Hydroxyacetone

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018/10/29 - 2018/11/02

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:

The following deviations were documented:
¨ The pH of the blank control should not change by more than 1.5 units. The change was 2.2 units, this was caused by the test design as a closed system. The pH increase was caused by CO2 shortage in the closed system. As normal growth was detected in the blank controls this was stated as uncritical.
¨ The stock solutions to be autoclaved were autoclaved for 20 minutes instead of the required 15 minutes. This can be considered uncritical, as the goal of sterility is achieved.
¨ The temperature during pre-culture incubation was in the range 21.8 – 24.5°C, and therefore not in the desired range (21.0 - 24.0°C). As all validity criteria were met and exponential growth was observed in the blank control this was stated as uncritical.
¨ The temperature during pre-culture incubation was in the range 21.8 – 24.5°C and therefore fluctuated by more than ± 2°C. As all validity criteria were met and exponential growth was observed in the blank control this was stated as uncritical.
¨ The pre-culture was prepared 5 days before the test start. As all validity criteria were met and exponential growth was observed in the blank control this was stated as uncritical.

The deviations were assessed and signed by the study director on 12. Nov. 2018.

Deviations:

yes

Remarks:

please refer to 'version/remarks'

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, 55116 Mainz

Specific details on test material used for the study:

The test item was stored in the test facility in a closed vessel at room temperature (20 ± 5°C)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 4.6 / 10 / 22 / 46 / 100 mg/L
- Sampling method: Before the start of incubation and every 24 hours the cell number was determined. The content of the test item in the test vessels was measured at the start and at the end of the test. For elimination of the algal cells before analytical determination at the end of the test, samples were centrifuged

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution containing 100.0 mg/L test item in buffered algal medium (demineralised water enriched with minerals but without algae) was prepared. This was done by mixing 92.4 μL/L test item (based on a density of 1.0828 ± 0.0002 g/cm3 at 20.0 ± 0.2 °C, determined in LAUS study 17100906G912) with the corresponding amount of algal medium (demineralised water enriched with minerals but without algae). The lower treatments were prepared by dilution of this stock solution with algal medium.
- Controls: Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a separate reference test.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: (SAG Strain Number): 86.81
- Source (laboratory, culture collection): MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen)
- Method of cultivation: 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.

ACCLIMATION
- Acclimation period: 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 (5000 lux, 21.8 – 24.5 °C). The resulting culture grew exponentially.
Before usage, the pre-culture was checked for the absence of cell aggregates and the cell number of culture was determined
- Culturing media and conditions (same as test or not): The algal medium corresponds to the OECD TG 201 medium, nutrient medium (demineralised water enriched with minerals):
Algal Medium
Stock Solution I 10.0 mL (NH4Cl 1500 mg, MgCl2*6H2O 1200 mg, CaCl2*2H2O 1800 mg, MgSO4*7H2O 1500 mg, KH2PO4 160 mg, H2O deionised ad 1000 mL)
Stock Solution II 1.0 mL (FeCl3*6H2O 64 mg, Na2EDTA*2H2O 100 mg, H2O deionised ad 1000 mL)
Stock solution III 1.0 mL (H3BO3 185 mg, MnCl2*4H2O 415 mg, ZnCl2 3 mg, CoCl2*6H2O 1.5 mg, CuCl2*2H2O 0.01 mg, Na2MoO4*2H2O 7 mg, H2O deionised ad 1000 mL)
Stock solution IV 1.0 mL (NaHCO3 50 g, H2O deionised ad 1000 mL)
H2O deionised ad 1000 mL
All solutions were sterilized before use. Stock solutions I and III and the deionised water were sterilized using an autoclave. Stock solutions II and IV were sterilized using filtration. To avoid a strong pH increase and CO2 shortage in the closed system, additional 6 mL of Stock Solution IV (sodium hydrogen carbonate solution (50 g/L)) was added to the major amount of algal medium (e.g. 90 - 99 % of the desired end volume). Then, pH was adjusted to 7.1 and the solution was filled up to 1000 mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

Before the start of incubation and every 24 hours, the cell number was determined

Test temperature:

22.0-23.3 °C

pH:

7.3-9.7, refer to table 1

Nominal and measured concentrations:

please refer to table 2

Details on test conditions:

TEST SYSTEM
- Test vessel: glass vessel
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, total volume 65 mL
- Initial cells density: 2.6 *10E3 cells/mL
- Control end cells density: please refer to table 3
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, corresponding to OECD TG 201 medium

OTHER TEST CONDITIONS
- Sterile test conditions: n/a, solutions were sterilized
- Photoperiod: continuous lighting
- Light intensity and quality: 5000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 2.2
- Range finding study: The concentrations to be tested are based on non GLP pre-tests
- Test concentrations: 4.6 / 10 / 22 / 46 / 100 mg/L nominal concentration

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a separate reference test.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

66.44 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.:

46.97 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

According to the guideline, NOEC is determined by comparing of the respective treatment with the blank control. Statistically insignificant variation is considered as “no observed effect”, although the EC10 which is read from the graph toxicity vs. concentration may lie lower

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other:

Remarks:

According to the guideline, NOEC is determined by comparing of the respective treatment with the blank control. Statistically insignificant variation is considered as “no observed effect”, although the EC10 which is read from the graph toxicity vs. concentration may lie lower

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

40.52 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

51.24 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities observed
- Unusual cell shape: no
- Colour differences: no
- Any stimulation of growth found in any treatment: yes at 4.6 mg/L and 22 mg/L treatment (please refer to table 5)
- 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: no

Results with reference substance (positive control):

- Results with reference substance valid? yes, in a seperate reference study (201803R301), under GLP conditions
- EC50: 0.79 mg/L based on growth rate and 0.39 mg/L based on yield (within the range of the laboratory (growth rate: EC50 = 0.73 - 1.10 mg/L, yield: EC50 = 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 3.2.1.

