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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020
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)
Version / remarks:
Raphidocelis subcapitata Growth Inhibition Test according to
OECD 201 (23 March 2006)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Active substance: 100%
Analytical monitoring:
yes
Details on sampling:
Chemical analysis of the test item concentration in the test solutions was performed to determine exposure concentration of the test item during the exposure period. Substance specific analysis was performed at an external test site.
After 0 h, 24 h, 48 h and 72 h exposure, 4 ml from each analytical test vessel of NC, A, B, C, D, E, F and G were sampled. The samples were filled into 8 mL-glass screw-cap vials and stabilized with 4 ml Acetonitrile. The samples were stored in the freezer (≤ -18 °C).
Of each sampled treatment, one of the analytical samples from 0 h, 24 h, 48 h and 72 h was sent to the analytical laboratory at the external test site for chemical analyses using and insulated box with thermal packs. The remaining samples were stored as retain samples in the freezer (≤ -18 °C) until finalisation of the study.
Vehicle:
no
Details on test solutions:
The test was performed with OECD TG 201 medium according to OECD 201 (2006), prepared as following:

Solution A1
Ammonium chloride p.a. NH4Cl 1.5 g
Magnesium chloride p.a. MgCl2 × 6 H2O 1.2 g
Calcium chloride dihydrate e.p. CaCl2 × 2 H2O 1.8 g
Magnesium sulfate heptahydrate p.a. MgSO4 × 7 H2O 1.5 g
Potassium dihydrogen phosphate p.a. KH2PO4 0.16 g
dissolved in 1000 mL deionised water

Solution A2
Iron (III) chloride hexahydrate p.a. Fe(III)Cl3 × 6 H2O 0.064 g
Sodium-EDTA dihydrate Na2EDTA × 2 H2O 0.100 g
dissolved in 1000 mL deionised water

Solution A3
Boric acid p.a. H3BO3 0.185 g
Manganese chloride tetrahydrate p.a. Mn(Cl2) × 4 H2O 0.415 g
Sodium molybdate dihydrate p.a. Na2MoO4 × 2 H2O 0.007 g
Zinc chloride p.a. ZnCl2 0.003 g
Cobalt chloride hexahydrate p.a. CoCl2 × 6 H2O 0.0015 g
Copper chloride dihydrate p.a. CuCl2 × 2 H2O 0.00001 g
dissolved in 1000 mL deionised water (for ZnCl2, CoCl2 and CuCl2, higher concentrated solutions
were prepared and the correspondent volumes used for A3).

Solution A4
Sodium hydrogen carbonate p.a. NaHCO3 50 g
dissolved in 1000 mL deionised water
Solutions A1 – A3 were autoclaved, and solution A4 was sterile-filtered (pore diameter 0.2 μm).
A 10-fold concentrated medium was prepared by using 100 mL of stock solution A1 and 10 mL
of stock solutions A2, A3 and A4, filling up to 1000 mL with ultrapure water and aerating for 50 min.
The 1-fold concentrated medium was prepared by diluting 200 mL of the 10-fold concentrated
medium with 1800 mL ultrapure water.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Raphidocelis subcapitata (Strain No. 61.81 SAG)
The test organism Raphidocelis subcapitata (Strain No. 61.81 SAG) originates from the Culture Collection of Algae at the University of Goettingen. The strain used for this study has been cultured in suspension culture at Hydrotox GmbH since February 2020. Twice a week, the stock suspension is inoculated into fresh Holm-Hansen medium (composition: 496 mg/L NaNO3, 39 mg/L K2HPO4, 75 mg/L MgSO4×7H2O, 36 mg/L CaCl2×2H2O, 58 mg/L Na2CO3, 10 mg/L Na2EDTA×2H2O, 3 mg/L citric acid, 3 mg/L iron citrate, 0.1144 mg/L H3BO3, 0.0724 mg/L MnCl2×4H2O, 0.0088 mg/L ZnSO4×7H2O, 0.0032 mg/L CuSO4×5H2O, 0.0010 mg/L Na2MoO4×2H2O, 0.0016 mg/L CoCl2× 6H2O) under axenic conditions to keep it in exponential growth.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
The test was performed with OECD TG 201 medium according to OECD 201 (2006)
Test temperature:
21 – 24 °C, constant within  2 °C).
pH:
The pH was 7.6 – 8.1 in the control and 7.7 – 9.1 in the test item treatment
Details on test conditions:
The purpose of this study was to investigate the effects of the test item on the growth of freshwater algae Raphidocelis subcapitata. A four-day old pre-culture was used as inoculum in the test. The algae were exposed to different concentrations of the test item in OECD TG 201 medium for 72 h.
A static test design was applied with an accompanying substance-specific chemical analysis to determine the test item exposure concentrations. The algal biomass was determined after 0 h, 24 h, 48 h and 72 h exposure by measuring the surrogate parameter chlorophyll fluorescence. In the present study, seven nominal concentrations of 11.7 – 35.0 mg/L test item were used. A preliminary range finding test without GLP was performed before start of this GLP-study.
Nominal concentrations of 1, 10 and 100 mg/L test item were tested and resulted in inhibition of 4.5 %, -8.0 %, 100 % for growth rate and 5.9 %, -36.6 %, 102.1 % for yield (72 h), respectively. A first main test with five nominal test item concentrations of 12.0, 20.4, 34.6, 58.8 and 100 mg/L resulted in inhibition of 5.5 %, 17.7.%, >100 %, >100% and >100% for growth rate and 4.5 %, 43.8 %, 100.7 %, 101.7 and 101.8 for yield (72 h), respectively. The concentration range for the reported test was therefore adjusted to 11.7 – 35.0. with a spacing factor of 1.20.
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
31.38 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
18.72 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The measured test item concentrations in the test item treatments were 101.2 – 109.0 % of the nominal concentrations
Validity criteria fulfilled:
yes
Conclusions:
The EC50 was determined to be at 31.38 g/ml, based on growth rate (72 h)
The EC10 was determined to be at 18.72 g/ml, based on growth rate (72 h)
Executive summary:

The EC50 was determined to be at 31.38 g/ml, based on growth rate (72 h)

The EC10 was determined to be at 18.72 g/ml, based on growth rate (72 h)

Description of key information

The EC50 was determined to be at 31.38 g/ml, based on growth rate (72 h)

The EC10 was determined to be at 18.72 g/ml, based on growth rate (72 h)

Key value for chemical safety assessment

EC50 for freshwater algae:
31.38 mg/L
EC10 or NOEC for freshwater algae:
18.72 mg/L

Additional information