sampled: control, 10, 32 and 100 mg/L
- Sampling: duplicate 10-mL samples from each sampled concentration
- Sampling frequency: at t = 0 h and t = 48 h
- Sampling storage: not applicable, immediate analysis
Details on test solutions:
- Preparation of test solutions started with a stock solution of 100 mg/L in test medium, applying a 10-minute stirring period.
- Preparation of lower test concentrations through subsequent dilutions of the stock in test medium.
- Since pH of stock solution was 9.3-9.7 (outside optimum range for testing with daphnids (6.0-8.5)), pH was adjusted to 8.0 with 1 N HCl in the final test to exclude possible pH related effects.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: water flea
- Strain: Straus, 1820
- Source: In-house laboratory culture with a known history
- Age at study initiation: < 24 h (from parental daphnids > 2 weeks old and < 4 weeks old)
- Method of breeding: start of each batch with newborn daphnids < 3 days old, by placing about 250 of them into 10 L of M7 (Elendt, 1990) in an all-glass culture vessel. Cultures were kept at 18-22°C, fed daily with algal suspensions, and after 7 d half of the medium was replaced twice a week. Maximum age of cultures = 4 weeks.
- Feeding during test: no
Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
250 mg CaCO3/L
Test temperature:
20.3-21.2°C (final test)
7.9-8.1 (final test)
Dissolved oxygen:
8.7-9.3 mg/L
Not applicable
Nominal and measured concentrations:
Nominal concentrations: Control + 10, 18, 32, 56 and 100 mg/L (final test)
Measured concentrations: 10 mg/L: ranged from 10.0 to 10.2; 32 mg/L: ranged from 32.1 to 33.5 mg/L; 100 mg/L: ranged from 103 to 106 mg/L; overall 100-106% of nominal concentrations
Details on test conditions:
- Test vessel: 100 mL, all-glass, fill-volume 80 mL
- Aeration: no
- Static test system
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

- ISO-medium (prepared using tap water purified by reverse osmosis (milli-RO):
CaCl2.2H2O: 293.8 mg/L
MgSO4.7H2O: 123.3 mg/L
NaHCO3: 64.8 mg/L
KCl: 5.8 mg/L
- Culture medium different from test medium: yes (M7 for cultures)

- Adjustment of pH: before the start of testing adjustment to pH 8.0 with 1 N HCl, no adjustment during test
- Photoperiod: 16L:8D
- Light intensity: no data

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Immobility (including mortality) at 24 and 48 h

- Range finding study: Control, 0.1-1.0-10-100 mg/L
- Pre-test: Control, 10, 100 mg/L
- Final test: Control, 10-18-32-56-100 mg/L
- Range finding study was conducted without pH adjustment
- Pre-test and final test: adjustment of pH before start of testing
- Range finding study and pre-test indicated that EC50 was between 10 and 100 mg/L
Reference substance (positive control):
potassium dichromate
Key result
48 h
Dose descriptor:
Effect conc.:
88 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: 95% CL = 73-110 mg/L
Details on results:
- In the range finding test (without pH adjustment) and the pre-test (with pH adjustment), some precipitate was observed in the 100 mg/L test vessels. This may have affected mortality (e.g., mechanical effect), because mortality at 100 mg/L in the final test, where no precipitate was observed, was slightly lower, i.e., 65% after 48 h versus 100 and 95% after 48 h in the range finding and pre-test, respectively.
- 24-h EC50 = 110 mg/L
- 48-h NOEC = 32 mg/L
Results with reference substance (positive control):
The 24h-EC50 (based on the initial concentration) of potassium dichromate was within the range 0.6 mg/L to 1.7 mg/L (0.93 mg/L).
Reported statistics and error estimates:
The EC50 was calculated from the probits of the percentages of affected daphnids and the logarithms of the corresponding test substance concentrations using the maximum likelihood estimation method (Finney, D.J., 1971: Probit analysis, Cambridge University Press, Cambridge, U.K., 3rd edition).
Validity criteria fulfilled:
Under the conditions of the present study, the substance did not induce acute immobilisation of Daphnia magna at 32 mg/L after 48 hours of exposure (NOEC). The 48-h EC50 was 88 mg/L based on nominal concentrations (95% confidence interval between 73 and 110 mg/L). Measured concentrations were between 100 and 106% of nominal concentrations. The test can be considered reliable (Klimisch 1).

Description of key information

The key study is an acute Daphnia magna study by Bouwman (2004) performed at NOTOX according to ISO Guideline 6341 15, EU Method C.2 and OECD Guideline 202. This Klimisch 1 study yielded a 48-h EC50 of 88 mg/L, which can be used for PNEC derivation.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
88 mg/L

Additional information

A single acute Daphnia magna study is available (Bouwman, 2004). Under the conditions of this study, which was conducted according to standard guidelines (e.g., OECD 202), the substance did not induce acute immobilisation at 32 mg/L after 48 hours of exposure (NOEC). The 24-h EC50 approximated the maximum concentration tested and was estimated at 110 mg/L based on nominal exposure concentrations (95% confidence interval between 85 and 180 mg/L). The 48-h EC50 was 88 mg/L based on nominal concentrations (95% confidence interval between 73 and 110 mg/L). Measured concentrations were between 100 and 106% of nominal concentrations. This study is considered reliable (Klimisch 1) and is considered the key study for this endpoint.