Chloroacetone

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-02-23 to 2021-03-26

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:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

acc. Council Regulation (EC) No. 266/2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling schedule: 0 hours and 72 hours

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 10.0 mg test item/L was freshly prepared with dilution water. For preparation the density was taken into account. The dispersion was agitated until the solution was visually clear (approx. 1 minute). Out of the stock solution 5 concentrations were tested in a geometric series with a dilution factor of 2.
- Controls: negtaive control, reference control
- Test concentration separation factor: 2
- Evidence of undissolved material: no

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: CCAP 278/4 (axenic)
- Source: laboratory culture; originally obtained from Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Dunstaffnage Marine Laboratory
Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounts 2567 – 5130 lux for 24 hours per day.

ACCLIMATION
A four days old preculture, prepared in dilution water and incubated under test conditions, was used as inoculum.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

not specified

Test temperature:

22.8 °C

pH:

8 - 9.49

Dissolved oxygen:

not specified

Salinity:

not applicable

Conductivity:

not specified

Nominal and measured concentrations:

nominal: 0.0625, 0.125, 0.250, 0.500 and 1.00 mg test item/L; measured: 0.0128, 0.0289, 0.0863, 0.164 and 0.442 mg/L (geometric mean measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: headspace flasks with aluminium tops with PTFE seals
- Type: closed
- Material, size, headspace, fill volume: glass, volume: 59 mL
- Agitation Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
- Initial cells density: nominal: approximately 5 x 10^3 - 10^4 cells/mL; actual: 5375 cells/mL
- No. of vessels per concentration: 3
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: 24 hours/day light
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120 μE*m^-2*s^-1; homogenicity within ± 15 % over incubation area; Fluorescent tubes L 36 W/865 (OSRAM), cool daylight

EFFECT PARAMETERS MEASURED :
- Chlorophyll a-fluorescence: The cell density was measured daily via Chlorophyll a fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was observed in the definitive test at the concentration of 1.00 mg/L.
- Microscopic evaluation: Microscopic evaluation of the cells at the start and the end of the incubation period was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes
- Test concentrations: 1.00 and 0.100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.029 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.099 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % Confidence Limits: 0.0950 – 0.108 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.064 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % Confidence Limits: 0.0571 – 0.0703 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.076 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % Confidence Limits: 0.0705 – 0.0795 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.035 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % Confidence Limits: 0.0300 – 0.0403 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.029 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: yes
Microscopic evaluation of the cells at the start and at the end revealed no morphological abnormalities.
- Observation of abnormalities: no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no significant stimulation of growth detected
- Any observations 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):

EC50 (growth rate, 72 h) = 0.903 mg/L (corresponding to 1.27 mg/L without headspace); EC50 (biomass 72 h) = 0.441 mg/L (corresponding to 0.786 mg/L without headspace)

Reported statistics and error estimates:

ECx determined based on sigmoidal dose-response (variable slope); NOEC/LOEC values were determined using Shapiro-Wilk´s Test on Normal Distribution, Levene´s Test on Variance Homogeneity (with Residuals), Trend analysis by Contrasts (Monotonicity of Concentration/Response) and Williams Multiple Sequential t-test Procedure

Table 1: Validity criteria

Validity Criterion	Required	This study
Increase of the cell growth in the control cultures	Exponentially, ≥ 16-fold corresponding to a specific growth rate of 0.92 day^-1	84-fold (specific growth rate 1.48 day^-1)
Mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures	≤ 35%	6.69%
Coefficient of variation of average specific growth rates during the whole test period in replicate control cultures	≤ 7%	1.49%

 

Table 2: Measured test item concentration in the definitive test with algae

Sampling date	Fresh media, o hours		Old media, 72 hours		Geometric mean measured concentration
Nominal test item concentration [mg/L]	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	[mg/L]
1.00	1.00	100	0.194	19	0.442
0.500	0.497	99	0.0543	11	0.164
0.250	0.250	100	0.0298	12	0.0863
0.125	0.131	105	0.00638	5	0.0289
0.0625	0.0586	94	0.00267	4	0.0128
Control	< LOQ		< LOQ		< LOQ
QC	106%		100%		-

Meas. conc. = measured concentration of the test item, dilution factors taken into account
% = percent of the of the nominal test item concentration
LOQ = limit of quantification of the analytical method (nominal 1.5 μg/L of the test item)
QC = Procedural recovery on 10 x LOQ-level, % recovery calculated to weighed amount of the test item, mean value 103 %

 

Table 3: Evaluation after 72 hours; statistically significant differences of growth rates and yield compared to control values are marked (s), not significant differences are marked (ns), n.d. = not determinable.

