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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
analytical verification of the test substance is missing, not performed according to GLP and OECD guidelines,
Qualifier:
according to
Guideline:
other: DIN 38419, Part9
GLP compliance:
no
Analytical monitoring:
not specified
Vehicle:
yes
Details on test solutions:
Cremophor max.5 mg/L
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Strain: SAG 86.81
- Source: Algensammlung Göttingen
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
21.3 °C
pH:
7.3 - 8.1
Nominal and measured concentrations:
Test concentrations (nominal): 1, 2.5, 5, 10, 25, 50 mg/L
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
34.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % CL 12.3 - 95.0 mg/L

growth rate:

EC10 (72h): 24.6 mg/L (95 % CL: 10.2 - 59.8 mg/L)

EC10 (96h): 13.3 mg/L (95 % CL: 5.5 - 31.8 mg/L)

EC50 (72h): 34.1 mg/L (95 % CL: 12.3 - 95.0 mg/L)

EC50 (96h): 18.4 mg/L (95 % CL: 7.7 - 43.9 mg/L)

biomass:

EC10 (72h): 23.9 mg/L (95 % CL: 5.0 - 112.3 mg/L)

EC10 (96h): 22.46 mg/L (95 % CL: 4.4 - 114.3 mg/L)

EC50 (72h): 33.2 mg/L (95 % CL: 5.7 - 192.2 mg/L)

EC50 (96h): 30.5 mg/L (95 % CL: 5.3 - 176.9mg/L)

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 18200055
- Expiration date of the lot/batch: 30 June 2020
- Purity: 98.3 %
Analytical monitoring:
yes
Details on sampling:
A stock solution with a nominal concentration of 100 mg/L was freshly prepared with dilution water. The dispersion was stirred at 1100 rpm at room temperature for ca. 15 minutes until the solution was visually clear. The stock solution was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative. Out of the stock solution six concentration levels were tested in a geometrical series with a dilution factor of √10.

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution was prepared by adding an appropriate amount of the test item (100 mg/L) to dilution water. The dispersion was stirred at 1100 rpm at room temperature for ca. 15 minutes until the solution was visually clear. The stock solution was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative. The stock solution and a further 5 test item concentrations were prepared by diluting the stock solution accordingly.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Dunstaffnage Marine Laboratory, Dunbeg, OBAN; Argyll PA37 1QA, Scotland, UK
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounts to 2590 - 5180 lux corresponding to 35 - 70 µE m-2 s-1 for 24 hours per day.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
38 mg/L as CaCO3
Test temperature:
22 - 23 °C
pH:
7.76 - 8.05 (test start)
8.00 - 9.40 (test end)
Conductivity:
141 µS/cm
Nominal and measured concentrations:
Nominal: 0.316, 1.0, 3.16, 10, 31.6 and 100 mg/L
Measured (0h): 0.312, 1.18, 3.15, 10.3, 33.3 and 102 mg/L
Measured (72h): 0.296, 0.976, 2.92, 9.32, 27.4 and 95.1 mg/L
measured > 80% nominal
Details on test conditions:
TEST SYSTEM
- Test vessel: sterile Erlenmeyer flasks, volume: 250 mL, sealed with cotton wool plugs with 100 mL test solution
- Initial cells density: 6442 cells/mL
- Control end cells density: 1540086 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guidelines
- Total organic carbon: < LOQ
- Alkalinity: 1 mmol/L
- Conductivity: 141 µS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH-value at the start of the exposure was measured out of one additional replicate of the test item concentrations and the control. At the end of the exposure it was measured from pooled replicates of the test item concentrations and control, respectively. The room temperature was measured continuously. Light intensity was measured prior to the start of the exposure.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: 24h light
- Light intensity and quality: 5281 - 6999 lx

EFFECT PARAMETERS MEASURED: Cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. Self-fluorescence was observed in the preliminary range finding test at the concentration of 120 mg/L. A separate replicate of each test item concentration was prepared without algae and treated like the other test replicates. The self-fluorescence of the measuring points between 24 and 72 hours was taken into account in the evaluation. Microscopic evaluation of the cells at the start and end of the exposure was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and aggregation of algae cells.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
14.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: analytically verified
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
10.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: analytically verified
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none
- Unusual cell shape: no
- Colour differences: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 0.799 mg/L
- validity range: 0.753 ± 0.544
Validity criteria fulfilled:
yes
Conclusions:
The test substance showed a 72h-EC50 of 14.3 and a 72h-EC10 of 10.4 mg/L. Thus, the test substance is harmful to aquatic invertebrates.

Description of key information

EC50(72h) = 14.3 mg/L (nominal, analytically verified) for growth rate inhibition of Pseudokirchneriella subcapitata (OECD 201)

EC10(72h) = 10.4 mg/L (nominal, analytically verified) for growth rate inhibition of Pseudokirchneriella subcapitata (OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:
14.3 mg/L
EC10 or NOEC for freshwater algae:
10.4 mg/L

Additional information

Two studies are available investigating the toxicity of triisubutyl phosphate to freshwater algae. However, one of the studies (BASF 1989) was considered to be not reliable due to methodological shortcomings (missing analytical verification of test concentrations, not performed according to GLP and current OECD guidelines). Thus, a new test was performed according to current quality criteria. This key study was performed according to GLP and OECD giudeline 201 using Pseudokirchneriella subcapitata as test organism (Herrmann 2019). Analytical verification of the test concentrations at the beginning and the end of the test showed > 80% of nominal at any measurement. All validity criteria of the test are fulfilled. The 72h-EC50 is determined to be 14.3 mg/L and the 72h-EC10 is determined to be 10.4 mg/L based on nominal concentrations.