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Toxicity to aquatic algae and cyanobacteria

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toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP compliant OECD 201 study. No deviation from guidelines, except that dissolved oxygen was not monitored. This study is therefore classified as Klimisch 2b
according to guideline
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
Analytical monitoring:
Details on sampling:
2.0 mL of the test fluid was sampled from the test containers of the 3 replicates of each concentration group at the start of exposure (0 hr) as well as at conclusion (72h later). At time of start, a 0.75 mL aliquot of each sample was directly analysed while at the conclusion, Selenastrum Capricornutum was centrifuged (3000rpm, 10 minutes), and 0.75 mL of the supernatant was used for analysis.
An equivalent volume of acetonitrile was added to each analysis specimen, and after mixing, analyzed by HPLC. The concentration of the test substance in each test fluid was quantitated from the ratio with the peak surface area of the standard solution.
Details on test solutions:
40mg of the test substance was weighed out, and after dissolving in dimethyl sulfoxide 240 mg and mixed after adding 80 mg of a dispersal agent (HCO-30). This was diluted with the culture medium to a set volume of 100 mL to reach a n-nitrosodiphenylamine concentration of 400 mg/L. A 3200 mg/L adjuvant fluid (dimethyl sulfoxide 2400 mg/L, HCO-30 800 mg/L) not containing the test substance was also prepared at the same time.
100 mL of culture medium at concentrations of 0.520, 0.800, 1.20, 1.80, 2.60, 4.00 mg/L were prepared in triplicate. Adjuvant stock solution was added to the different culture medium so that adjuvant concentration was constant (32 mg/L) for each concentration (reaching the same total amount of liquid in each replicate). The control group was prepared using the culture medium only and the adjuvant control group was prepared including the adjuvant only (adjuvant concentration 32 mg/L).
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Thespecies used was Selenastrum Capricornutum, a type of single-cell green algae. This variety was obtained from the American Type Culture Collection on June 20, 1996, and the ATCC22662 strain has been aseptically and successively cultured at this laboratory. Prior to the start of the experiment, the Selenastrum Capricornutum supplied to the test was cultured for 3 days before the start of exposure under the same conditions under the test conditions.
Test type:
Water media type:
Limit test:
Total exposure duration:
72 h
Post exposure observation period:
Not mentioned
Test temperature:
22.3-23.°3°C (min-max)
7.8-8.5 (min-max)
Dissolved oxygen:
Not measured
Nominal and measured concentrations:
Nominal concentrations: 0.52 ; 0.8 ; 1.2 ; 1.8 ; 2.6 ; 4 mg/L (+ control + solvent control)
Measured concentrations at start: 0.528 ; 0.829 ; 1.26 ; 1.99 ; 2.57 ; 4.51 mg/L (did not deviate by more than 20% from the nominal concentrations)
Measured concentrations at end: 0.015 ; 0.1 ; 0.265 ; 0.637 ; 1.13 ; 2.11 mg/L

HPLC analysis demonstrates that the loss of product within 3 days are the results of n-nitrosodiphenylamine degradation into two products. Since it is attributed to the susceptibility of the molecule to photolysis, degradation is considered as a normal process that will also occur in the wild. Nominal concentrations are therefore used for calculation.
Details on test conditions:
- Exposure Form: Static method, oscillating culture (100rpm)
- Exposure Period: 72 hours
- Test fluid volume: 100 mL (OECD culture medium)
- Number of replicates: 3 containers per concentration, including control and solvent control
- Initial Cell Concentration: 1×104 cells/mL
- Testing temperature: 23±1°C
- Illumination: Continuous lighting at 4000~5000 lux (Variation within ±20%, near the flask liquid surface)
- Medium prepared as indicated in OECD test guidelines for both before culturing and testing.

Test Container, Algae Culture Testing Equipment and Devices, etc.
- Testing Container: 30 mL glass triangular flask (with breathable silicone plug)
- Algae Culturing and Testing Equipment: Model AGP-150RL made by Ito Corporation
- Optical Microscope: Model FHT Made by Olympus Optical
- Particle Counting Equipment: Model CDA-500 Toa Medical Electronics
- Electrolytic Fluid Used by the Particle Counter: Cellpak Made by Toa Medical Electronics
- pH Meter: Model 900 Tabletop pH meter/Ion meter made by Orion
- Thermometer: Tasco Japan Co., Ltd Model TNA-120
- Illumination Meter: Model IM-2D Made by Topcon
Reference substance (positive control):
Potassium dichromate
72 h
Dose descriptor:
Effect conc.:
> 4 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
72 h
Dose descriptor:
Effect conc.:
2.2 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Specific growth rate of algae exposed to n-nitrosodiphenylamine up to 4.2 mg/L for 72h was not inhibited by more than about 30%. EC10 was graphically determined at 2.4 mg/L.
Results with reference substance (positive control):
The 50% 72-hour growth inhibition concentration (EbC50) with the standard substance (potassium dichromate, reagent grade) was 0.41 mg/L. (Linear Regression Analysis).
Reported statistics and error estimates:
The mean growth rate (µ) was calculated by the following formula from the mean cell concentration by an exponentially growing culture.

µ = ln (N0h/N72h)/3

N0h: Actually-measured cell concentration at 0h (cells/mL)
N72h: Actually-measured cell concentration at 72h (cells/mL)

The mean percentage drop in each concentration plot was calculated by the following formula from the mean specific growth rate (µ):

I(%) = (I0h-I72h)/I0h*100

Since ECx were not calculated in the report by Mitsubishi, ARKEMA performed the calculations from the raw data (see attached file "ARKEMA calculations from raw data of 6B682G Mitsubishi report.pdf"
Validity criteria fulfilled:
Selenastrum capricornutum were exposed to graded series of n-nitrosodiphenylamine for 72 hours. Inhibitions of specific growth rates (SGR) were calculated against the SGR of the solvent control group. EC50 was above 4 mg/L and EC10 was estimated at 2.2 mg/L
Executive summary:

Selenastrum capricornutum were exposed to graded series of n-nitrosodiphenylamine (dissolved in DMSO+HCO-30) for 72 hours, with both control and solvent control groups. Nominal concentrations were used for the ECx calculations of specific growth rate (SGR) inhibition over the 72-h period. SGR from the control group and the solvent control group did not differ. SGR inhibition in the tested concentrations were calculated against the SGR of the solvent control group. SGR inhibition were then plotted against the nepierian logarithm of the tested concentrations. EC50 was above 4 mg/L and EC10 was estimated at 2.2 mg/L

Description of key information

EC50 was superior to the highest concentration tested (4 mg/L). EC10 was 2.2 mg/L

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
2.2 mg/L

Additional information