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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:
2017-05-24 to 2017-11-30; Experimental phase: 06 - 15 June 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Source and lot/batch No.of test material: M-150801 (according to CoA from 14 March 2017)
- Expiration date of the lot/batch: 2018-04-07
- Purity test date: 13.03.2017

- Storage condition of test material: Keep only in the original container. Protect from heat and direct sunlight. Store at max. +30°C
- Stability under test conditions: Not stable
Analytical monitoring:
Details on sampling:
Substance specific analysis was performed at the test site of biochemA GmbH.
- Concentrations: 0.0 (Sample code: NC), 1.1 (Sample code: A), 2.8 (Sample code: B), 7.4 (Sample code: C), 19.2 (Sample code: D), and 50.0 (Sample code: E)
- Sampling method: The samples were filled into 2 mL microcentrifuge tubes and stored in the freezer IN 434 (≤ -18 °C).

The sampling and documentation was conducted according to the following specification:
• At the start of the test (0 h), the test solutions NC, A, B, C, D and E were sampled before division to the test vessels (each concentration: 3 samples of 1.5 mL).
• After 24 h and 48 h exposure, both additional vessels of NC, A, B, C, D and E were sampled (each concentration: 3 samples of 1.5 mL).
• At the end of the test (72 h), both additional vessels and 2 replicates of the test vessels from NC, A, B, C, D and E were sampled (each concentration: 3 samples of 1.5 mL).
Of each sampled treatment, one of the samples from 0 h, 24 h, 48 h and 72 h was sent to the analytical laboratory at the test site biochemA. The remaining samples were stored as retain samples in the freezer until finalization of the study.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solution was prepared by adding 55.4 mg test item (including a factor of 1.11 to take into account the dilution caused by addition of the algal inoculum) to 1000 mL test medium and stirring for 1 h at room temperature. This stock solution was used as highest
concentration in the test.
- Differential loading: nominal loading rates of 0, 1.1, 2.8, 7.4, 19.2, 50.0 mg/L.
- Controls: yes
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 Göttingen and was cultivated in suspension culture. The strain used for this study has been cultured at Hydrotox GmbH since May 2016. 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 CaCl2x2H2O, 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 MnCl2x4H2O, 0.0088 mg/L ZnSO4x7H2O, 0.0032 mg/L CuSO4x5H2O, 0.0010 mg/L Na2MoO4x2H2O, 0.0016 mg/L CoCl2x6H2O) under axenic conditions to keep it in exponential growth.

Before the start of the test, 1.32 mL of the algae stock suspension was diluted with 48.68 mL OECD TG 201 medium to obtain a concentration of 5 x 10^4 cells/mL. This pre-culture was incubated for 4 d at 22.4 — 23.0 °C and 70.7 μE/m²s +/- 5.3% continuous lighting. For the start of the test, the cell concentration was determined using the Coulter Counter, resulting in 97.627 x 10^5 cells/mL. To obtain a nominal inoculum concentration of 7 x 10^4 cells/mL, 1.795 mL pre-culture was diluted into 250 mL OECD TG 201 medium.
Test type:
Water media type:
Limit test:
Total exposure duration:
72 d
Remarks on exposure duration:
The algal biomass was determined after 0h, 24 h, 48 h and 72 h exposure by measuring the surrogate parameter chlorophyll fluorescence.
Test temperature:
The temperature during the exposure was 22.5 - 22.8 0C
The pH was 7.7 - 8. in the control and 7.5 - 8.0 in the test item treatment
Dissolved oxygen:
not applicable
Nominal and measured concentrations:
- nominal concentrations of 1.1, 2.8, 7.4, 19.2, 50.0 mg/L.
- measured concentrations of 0.33, 0.59, 1.38, 4.52, 12.24 mg/L.
Details on test conditions:
- Test vessel: Erlenmeyer glass flasks, Schott, Mainz
- Fill volume: 100 mL
- Aeration: no
- Initial cells density: 7 x 10^4 cells/mL
- Control end cells density: 5 x 10^5 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

- Medium used: OECD TG 201 medium according to OECD 201 (2006)

- Adjustment of pH: yes
- Light intensity and quality: 70.7 μE/m²s +/- 5.3%

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter Counter Z2, Beckman Coulter, Krefeld
- Chlorophyll measurement: 96-well micro-plates, No. 655 101, Greiner bio-one, Frickenhausen; Fluorescence Microplate Reader, Tecan infinite F200, Tecan Group Ltd. Männedorf, Switzerland

