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Ecotoxicological information

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:
2019-10-14 - 2019-10-18 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
under GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
OECD Guideline for the Testing of Chemicals No. 201, adopted 23. Mar. 2006, Annex 5 corrected: 28 July 2011 “Freshwater Alga and Cyanobacteria, Growth Inhibition Test”
Deviations:
yes
Remarks:
but not affecting the validity or integrity of the study
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Commission Regulation (EU) No. 2016/266 amending Regulation (EC) No. 440/2008, Annex IV, Method C.3: “FRESHWATER ALGAE AND CYANOBACTERIA, GROWTH INHIBITION TEST,” adopted 07. December 2015
Deviations:
yes
Remarks:
but not affecting the validity or integrity of the study
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
The test item was stored in the test facility in a closed vessel in a fridge at 2.5 – 8.3 °C, away from light and humidity.
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 / 1 / 3.2 / 10 / 32 / 100 mg/L (nominal)
- Miscellaneous: On each sampling day, validity of calibration was controlled by measuring QC samples (1 mg/L). Before the first measurement at the start of the test, a new calibration was performed.
Vehicle:
no
Remarks:
test medium was used
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
A saturated solution was prepared for the test. This was done by weighing the real load of 100.0 mg/L, adding the corresponding amount of algal medium (demineralised water enriched with minerals but without algae) and shaking vigorously for 24 hours. The resulting solution was filtrated through 0.45 µm PTFE filters and the filtrate was used as test medium of the highest test concentration of nominal 100 mg/L. The lower treatments (1.0 / 3.2 / 10 / 32 mg/L nominal) were prepared by dilution of this filtrate with algal medium.
- Controls: algal medium / potassium dichromate
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Unicellular freshwater green alga
- Strain: Desmodesmus subspicatus, SAG Strain Number 86.81
- Source (laboratory, culture collection): The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sci-encebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.
- Age of inoculum (at test initiation): For the pre-culture an aliquot of the permanent culture was brought into nutrient medium and incubated under continuous lighting for 96 hours under test conditions. The resulting culture grew exponentially.
Before usage, the 5 days old pre-culture was checked for the absence of cell aggregates and the cell number of the culture was determined.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
none
Test temperature:
20.4 – 22.8 °C
pH:
7.4 - 8.4
Nominal and measured concentrations:
Nominal: 0 / 1.0 / 3.2 / 10 / 32 / 100 mg/L
Measured, t=0h: n.d. / 0.32 / 0.83 / 2.62 / 7.55 / 25.02 mg/L
Measured, t=72h: n.d. / 0.21 / 0.52 / 1.33 / 4.05 / 12.53 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: glass flasks
- Type (delete if not applicable): open, covered with perforated plastic foil acting as a stopper
- Material, size, headspace, fill volume: glass flasks total volume 65 mL, filled with 45 ±1 mL
- Renewal rate of test solution (frequency/flow rate): none
- Initial cells density: 2300 cells/mL
- Control end cells density: 425073 cells/mL
- No. of vessels per concentration (replicates): 3 replicates for each treatment
- No. of vessels per control (replicates): 6 replicates for the blank control
- No. of vessels per vehicle control (replicates): n/a

GROWTH MEDIUM
- Standard medium used: Yes, the algal medium corresponds to the OECD TG 201 medium.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 4440 - 8880 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter
- Other: pH; appearance of the algae

TEST CONCENTRATIONS
The concentrations to be tested are based on non GLP pre-tests.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a separate reference test.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.26 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 0.26 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.65 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
< 0.26 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
4.25 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.26 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8.95 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.11 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: none
Results with reference substance (positive control):
- Results with reference substance valid? yes, the 72h-EC50 values of potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) laid within the range of the laboratory (growth rate 0.65 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).
Reported statistics and error estimates:
Calculation of results was performed with the help of validated software (Microsoft Excel®). The estimation of the biological data was accomplished using the software ToxRat® Professional, version 3.3.0.
Validity criteria fulfilled:
yes
Conclusions:
- NOEC (Growth Rate) 72 h: 0.26 mg/L
- NOEC (Yield) 72 h: <0.26 mg/L
- LOEC (Growth Rate) 72 h: 0.65mg/L
- LOEC (Yield) 72 h: ≤0.26 mg/L
- 72h ErC10: 4.25 mg/L
- 72h EyC10: 0.26 mg/L
- 72h ErC50: 8.95 mg/L
- 72h EyC50: 2.11 mg/L
Executive summary:

The study was conducted under GLP according to OECD guideline 201 and EU method C.3 on the registered substance itself. One valid experiment was performed using 5 concentrations ranging from 1.0 to 100 mg/L (nominal).

Due to the poor water solubility of2-hydroxy-1-[4-(4-(2-hydroxy-2-methylpropionyl)phenoxy)phenyl]-2-methylpropan-1-one, a saturated solution was prepared as test medium of the highest test concentration bydirect weighting, prolonged stirring and subsequent filtration according to OECD Guidance document No. 23. The lower treatments were prepared by dilution of this filtrate with dilution water.

Incubation time (test system Desmodesmus subspicatus) was 72 hours.

The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate µ and the yield[1]were determined from the cell number at the respective observation times.

At all concentrations (except 1.0 mg/L (nominal) for growth rate), significant inhibition of algal growth was observed.

At the beginning and at the end of the test, the content of the test item in the test solutions was determined using HPLC-UV determination. The concentrations determined at the start of the test ranged from 24% to 32 % of the nominal concentration. At the end of the test the determined concentrations ranged from 13% to 21 % of the nominal concentration. Therefore, the determination of the results was based on the geometric mean of the measured concentrations.

The 72h-EC50values of potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) were determined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.65 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

The following results for the test item 2-hydroxy-1-[4-(4-(2-hydroxy-2-methylpropionyl)phenoxy)phenyl]-2-methylpropan-1-one were determined:

Endpoint

NOEC

LOEC

EC10

EC50

Growth Rate

0.26 mg/L

0.65mg/L

4.25 mg/L

8.95 mg/L

Yield

< 0.26 mg/L

≤ 0.26 mg/L

0.26 mg/L

2.11 mg/L

Description of key information

Freshwater algae Desmodesmus subspicatus (OECD 201; GLP):

Parameter

Value [meas. (geom. mean)]

95 % confidence interval

NOEC (Growth Rate) 72 h

0.26 mg/L

--

NOEC (Yield) 72 h

<0.26 mg/L

--

LOEC (Growth Rate) 72 h

0.65mg/L

--

LOEC (Yield) 72 h

≤0.26 mg/L 

--

72h ErC10

4.25 mg/L

3.63 mg/L – 4.97 mg/L

72h EyC10

0.26 mg/L

0.20 mg/L – 0.34 mg/L

72h ErC50

8.95 mg/L

7.45 mg/L – 10.71 mg/L

72h EyC50

2.11 mg/L

1.85 mg/L – 2.40 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
8.95 mg/L
EC10 or NOEC for freshwater algae:
0.26 mg/L

Additional information

A reliability score of 1 was assigned for the GLP guideline study, in accordance with the criteria for assigning data quality in line with the principles described by Klimisch (1997).