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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23.07.2018 - 20.02.2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Based on the results of a preliminary non-GLP range finding test, the following concentrations (loading rates) in a geometrical series were tested in the definitive test:
100, 31.6, 10.0, 3.16, and 1.00% of the filtrate of a stock solution at a loading rate of 100 mg/L (spacing factor: square root of 10).

Test solutions

Details on test solutions:
The growth medium was OECD medium as described in the test guideline, modified to optimisegrowth of Raphidocelis subcapitata. The OECD growth medium
was prepared with a 10-fold concentrated iron (Fe)-EDTA complex (see bold font in recipe below) compared to the original recipe according to guideline
OECD 201. The growth rates of the algae in pre-culture were not sufficient in original OECD medium but satisfactory in the modified medium

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Study design

Test type:
static
Water media type:
freshwater
Total exposure duration:
72 h
Remarks on exposure duration:
The test item was applied once at start of the test.

Test conditions

Test temperature:
Mean: 22.9 °C
Minimum value: 22.9 °C, Maximum value: 23.8 °C
Number of measurements: 79
The temperature was within the required range of 21–24 °C, controlled at ±2 °C, as required in the guideline and the study plan.
pH:
pH of test medium: 8.2
pH in test solutions: 7.5 - 9.5
pH values at the beginning (pH0) and pH values at the end (pH72) of the test:

Nominal concentration pH(0) 8.1 pH(72)
% of filtrate at loading rate
of 100 mg test item / L

Control 8.1 9.5
1.00 8.1 9.0
3.16 8.1 9.0
10.0 8.1 8.4
31.6 7.9 8.2
100 7.5 8.0

The pH-drift observed in the control was <1.5 pH units, as required in the guideline and the study plan.
Nominal and measured concentrations:
Nominal concentration Measured concentration Measured concentration Geometric mean concentration
Test period: 0 h Test period: 72 h
%* or mg test item / L mg test item / L mg test item / L mg test item / L

0.00 n.d. n.d. n.d.
1.00 0.991 0.673 0.817
3.16 3.06 1.31 2.00
10.0 10.1 7.38 8.63
31.6 32.8 29.5 31.1
100 100 101 100

*% of saturated solution at a loading rate of 100 mg test item / L

At time 0 hours the measured concentrations ranged between 97.0–104% of the nominal concentrations (expressed in mg/L assuming full solubility of the test item).

After 72 hours exposure, the two highest test concentrations remain between 93 and 101% of nominal, while the lower concentration levels decreased to 41.0–74.0% of the nominal concentrations (expressed in mg/L assuming full solubility of the test item) Since the test item concentrations did not remain stable within ±20% of the initially measured concentrations throughout the exposure period, the biological results are additionally expressed based on geometric mean of the concentrations measured at start (0 h) and end of exposure (72 h).
Details on test conditions:
After temperature adaptation of the test solutions, 2.101 mL of the algal pre-culture (Raphidocelissubcapitata) were added to volumetric flasks containing
1000 mL of the test solutions, respectively to achieve an algal cell concentration of approximately 0.5×104 cells/mL. These manipulations were performed
under a laminar flow box. The test solutions were homogenised by manual shaking 15 times overhead, before adding 100±5 mL of the test media to each
test vessel to ensure a homogeneous distribution of the algal cells.

The test vessels were placed onto a shaker under light and temperature controlled conditions. The medium was sterilised by sterile filtration (pore size 0.2 μm)
before use. All glassware and materials used for testing purposes were sterilised for at least 3 hours at ≥150 °C using a heating furnace or autoclaved for
20 minutes at 120°C with a maximum pressure of 1.4 bar, except preparation flask (volumetric flask) used for preparation of test solution.
Reference substance (positive control):
yes
Remarks:
A reference test using potassium dichromate (K2Cr2O7) as reference item were performed in August 2018 (ECT study number: IAO1804). Standard: Growth rate ErC50 (0–72 h): 0.953 (0.919 –0.988 mg/L; 95%-CL)

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
14.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
At start, cell numbers were determined microscopically using a counting chamber.After 24, 48 and 72 hours, the cell numbers were determined by measuring the fluorescence intensity in 4 samples of 200 μL of test solution per replicate using a fluorometer
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.817 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
46.87 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Results with reference substance (positive control):
A reference test using potassium dichromate (K2Cr2O7) as reference item were performed in August
2018 (ECT study number: IAO1804).

Result: Standard: Growth rate ErC50 (0–72 h): 0.953 (0.919 – 0.988 mg/L; 95%-CL)

Based on an international ring test (ISO (2004). Water Quality – Freshwater algal growth inhibition test with unicellular green algae. European Standard
EN ISO 8692, October 2004.) mentioned in OECD guideline 201, the ErC50 (72h)-value for potassium dichromate obtained from different laboratories
was 1.19 mg/L with a standard deviation of 0.27 mg/L.

The ErC50 value for the toxic reference item, potassium dichromate, was determined as 0.953 mg/L.
This value is within the range of the international ring test (ISO (2004)). Therefore, the results of this
reference test are acceptable and the test conditions are reliable.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
All validity criteria were fulfilled as required by the study plan:
Executive summary:

The EC50 for yield of Raphidocelis subcapitata was stated to be 16.7% of filtrate at a loading rate of

100 mg test item/L, equivalent to 14.8 mg DCHA-Oleat/L based on measured concentrations.

The NOEC based on nominal concentration appeared to be 1.00% of filtrate at a loading rate of 100 mg

test item/L, equivalent to 0.817 mg DCHA-Oleat/L based on measured concentrations.

The EC50 for growth rate of Raphidocelis subcapitata was stated to be 51.4% of filtrate at a loading

rate of 100 mg test item/L, equivalent to 46.87 mg DCHA-Oleat/L based on measured concentrations.

The NOEC based on nominal concentration appeared to be 3.16%of filtrate at a loading rate of 100 mg

test item/L, equivalent to 2.00 mg DCHA-Oleat/L based on measured concentrations.