A preliminary test without GLP was performed before start of this GLP-study. Three water accommodated fractions (WAF) with the loading rates 35, 100 and 1000 mg/L were tested, since the water solubility of the test item was described as low according to the data sheet from 28 March 2017. All three loading rates resulted in inhibition rates of 100% for yield and growth rate. Chemical analysis showed recovery rates at 0 h in the same range as the nominal concentrations and a decrease in test item concentration after 72 h. According to these results, the test item is regarded as water soluble.
- Spacing factor for test concentrations: 2.6
- Range finding study: yes
- Test concentrations: 35, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
potassium dichromate
Key result
72 h
Dose descriptor:
Effect conc.:
4.24 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
72 h
Dose descriptor:
Effect conc.:
3.77 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
72 h
Dose descriptor:
Effect conc.:
4.52 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
72 h
Dose descriptor:
Effect conc.:
1.38 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities observed.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- ErC50: 0.95 mg/L (CL 95 %: 0.61 - 1.47 mg/L)
- Other: EyC50: 0.06 mg/L (CL 95 %: 0.0 - 1.29 mg/L)
Reported statistics and error estimates:
The effect concentrations after 72 h exposure are calculated with the statistical software ToxRat Professional 3.2.1.

Table 2: Analytically measured test item concentrations after 0 h, 24 h, 48 h and 72 h exposure.

Nominal test concentration [mg/L]  Test item concentration [mg/L] 

Geometric mean


0 h 24 h 48 h 72 h 0 -72 h
1.1 0.77  BLQ  BLQ  BLQ 0.33
2.8 2.09 0.91  BLQ  BLQ 0.59
7.4 5.51 2.44 1.08  BLQ 1.38
19.2 15.10 6.57 2.92 1.44 4.52
50.0 39.70 17.40 8.46 3.84 12.24

BLQ: Below limit of quantification (LOQ)

* For measurements which are < LOQ, the value of LOQ/2, i.e. 0.25 mg/L, was used for calculation.

Validity criteria fulfilled:
The 72-h exposure of Pseudokirchneriella subcapitata to OO-tert-butyl monoperoxymaleate results in an EC50 value of 4.24 mg/L (measured concentration).
Executive summary:

In a 72-hour algal growth inhibition test, cultures of Pseudokirchneriella subcapitata (Strain No. 61.81 SAG) were exposed to OO-tert-butyl monoperoxymaleate at nominal concentrations of 0 (control), 1.1, 2.8, 7.4, 19.2, 50.0 mg/L and measured concentrations of 0, 0.33, 0.59, 1.38, 4.52 and 12.24 mg/L (geometric mean) under static conditions in accordance with the OECD Guideline 201 (23 March 2006), OECD 23 (2000) and under GLP. The % growth inhibition in the treated algal culture as compared to the control ranged from -0.3 % at a concentration of 0.33 mg/L to 109.7% at a concentration of 12.24 mg/L.

This toxicity study is classified as acceptable and satisfies the guideline requirements for an algae growth inhibition toxicity study.  


Results Synopsis

Test Organism: Pseudokirchneriella subcapitata (Strain No. 61.81 SAG)

Test Type: Static 

Specific Growth Rate:

72 hr EC50: 4.24 mg/L                  95 % C.L.: n.d.;

72 hr EC10: 3.77 mg/L                  95 % C.L.: 0.0 – n.d.;

72 hr LOEC: 4.52 mg/L;

72 hr NOEC: 1.38 mg/L.  


Endpoint(s) Effected:  Specific growth rate

Description of key information

For the static algae growth inhibition test conducted with Pseudokirchneriella subcapitata, applying nominal concentrations of 0 (control), 1.1, 2.8, 7.4, 19.2, 50.0 mg OO-tert-butyl monoperoxymaleate/L, results based on growth rate after 72 h were as follows:

- EC50: 4.24 mg/L (95 % C.I.: n.d.);

- EC10: 3.77 mg/L (95 % C.I.: 0.00 – n.d.)

- LOEC: 4.52 mg/L;

- NOEC: 1.38 mg/L.

As the measured test item concentrations are not within ± 20 % of the nominal concentrations, according to OECD 201 (2004) and OECD 23 (2000), all results are given in relation to the analytically measured test item concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:
4.24 mg/L
EC10 or NOEC for freshwater algae:
3.77 mg/L

Additional information