Table 4: Growth Rate, Yield
Nominal Concentration in mg/L	Parameter	Growth Rate (0 -72 h) [per day]	Yield (0 - 72 h) [cell concentration/mL]
Blank control	Mean	1.70	433533
	SD	0.07	84567
4.6	Mean	1.82	613767
	SD	0.06	100820
10	Mean	1.65	399247
	SD	0.18	193356
22	Mean	1.79	569780
	SD	0.06	103056
46	Mean	1.58	353867
	SD	0.25	228089
100	Mean	0.11	1460
	SD	0.20	1948
SD = Standard Deviation

Table 5: Inhibition Values Nominal concentration in mg/L % Inhibition Growth Rate (0-72h)       Yield (0-72h) Blank control 0 0 4.6 -6.9 -41.6 10 3.0 7.9 22 -5.4 -31.4 46 7.0 18.4 100 93.3 99.7 Negative inhibition values indicate a stimulation of algal growth compared to the blank control.

Validity criteria fulfilled:

yes

Remarks:

Please refer to 'Overall Remarks'

Conclusions:

The study was performed using 5 concentrations ranging from 4.6 to 100 mg/L. To avoid volatilization of the test item, the study was performed in closed vessels. Incubation time (test system Desmodesmus subspicatus) was 72 hours. Significant inhibition of algal growth was observed in the highest concentration (100 mg/L nominal). No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.

Executive summary:

Toxicity to algae was determined following OECD Guideline 201 applying GLP criteria. The study was performed using 5 concentrations ranging from 4.6 to 100 mg/L. To avoid volatilization of the test item, the study was performed in closed vessels. 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 and the yield were determined from the cell number at the respective observation times. Significant inhibition of algal growth was observed in the highest concentration (100 mg/L nominal). At the start and at the end of the test, the content of the test item in the test solutions was determined using HPLC-DAD. The measured concentrations lay between 100 % and 105 % of the nominal concentrations at the beginning of the test. During validation the test item was shown to be stable in test medium without algae up to 72 hours. However, at the end of the test no test item could be detected for any test concentration. This observation can only be explained that the measurable test item concentration is biased by the presence of the test organism (adsorption or ingestion onto the algae cells). Thus, the test item was present but not measurable in the test solutions. As the test substance was biologically available, and the initial values well conformed the the nominal values, the biological results are related to the nominal values. This procedure is in agreement with the OECD guideline No. 201. The 72h-EC50 values 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 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

The following results for the test item were determined:

Table 8: Results of the test item
Endpoint	NOEC	LOEC	EC10	EC50
Growth Rate	46.00 mg/L	100.00 mg/L	46.97 mg/L	66.44 mg/L
Yield	46.00 mg/L	100.00 mg/L	40.52 mg/L	51.24 mg/L

 

Description of key information

D. subspicatus, static, 72 h , OECD 201, GLP, EC50 = 66.44 mg/L (growth rate)

Key value for chemical safety assessment

EC50 for freshwater algae:

66.44 mg/L

EC10 or NOEC for freshwater algae:

46.97 mg/L

Additional information

Toxicity to algae was determined following OECD Guideline 201 applying GLP criteria. The study was performed using 5 concentrations ranging from 4.6 to 100 mg/L. To avoid volatilization of the test item, the study was performed in closed vessels. Incubation time (test systemDesmodesmus 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 and the yield were determined from the cell number at the respective observation times. Significant inhibition of algal growth was observed in the highest concentration (100 mg/L nominal). At the start and at the end of the test, the content of the test item in the test solutions was determined using HPLC-DAD. The measured concentrations lay between 100 % and 105 % of the nominal concentrations at the beginning of the test. During validation the test item was shown to be stable in test medium without algae up to 72 hours. However, at the end of the test no test item could be detected for any test concentration. This observation can only be explained that the measurable test item concentration is biased by the presence of the test organism (adsorption or ingestion onto the algae cells). Thus, the test item was present but not measurable in the test solutions. As the test substance was biologically available, and the initial values well conformed the the nominal values, the biological results are related to the nominal values. This procedure is in agreement with the OECD guideline No. 201. The 72h-EC50 values 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 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

The following results for the test item were determined:

Table 1: Results of the test item
Endpoint	NOEC	LOEC	EC10	EC50
Growth Rate	46.00mg/L	100.00mg/L	46.97mg/L	66.44mg/L
Yield	46.00mg/L	100.00mg/L	40.52mg/L	51.24mg/L