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EC number: 257-913-4 | CAS number: 52434-90-9
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- Nanomaterial specific surface area
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- Endpoint summary
- Stability
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- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
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- Biotransformation and kinetics
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- Irritation / corrosion
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Toxicity to aquatic algae and cyanobacteria
Administrative data
Link to relevant study record(s)
- Endpoint:
- toxicity to aquatic algae and cyanobacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 16 June 2022 to 30 June 2022
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
- GLP compliance:
- yes (incl. QA statement)
- Analytical monitoring:
- yes
- Details on sampling:
- The test concentrations along with the negative control and the vehicle control were analyzed for test item at the beginning and at the end of the test.
For analysis, duplicate composite samples were drawn from prepared test concentrations, and from the negative and the vehicle controls.
Analysis was conducted using a validated analytical method. The criteria for acceptance of the results of test concentration analysis were 80 to 120% of nominal concentration with ≤20% RSD of analyzed concentration. - Vehicle:
- yes
- Remarks:
- Acetone
- Details on test solutions:
- Test item stock of 1 mg/mL was prepared by mixing 1000 mg of test item initially mixed with 0.1 mL of acetone and made up the volume to 1000 mL using growth medium in a volumetric flask and sonicated for 30 minutes. Growth medium with 0.01% acetone was prepared and used for preparation of test concentrations. 0.7 mL of acetone was mixed and made up to 7 L using growth medium in a pre-marked glass container. To prepare test concentrations of 50, 80, 128, 204.8 and 327.7 mg/L, 50, 80, 128, 204.8 and 327.7 mL of test item stock was mixed and made up to 1 L in a volumetric flask using growth medium with 0.01% acetone, respectively.
1 L of growth medium containing 0.01% acetone was used as vehicle control (G2).
For negative control (G1), required volume of algal cell suspension was made up to 1 L using growth medium.
Algal cell suspension of 3.6 mL was added before final volume of 1 L was made up for each test concentration, negative and vehicle control to achieve an initial cell density of 1x10^4 cells/mL.
Each replicate was prepared by dispensing 150 mL of the previously prepared test solutions. - Test organisms (species):
- Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
- Details on test organisms:
- TEST ORGANISM
- Common name: Raphidocelis subcapitata (formerly Pseudokirchneriella subcapitata) ATCC® 22662. This algal species was used as the test system as it is a recommended algal species as per the guideline.
- Source: American Type Culture Collection, P.O. Box 1549, Manassas, VA 20108, USA
ACCLIMATION
- Culturing media and conditions: Algal cells were maintained at the test facility through sub-culturing once in 5 to 10 days from the continuous culture into fresh medium in a conical flask. These conical flasks were incubated between
21 and 25°C (±2°C) under a cool white fluorescent continuous illumination of 8000±1600 Lux. - Test type:
- static
- Water media type:
- freshwater
- Limit test:
- no
- Total exposure duration:
- 72 h
- Test temperature:
- 22.1 to 22.6 °C
- pH:
- 8.11 to 8.18
- Nominal and measured concentrations:
- Nominal concentrations: 50 mg/L, 80 mg/L, 128 mg/L, 204.8 mg/L and 327.7 mg/L
- Details on test conditions:
- TEST SYSTEM
- Test vessel: Conical flasks, closed using cotton.
- Initial cells density of inoculum at start of exposure: 240 x10^4/mL.
- No. of vessels per concentration (replicates): 3
- No. of vessels per negative control (replicates): 6
- No. of vessels per vehicle control (replicates): 6
GROWTH MEDIUM
- Standard medium used: Yes.
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Sterilized water.
- Culture medium: Growth medium (test medium) was algal medium as described in Annex 3 of the OECD test guideline 201 (OECD 2006).
OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the medium was 8.19 during definitive test. This medium was equilibrated with carbon dioxide by keeping it in laminar hood for two hours.
- Light intensity and quality: Cool white fluorescent continuous illumination of 8000±1600 Lux.
EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: The cell growth was measured at 24, 48 and 72 hours after the initiation of the test using a haemocytometer (microscopic observation).
- Other: The concentration-effect relationship was determined using two factors namely growth rate and yield at the end of the test (72 hours).
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.6
- Range finding study: A range finding test was conducted to select the test concentrations for the definitive test with a negative control, vehicle control and test item concentrations at 0.01, 0.1, 1, 10 and 100 mg/L. Each group had a single replicate.
- Results used to determine the conditions for the definitive study: During range finding study, the test item exhibited 0, 0, 0, 0 and 20.02 percent cell reduction at the tested concentrations of 0.01, 0.1, 1, 10 and 100 mg/L at 72 h. - Reference substance (positive control):
- yes
- Remarks:
- Potassium dichromate (K2Cr2O7)
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- 170.37 mg/L
- 95% CI:
- >= 157.468 - <= 184.327
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Duration:
- 72 h
- Dose descriptor:
- EC20
- Effect conc.:
- 108.119 mg/L
- 95% CI:
- >= 99.931 - <= 116.977
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Duration:
- 72 h
- Dose descriptor:
- EC10
- Effect conc.:
- 85.243 mg/L
- 95% CI:
- >= 78.788 - <= 92.227
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- 95.183 mg/L
- 95% CI:
- >= 86.498 - <= 104.739
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- yield
- Duration:
- 72 h
- Dose descriptor:
- EC20
- Effect conc.:
- 61.649 mg/L
- 95% CI:
- >= 56.024 - <= 67.838
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- yield
- Duration:
- 72 h
- Dose descriptor:
- EC10
- Effect conc.:
- 49.126 mg/L
- 95% CI:
- >= 44.644 - <= 54.058
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- yield
- Duration:
- 72 h
- Dose descriptor:
- LOEC
- Effect conc.:
- 80 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Key result
- Duration:
- 72 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 50 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Details on results:
- The average cell biomass was 80.75, 51.67, 22.25, 6.17 and 1.42 (x 10^4 cells/mL) at the tested concentrations 50, 80, 128, 204.8 and 327.7 mg/L respectively.
