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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:
1 (reliable without restriction)
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
Analytical monitoring:
Details on sampling:
0.75 mL of each test fluid was sampled into measurement vials at the start of exposure and the conclusion of exposure for all concentration groups (however, 1 test container each), and analysed by HPLC after having been spiked with an equivalent volume of acetonitrile. The test material concentration of each test fluid was quantitated from the ratio to the peak surface area of the test solution.
Details on test solutions:
150 mg of the test substance was weighed out and after dissolved into 150 mg DMSO and 350 mg HCO-30. This was diluted with the dilution water to a set volume of 1000 mL (stock solution with a test substance concentration of 150 mg/L). At the same time, an adjuvant stock solution not containing the test substance (DMSO 150 mg/L, HCO-30 350 mg/L) was prepared.
The dilution water was placed in a 500 mL flask, and after adding the tested substance stock solution according to each concentration, the adjuvant stock solution was added to a set rhe same adjuvant concentration (100 mg/L) in all flasks.
A control without substance and without adjuvant, and an adjuvant control group (100 mg/L) were also tested. The control group used only the dilution water.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test used immature Daphnia Magna within 24 hours after hatching. This variety was obtained from the National Institute of Environmental Studies on July 18, 1995, and has been successively cultured at this laboratory.

Daphnia Magna Culturing Method to Obtain Tested Larva
Female adults holding larva that appeared healthy and sufficiently large on gross examination were selected in the succession, transferred to a separately prepared beaker, and the next day the larvae produced were separated to a different beaker. These larvae (born January 14, 1997) were treated as the parents of the tested Daphnia Magna, and where raised under the following conditions. Once reaching maturity and producing larvae, these larvae were removed 2 times per week. At the 3rd week, female adults with larvae in their brood chambers were selected from a batch that had a mortality rate of 10% over the previous 2 weeks and that had not produced dormant eggs or males, and the larvae produced the next day (within 24 hours) were used in the test (Exposure started on February 4, 1997).

- Culture Concentration: When larval 35 or 70/3L culture water
After maturity 36/3L culture water
- Water Temperature: 20±1°C
- Illumination: Indoor light, 16L:8D
- Feed: Chlorella vulgaris
- Feed volume: 0.1-0.2 mg C (organic carbon content) per day per Daphnia magna
Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
Post exposure observation period:
65 mg/L equivalent CaCO3
Test temperature:
20°C (19.9-20.3 min-max)
7.7 - 8.1 (min-max)
Dissolved oxygen:
6.7-8.2 (min-max)
10 mg/L sodium
Nominal and measured concentrations:
Nominal: 3 ; 5.4 ; 9.3 ; 18 ; 30 mg/L (+ control + solvent control)
Measured concentrations (geometrical mean): 2.98 ; 5.29 ; 9.05 ; 18.2 ; 26.1 mg/L

Measured concentrations did not deviate by more than 20% from the nominal concentrations. Therefore, nominal concentrations were used for the data analysis.
Details on test conditions:
- Exposure Form: Static method.
- Exposure Period: 48 hours
- Test fluid volume: 100 mL (1 L glass beaker)
- Number of replicates: 4 per concentration, control or solvent control
- Number of test organisms: 20 per concentration (5 per replicate)
- Test water temperature: 20±1°C
- Illumination: Indoor light, 16 hours lit/8 hours dark
- Feeding: No feeding

Dilution Water
De-chlorinated water (Yokohama municipal tap water processed with activated charcoal, removing residual chlorine, etc., and thereafter fully aerated) was used. The hardness of the diluted water was 65mg/L (CaCO3 conversion), and the pH was 8.2.
Reference substance (positive control):
potassium dichromate
48 h
Dose descriptor:
Effect conc.:
10.1 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: CI = 8.7-11.9
Details on results:
The swimming impediment rate at 24 hours after start of exposure was 0% in the control group, adjuvant control plot, as well as the 3.00-9.30 mg/L groups, 95% in the 18.0 mg/L group, and 100% in the 30.0 mg/L group.
The swimming impediment rate at 48 hours after start of exposure was 0% in the control group, adjuvant control group, as well as the 3.00 mg/L group, 5% in the 5.40 mg/L group, 30% in the 9.30 mg/L group, and 100% in the 18.0 and 30.0 mg/L groups.
In addition, no Daphnia Magna were observed floating in the water surface in the control group.
The Median Immobilization Concentration (EC50) at 24 hours exposure was 13.5 mg/L (Binomial method), and the 95% reliability limits were 9.30-18.0 mg/L.
The Median Immobilization Concentration (EC50) at 48 hours exposure was 10.1 mg/L (Probit method), and the 95% reliability limits were 8.73-11.9 mg/L.
The No Observable Effect Concentration (NOEC) at 24 hours of exposure was determined to be 9.30 mg/L, and the NOECi at 48 hours of exposure 3.00 mg/L.
In addition, the 100% impediment concentration was 30 mg/L at 24 hours, and 18.0 mg/L at 48 hours.
Results with reference substance (positive control):
The 48-hour Median Immobilization Concentration (EC50) with the standard substance (potassium dichromate, reagent grade) was 0.23 mg/L.
Reported statistics and error estimates:
The swimming impediment rate (%) was derived from the number of swimming-impeded Daphnia magna in each concentration plot and the number of test organisms (20), and the Median Immobilization Concentration (EiC50) was calculated by the Probit method. The 95% reliability limit was also calculated at the same time.
In addition, the maximum concentration at which the Daphnia magna swimming was not impeded was treated as the No Observed Effect Concentration (NOEC), and the minimum concentration at which 100% of the Daphnia magna were swimming-impaired was treated as the 100% impediment minimum concentration.

Cumulative number of immobilized Daphnia magna after 48 hours exposure:

Control: 0

Solvent control: 0

3 mg/L: 0

5.4 mg/L: 1 (5%)

9.3 mg/L: 6 (30%)

18 mg/L: 20 (100%)

30 mg/L: 20 (100%)

Validity criteria fulfilled:
Daphnia magna were exposed to a graded series of nitrosodiphenylamine concentrations for 48 hours. The LC50 was 10.1 mg/L.
Executive summary:

Daphnia magna were exposed in quadruplicate to a graded series of n-nitrosodiphenylamine concentrations for 48 hours, according to OECD 202 guidelines (1984). Nominal concentrations were 3; 5.4 ; 9.3 ; 18 and 28 mg/L. Measured concentrations were 2.98 ; 5.29 ; 9.05 ; 18.2 ; 26.1 mg/L. They did not deviate by more than 20% from the nominal concentrations. Therefore, nominal concentrations were used for data analysis. Replicates were daily checked for D. magna immobilization. After 48 hours, estimated LC50 was 10.1 mg/L.

Description of key information

LC50 of Daphnia magna exposed to graded concentrations of n-nitrosodiphenylamine was 10.1 mg/L (MITI, 1997). LeBlanc (1980) study was disregarded since it contains many flaws that do not make the results relevant. LC50 found by LeBlanc was 7.8 mg/L, which is close to the result of MITI.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
10.1 mg/L

Additional information