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Ecotoxicological information

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:
11 - 13 Mar 2009
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
other: Daphnia sp., Acute immobilization Test stipulated in the "Testing Methods for New Chemical substances" (21/11/2003, No. 1121002, Pharmaceutical and Food Safety Bureau, MHLW; 13/11/2003, No. 2, Manufacturing Industries Bureau, METI No. 031121002
GLP compliance:
Analytical monitoring:
Details on sampling:
- Concentrations: All test concentrations were measured at test start, before and after medium renewal and at test end.
- Sampling method: Test samples were taken from the middle layer of the test vessels.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test sample and dilution water were mixed and stirred by magnetic stirrer for about 48 hours under about 20 °C to prepare each test concentration (loading rate). The prepared solutions were settled for one hour after cease of stirring, and then the solution taken out from the middle layer of the settled solution was filtrated with a 0.4 µm glass fiber filter by suction. After the filtration, the filtrate was stirred for about 30 minutes under about 20 °C in order to recover decreased dissolved oxygen concentration by suction and then used as test solution. The prepared test solution was divided into each test vessel.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Water flea
- Source: Laboratory breed. Daphnia magna (Clone A) originally came from the University of Sheffield, UK.
- Age at study initiation: < 24 h
- Method of breeding: The laboratory culture of the parents was held under the same conditions as in the test.
- Feeding during test: None

- Acclimation period: Not stated
- Acclimation conditions: The parents to obtain young daphnids were bred in the same quality of water (dechlorinated tap water), water temperature (20 ± 1 °C), and photoperiod (16 h light/8 h dark) as used in the test.
- Type and amount of food: ChIorella vulgaris of 0.1 to 0.2 mg C/day and daphnia was fed to the parents
- Feeding frequency: once daily
- Health during acclimation: Parents used for the test were from the same lot and bred more than 14 days, no mortality occurred.

Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
42 mg/L as CaCO3
Test temperature:
20.0 - 20.2 °C
7.3 - 7.8
Dissolved oxygen:
8.4 - 8.9 mg/L
Nominal and measured concentrations:
Nominal: 0, 35, 46, 59, 77, 100 mg/L
Measured: < LOQ, 3.7, 5.3, 6.4, 8.6, 9.5 mg/L (geometric mean)
Details on test conditions:
- Test vessel: beakers
- Type: closed
- Material, fill volume: glass, 100 mL
- Aeration: None
- Renewal rate of test solution: every 24 h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

- Source/preparation of dilution water: Dechlorinated and aerated tap water
- Total organic carbon: 0.4 mg/L
- Particulate matter: < 1 mg/L
- Metals (mg/L): B 0.08, Al 0.042, Na 14.9, K 1.8, Ca 11.5, Mg 3.3, the other metals analysed were in the range from < 0.001 to < 0.01
- Pesticides: < 0.0001 to 0.0005 mg/L
- Chlorine: < 0.02 mg/L
- Alkalinity: 29 mg/L
- Ca/Mg ratio: 3.5:1
- Conductivity: 17.1 mS/m
- Culture medium different from test medium: no
- Intervals of water quality measurement: Dissolved oxygen, pH and temperature were measured at test start, before and after renewal and at test end. Appearance of the test solutions was observed at test start and before renewal.

- Photoperiod: 16 h light/8 h dark, artificial light of white fluorescent lamp

EFFECT PARAMETERS MEASURED (with observation intervals if applicable)
- Immobility and symptoms were observed 24 and 48 h after test start.

- Spacing factor for test concentrations: 1.3
- Range finding study
- Test concentrations: Nominal: 5.0, 30, 60 and 100 mg/L; the 5.0 and 60 mg/L test levels were analytically verified; at test start: 0.56 and 5.3 mg/L, after 48 h: < LOQ, 3.9 mg/L
- Results used to determine the conditions for the definitive study: 30% immobilisation and reduced activity at nominal 100 mg/L; since it was expected that the measured concentrations of the test item in the test solutions decrease over 48 h, the definitive study was conducted under semi-static conditions with renewal of the test medium after 24 h.
Reference substance (positive control):
Potassium dichromate
Key result
48 h
Dose descriptor:
Effect conc.:
9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: filtered test solution
Basis for effect:
Remarks on result:
other: 95% CL = 8.2 to 11 mg/L
Details on results:
- Behavioural abnormalities: Lethargic immobilisation, reduced activity
- Other biological observations: Immobility recorded at 24 and 48 hours
- Mortality of control: none
- Other adverse effects control: none
- Appearance of test solutions: Clear and colourless throughout the test period
- Measured test item concentrations: 3.8 to 11 mg/L at test start and after renewal, 3.0 to 9.5 mg/L before renewal and at test end.
Results with reference substance (positive control):
- Results with reference substance valid? Yes.
- Test date: 02 Feb - 04 Feb 2009
- EC50 (48 h): 0.28 mg/L (range of laboratory data: 0.13 - 0.36 mg/L)
Reported statistics and error estimates:
The EC50 (48 h) value was calculated by Moving average analysis. The EC50 (24 h) was estimated using a program running on MS Excel, developed by our laboratory.

Table 1: Cumulative Immobilisation of D. magna

Measured concentration (mg/L)

Immobility (%)

24 h

48 h



















Validity criteria fulfilled:
The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL50 value of 88 mg/L .
Executive summary:

This study was performed according to the following test methods and guidance document. (1) Daphnia sp., Acute Immobilization Test stipulated in the "Testing Methods for New Chemical Substances" (November 21, 2003; No. 1121002, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; November 13, 2003, No.2, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 031121002, Environmental Policy Bureau, Ministry of the Environment) (2) OECD Guidelines for Testing of Chemicals, Section 2 : Effects on Biotic Systems, 202 "Daphnia sp., Acute Immobilisation Test (Guideline 202, April 13, 2004)" (3) OECD Guidance Document 23 "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures" (September 2000) .

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-hour EL50 value of 88 mg/L and 48-hour EC50 (Median Effective Concentration) 9.0 mg/L (95% confidence interval: 8.2 to 11 mg/L)

Description of key information

EC50 (48 h) = 9 mg/L for Daphnia magna (OECD 202)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
9 mg/L

Additional information

For short-term toxicity to aquatic invertebrates, one test with Daphnia magna according to OECD 202 and GLP is available (key study: Showa Denko, 2009). Daphnia magna was exposed to analytically verified concentrations of the test item under semi-static conditions. After 48 h of exposure, the cumulative mortality was 60% at 9.5 mg/L. The EC50 (48 h) was determined to be 9.0 mg/L (measured).