The concentration-effect relationship was determined using two factors namely growth rate and yield at the end of the test (72 hours). The inhibition of growth rate was 0, 10.11, 29.30, 58.54 and 92.01% and the inhibition of yield was 0.0, 36.30, 73.28, 93.50 and 99.48% at the nominal concentrations 50, 80, 128, 204.8 and 327.7 mg/L respectively, when compared to the vehicle control. Observed cells were found to be morphologically normal.
The test item concentration analysis in all test concentrations showed that the mean percent recovery with the nominal concentration was from 94.592 to 107.275% at the start of the test (RSD was 0.085 to 0.491%) and 81.644 to 95.151% at the end of the test (72 hours) (RSD was 0.262 to 0.777%) indicating that the results were within the acceptable limit (80 to 120% of the nominal concentration with an RSD of < 20%).
The validity criteria of the test are fulfilled as:
- There was an increase in cell concentration of the negative control culture by a factor of 79.54, which is more than the required factor limit of at least 16 at the end of the test.
- The mean coefficient of variation for section-by-section specific growth rates in the negative control cultures during the test was 23.94%, which is within the required limit of 35%.
- The coefficient of variation of average growth rate between replicate cultures of negative control was 0.28%, which is within the required limit of 7%. - Results with reference substance (positive control):
- The test with reference substance Potassium dichromate (K2Cr2O7) has met all the validity criteria.
ErC50 (growth rate) = 0.6500 mg/L (95% Cl: 0.28890-1.38270)
EyC50 (yield) = 0.2130 mg/L (95% Cl: 0.16640-0.27350) - Reported statistics and error estimates:
- The EC10, EC20 and EC50 with 95% confidence limits will be determined by calculating the ErC and EyC by Probit analysis of Finney (1978) using a validated computer program.
For estimation of the NOEC, for effects of the test item on growth rate, analysis of variance (ANOVA) technique was used. - Validity criteria fulfilled:
- yes
- Conclusions:
- The test item AP 729 was tested for its effect on growth of freshwater unicellular green alga Raphidocelis subcapitata (formerly Pseudokirchneriella subcapitata) for 72 hours. The following EC50 values were found: ErC50 = 170.37 mg/L, EyC50 = 95.183 mg/L.
- Executive summary:
The effects of AP 729 were tested according to OECD Test Guideline 201 on the growth of freshwater unicellular green alga Raphidocelis subcapitata (formerly Pseudokirchneriella subcapitata) for 72 hours.
The algae were exposed to the test item at the test concentrations of 50, 80, 128, 204.8 and 327.7 mg/L (factor of 1.6) along with a negative control and vehicle control. Six replicates were maintained for the negative control and the vehicle control, and three replicates for the test concentrations. The cell growth was measured at 24, 48 and 72 hours after the initiation of the test using a haemocytometer (microscopic observation).
The concentration-effect relationship was determined using two factors, namely growth rate and yield at the end of the test (72 hours). The inhibition of growth rate was 0, 10.11, 29.30, 58.54 and 92.01% and the inhibition of yield 0.0, 36.30, 73.28, 93.50 and 99.48% at the nominal concentrations 50, 80, 128, 204.8 and 327.7 mg/L respectively, when compared to the vehicle control.
The test item concentration analysis in all test concentrations showed that the mean percent recovery with the nominal concentration was from 94.592 to 107.275% at the start of the test (RSD was 0.085 to 0.491%) and 81.644 to 95.151% at the end of the test (72 hours) (RSD was 0.262 to 0.777%) indicating that the results were within the acceptable limit (80 to 120% of the nominal concentration with an RSD of < 20%).
Observed cells were found morphologically normal in all the tested concentrations.The endpoint values for growth rate and yield at 72 hours, based on nominal concentration, are presented below in Table 1:
Table 1 At 72 hours
EC values (mg/L)
95% Cl
ErC50
170.37
157.468-184.327
ErC20
108.119
99.9315-116.9766
ErC10
85.243
78.788-92.2266
EyC50
95.183
86.498-104.7392
EyC20
61.6487
56.02383-67.83842
EyC10
49.126
44.64367-54.05836
LOEC
80
NA
NOEC
50
NA
Reference
Description of key information
The effects of AP 729 were tested according to OECD Test Guideline 201 on the growth of freshwater unicellular green alga Raphidocelis subcapitata (formerly Pseudokirchneriella subcapitata) for 72 hours.
The algae were exposed to the test item at the test concentrations of 50, 80, 128, 204.8 and 327.7 mg/L along with a negative control and vehicle control. Six replicates were maintained for the negative control and the vehicle control, and three replicates for the test concentrations.
The concentration-effect relationship was determined using two factors, namely growth rate and yield at the end of the test.
The validity criteria of the test were fulfilled.
The following EC50 values were found: ErC50 = 170.37 mg/L, EyC50 = 95.183 mg/L.
Key value for chemical safety assessment
- EC50 for freshwater algae:
- 170.37 mg/L
- EC10 or NOEC for freshwater algae:
- 50 mg/L
Additional information
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