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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
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
Analytical monitoring:
yes
Details on sampling:
After 0 h, 24 h, 48 h and 72 h exposure, the additional vessels for chemical analysis of NC, A, B,
C, D and E were sampled; 2 samples of 0.75 mL per treatment group.
The samples are filled into 2 mL glass vials. To each vial, 0.25 mL acetonitrile was added and the
samples were stored in the freezer at ≤ -18 °C.
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 ultrapure 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 ultrapure 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 ultrapure water (for ZnCl2, CoCl2 and CuCl2, higher concentrated solutions were prepared and the corresponding volumes used for A3).

Solution A4
Sodium hydrogen carbonate p.a. NaHCO3 50 g
dissolved in 1000 mL ultrapure 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 as well as
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 100 mL of the 10-fold concentrated medium with 900 mL ultrapure water for the stock solution and 200 mL of the 10-fold concentrated medium with 1800 mL ultrapure water for the dilution on the test day.

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
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 January 2022. 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.

Quality assurance takes place at regular intervals by testing the sensitivity of the test organisms to the reference item potassium dichromate (Honeywell GmbH, Seelze, Germany, Lot: H1030). The recent quality testing was performed in February 2022 (GLP 1717) which resulted in an ErC50 (72 h) of 1.03 mg/L (CL 95 %: 0.93 – 1.13 mg/L; interlaboratory test result for ErC50: mean of 1.19 mg/L with SD of 0.27 mg/L) and an EyC50 (72 h) of 0.63 mg/L (CL 95 %: 0.44 – 0.89 mg/L).

Before the start of the test, 1.315 mL of the algae stock suspension was diluted with 48.685 mL test medium to obtain a concentration of 5 × 104 cells/mL. This pre-culture was
incubated for 4 d at 22.7 – 23.0 °C and 90.2 μE m-² s-1 ± 6.3 % continuous lighting. For the start of the test, the cell concentration was determined using the Coulter Counter, resulting in
90.183 × 105 cells/mL. To obtain a nominal inoculum concentration of 7 × 104 cells/mL, 1.940 mL pre-culture was diluted into 250 mL test medium.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.0 – 23.1 °C
pH:
7.7 – 7.9 in control
6.4 – 7.8 in treatment
Nominal and measured concentrations:
In the control, the test item was not detected. The measured test item concentrations in the test
item treatments were 93.0 – 106.7 % of the nominal concentrations.

As the measured test item concentrations are within ± 20 % of the nominal concentrations,
according to OECD 201 (2006), all results are given in relation to the nominal test item
concentrations.
Details on test conditions:
Test vessels: Erlenmeyer glass flasks 100 mL, Schott, Mainz
Screw-cap glass bottles 100 mL, 500 mL, 1000 mL, Schott, Mainz
Adjustable micropipette 200 μL, 1000 μL, 5000 μL, Eppendorf, Wesseling/Berzdorf
Graduated pipettes 10 mL, 20 mL, Brand, Wertheim
Scale LP 6200 S, Sartorius AG, Göttingen
Analytical balance BP 221S, Sartorius AG, Göttingen
Magnetic stirrer, MONO direct with stir bars, H+P Labortechnik AG, Oberschleissheim
pH-electrode SenTix® 940, conductivity measuring cell TetraCon® 925 and multi-parameter
instrument inoLab Multi 9430 IDS, WTW, Weilheim
Shaking incubator INFORS Multitron Pro, INFORS GmbH, Einsbach
Thermometer IP65 LT-102, Dostmann electronic GmbH, Wertheim-Reichholzheim
Thermometer Traceable Ultra, VWR International GmbH, Darmstadt
Coulter Counter Z2, Beckman Coulter, Krefeld
Fluorescence Microplate Reader, Tecan infinite F200, Tecan Group Ltd. Männedorf, Switzerland
96-well micro-plates, No. 655 101, Greiner bio-one, Frickenhausen
Microscope with ocular 10× and objectives 10× und 40×, Olympus, Hamburg
Erythrocytometer, Neubauer type, 0.1 mm depth, Brand, Wertheim
Total carbon analyser TOC-L with autosampler ASI-L, Shimadzu, Duisburg

The test vessels were sealed with stainless steel caps permeable to air and kept in the light incubator
(specified temperature: 21 – 24 °C, constant within +/- 2 °C). They were placed on the shaking table
with adhesive mats and continuously shaken with 100 rpm and an orbital diameter of 5 cm.
The test vessels were continuously illuminated by LEDs (warm white) with a light intensity of
90.2 μE m-² s-1 ± 6.3 %. The pH was measured at the start (0 h) and at the end (72 h) of the test.
The temperature was measured continuously.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
127.1 mg/L
95% CI:
>= 107.5 - <= 150.3
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
18.5 mg/L
95% CI:
>= 12 - <= 28.5
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
39.6 mg/L
95% CI:
>= 32.3 - <= 48.5
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
9.2 mg/L
95% CI:
>= 6.1 - <= 13.6
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
yield
Details on results:
The morphology of the algal cells in the test item treatments as well as in the control showed no
obvious abnormality.
The biomass (cell concentration) in the control has increased by a factor of 23.2 and therefore
≥ 16 during the test period.
The mean coefficient of variation for section-by-section growth rate (day 0 – 1. 1 – 2 and 2 – 3)
in the control is 7.8 % and therefore ≤ 35 %.
The coefficient of variation of average specific growth rate during the test period in the control
replicates is 2.7 % and therefore ≤ 7 %.
Therefore the validity criteria are fulfilled.
Validity criteria fulfilled:
yes
Conclusions:
A static algal growth inhibition test with Raphidocelis subcapitata according to OECD 201
(23 March 2006, corrected 28 July 2011) was conducted to investigate the effects of the test item. The measured ErC50 is 127.1 g/l and the ErC10 18.5 mg/l. The NOEC of 10 mg/l can be regarded as chronic value.

Description of key information

A static algal growth inhibition test with Raphidocelis subcapitata according to OECD 201
(23 March 2006, corrected 28 July 2011) was conducted to investigate the effects of the test item. The measured ErC50 is 127.1 g/l and the ErC10 18.5 mg/l. The NOEC of 10 mg/l can be regarded as chronic value.

Key value for chemical safety assessment

EC50 for freshwater algae:
127.1 mg/L
EC10 or NOEC for freshwater algae:
10 mg/L

Additional information