Geometric mean measured test item concentration [mg/L]	Replicate	Growth Rate	Inhibition of Growth Rate	Yield	Inhibition of Yield
[mg/L]	No.	[d-1]	[%]	[cells/mL]	[%]
0.442	1	n.d.	100	n.d.	100
	2	n.d.	100	n.d.	100
	3	n.d.	100	n.d.	100
	Mean	(s) n.d.	100	(s) n.d.	100
0.164	1	0.083	94	1525	100
	2	-0.003	100	-44	100
	3	-0.073	100	-1056	100
	Mean	(s) 0.003	100	(s) 142	100
0.0863	1	1.09	26	136753	70
	2	1.11	25	143038	68
	3	1.15	23	161178	64
	Mean	(s) 1.11	25	146990	67
0.0289	1	1.48	0	447581	0
	2	1.48	0	445162	1
	3	1.44	2	404184	10
	Mean	(ns) 1.47	1	432309	4
0.0128	1	1.55	-5	549206	-22
	2	1.51	-2	498894	-11
	3	1.52	-3	505736	-13
	Mean	(ns) 1.53	-3	(ns) 517945	-16
Control	1	1.45		412199
	2	1.50		476414
	3	1.49		418644
	4	1.45		416377
	5	1.50		476707
	6	1.47		439810
	Mean	1.48		448359

Validity criteria fulfilled:

yes

Conclusions:

In a Growth Inhibition Study according to OECD 201 with Pseudokirchneriella subcapitata the EC50-value for inhibition of growth rate (ErC50) after 72 hours was 0.0993 (0.0950 – 0.108) mg/L. The EC10-value for inhibition of growth rate (ErC10) after 72 hours was 0.0758 (0.0705 – 0.0795) mg/L. The NOEC for growth rate was 0.0289 mg/L. All results are based on geom. mean measured concentrations.

Executive summary:

The toxic potential of the test item to freshwater green algae was assessed in a Growth Inhibition Study according to OECD 201 and the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3 with Pseudokirchneriella subcapitata over a period of 72 hours. The study was conducted under static conditions in a closed system without headspace with an initial cell density of 5375 cells/mL. A stock solution with a nominal concentration of 10.0 mg test item/L was freshly prepared with dilution water. Out of the stock solution five concentration levels were tested in a geometrical series with a dilution factor of 2: 0.0625, 0.125, 0.250, 0.500 and 1.00 mg test item/L, corresponding to the geometric mean measured test item concentrations of 0.0128, 0.0289, 0.0863, 0.164 and 0.442 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. The test item concentrations and the control of the test item  were determined in fresh media (0 hours) and at the end of the exposure interval (72 hours) via GC-MS. At the start of the exposure the measured concentrations were in the range of 94 % to 105 % of the nominal values. At the end of exposure after 72 hours, the measured concentrations were in the range of 4 % to 19 % of the nominal values. The geometric mean measured concentration was calculated.  As a result, an EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 0.0993 (0.0950 – 0.108) mg/L and 0.0638 (0.0571 – 0.0703) mg/L, respectively. The EC10-values for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 0.0758 (0.0705 – 0.0795) mg/L and 0.0347 (0.0300 – 0.0403) mg/L, respectively. The NOEC for both growth rate and yield was 0.0289 mg/L. All results are based on geom. mean measured concentrations.

Description of key information

In a Growth Inhibition Study according to OECD 201 with Pseudokirchneriella subcapitata the EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 0.0993 (0.0950 – 0.108) mg/L and 0.0638 (0.0571 – 0.0703) mg/L, respectively. The EC10-values for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 0.0758 (0.0705 – 0.0795) mg/L and 0.0347 (0.0300 – 0.0403) mg/L, respectively. The NOEC for both growth rate and yield was 0.0289 mg/L. All results are based on geom. mean measured concentrations (reference 6.1.5-1).

Key value for chemical safety assessment

EC50 for freshwater algae:

0.099 mg/L

EC10 or NOEC for freshwater algae:

0.076 mg/L

Additional information

The toxic potential of the test item to freshwater green algae was assessed in a Growth Inhibition Study according to OECD 201 and the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3 with Pseudokirchneriella subcapitata over a period of 72 hours. The study was conducted under static conditions in a closed system without headspace with an initial cell density of 5375 cells/mL. A stock solution with a nominal concentration of 10.0 mg test item/L was freshly prepared with dilution water. Out of the stock solution five concentration levels were tested in a geometrical series with a dilution factor of 2: 0.0625, 0.125, 0.250, 0.500 and 1.00 mg test item/L, corresponding to the geometric mean measured test item concentrations of 0.0128, 0.0289, 0.0863, 0.164 and 0.442 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. The test item concentrations and the control of the test item  were determined in fresh media (0 hours) and at the end of the exposure interval (72 hours) via GC-MS. At the start of the exposure the measured concentrations were in the range of 94 % to 105 % of the nominal values. At the end of exposure after 72 hours, the measured concentrations were in the range of 4 % to 19 % of the nominal values. The geometric mean measured concentration was calculated.  As a result, an EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 0.0993 (0.0950 – 0.108) mg/L and 0.0638 (0.0571 – 0.0703) mg/L, respectively. The EC10-values for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 0.0758 (0.0705 – 0.0795) mg/L and 0.0347 (0.0300 – 0.0403) mg/L, respectively. The NOEC for both growth rate and yield was 0.0289 mg/L. All results are based on geom. mean measured concentrations.