1-(2,4-dichlorophenyl)ethan-1-one

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:

From 26 March 2018 to 29 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 6.25, 12.5, 25, 50 and 100 % saturated solutions.
- Sampling method: Samples were taken from the test solutions at the applied concentration levels and from the control solution at the beginning and at the end of the experiment.
- Sample storage conditions before analysis: not required.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Because the Test Item was not readily soluble in water by direct addition, a test solution was prepared using a saturated solution method. The solution was prepared by dispersing/dissolving 100 mg/L Test Item into the test medium (OECD Medium) two days before the start of the experiment. This solution was shaken for approximately 24 hours at approximately 30°C (Day - 2) and then was equilibrated for approximately 24 hours at approximately 20°C (Day - 1). Any non-dissolved test materials were removed by filtration through a fine (0.22 μm) filter to give the 100% saturated solution. The analysis of the prepared solution showed a solution of 93.3 mg/L, so the method was considered valid for the study.
- Differential loading: nominal loading rate of 100 mg/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Specie: Pseudokirchneriella subcapitata
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source (laboratory, culture collection): The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Germany.
- Method of cultivation: The algae were cultured under standarised conditions, according to OECD 201 in the Ecotoxicological Laboratory of Citoxlab Hungary Ltd. The pre-culture, which is intended to give a quantity of algae suitable for the inoculation of test cultures, was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still exponentially growing (after an incubation period of 3 days). If the algal cultures contained deformed or abnormal cells, they were discarded.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

22.5 – 22.8 °C

pH:

7.32 – 8.71

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 6.25, 12.5, 25, 50 and 100% saturated solutions.
Measured concentrations: 0 (control), 4.59, 9.75, 19.6, 42.1 and 79.9 mg/L (geometric mean).

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Material, size, headspace, fill volume: 250 mL Erlenmeyer flasks, continuously shaken by a laboratory orbital shaker to keep algae in suspension. The flasks were covered with air-permeable stoppers.
- Aeration: No
- Initial cells density: 1E+04 algal cells per mL test medium
- Control end cells density: The cell density in the control cultures increased by the factor of 69.0 within three days.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS: Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: Continuously illuminated.
- Light intensity and quality: 7594 lux (equivalent to ~103 μE/m2/s), which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed ± 15 % and therefore provided equal conditions for each test vessel.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber. Microscopic observation of the algal cells in test concentration level and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.

TEST CONCENTRATIONS
Range finding study: A concentration range-finding test was conducted to determine the approximate toxicity of the test item so that appropriate test concentrations could be selected for use in the definitive test.
- Test concentrations: 0, 0.1, 1, 10 and 100 mg/L (2 replicates per each test concentration and 3 replicates in the control group, 72 hours)
- Results used to determine the conditions for the definitive study: At the highest applied concentration (100% saturated solution) chuff cells were observed. In the other applied test concentrations all cells were normal. Because a toxic response was observed during the preliminary range-finding test, five test concentrations in a geometric series (factor 2.0) and one Control were tested in the main experiment. The concentrations in the definitive test were based on the results of the preliminary range-finding test and were: 6.25, 12.5, 25, 50 and 100 % saturated solutions.

Reference substance (positive control):

yes

Remarks:

(Potassium dichromate is tested at least twice a year by CitoxLab)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

14.51 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 13.16 – 15.99 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.59 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.:

9.21 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL: 8.43 –10.06 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.59 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.87 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% CL: 8.19 – 9.60 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.59 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Chuff cells were observed at the tested concentration of 9.75, 19.6, 42.1 and 79.9 mg/L. There was no morphological deviation observed in the tested concentrations of 4.59 mg/L and in the Control during the study, all these cells were normal.
- 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
Potassium dichromate was tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study (Study Code: 17/367-022AL) with the reference item Potassium dichromate is (Batch Number: A0345704): 04-07/12/2017
The 72h ErC 50: 0.88 mg/L, (95 % confidence limits: 0.81 – 0.96 mg/L)
The 72h EbC 50: 0.63 mg/L, (95 % confidence limits: 0.58 – 0.69 mg/L)
The 72h EyC 50: 0.53 mg/L, (95 % confidence limits: 0.49 – 0.58 mg/L)
These values are within the range of laboratory ring test data.

Reported statistics and error estimates:

The section-by-section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test, days 0-1, 1-2 and 2-3) and to demonstrate exponential growth for the entire study period. The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).
The ErC10, 20, 50, EbC10, 20, 50 and EyC10, 20, 50 values of the Test Item and their confidence limits were calculated using Probit analysis by TOXSTAT software. Statistical comparisons of biomass, average specific growth rates and yield in controls and in the treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software. For the determination of the LOEC and NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test.

Table 2. Summary of results.

Parameter (0-72 h)	Growth rate (r) [mg/L]	Yield (y) [mg/L]	Biomass (b) [mg/L]
EC10	6.70	5.38	4.96
95 % conf. limits	5.73 – 7.85	4.75 –6.09	4.30 – 5.73
EC20	8.74	6.39	6.14
95 % conf. limits	7.68 – 9.94	5.76 – 7.08	5.45 – 6.90
EC50	14.51	8.87	9.21
95 % conf. limits	13.16 – 15.99	8.19 – 9.60	8.43 –10.06
NOEC	4.59	4.59	4.59
LOEC	9.75	9.75	9.75

Table 3. Calculation of the exposure concentrations.

Nominal concentrations (% saturated)	Measured concentrations  (mg/L)		Geometric mean (mg/L)
	0h	72h
Control	n.d.	n.d.	---
6.25	5.91	3.57	4.59
12.5	11.8	8.05	9.75
25	23.6	16.3	19.6
50	47.3	37.4	42.1
100	93.3	68.4	79.9

Table 4. Growth Rates (µ) and Percentage Inhibition of µ during the Test Period.

Concentrations		Growth ratemand % inhibition ofm
Nominal (% saturated)	Geometric Mean Measured (mg/L)	0–24 h		0–48 h		0–72 h
		m	%	m	%	m	%
Control	n.d.	^0.0589	0.0	0.0611	0.0	0.0588	0.0
6.25	4.59	0.0569	3.4	0.0601	1.6	0.0586	0.3
12.5	9.75	0.0458	22.2	0.0424*	30.6	0.0450*	23.5
25	19.6	0.0193*	67.3	0.0144*	76.4	0.0096*	83.6
50	42.1	0.0096*	83.6	0.0096*	84.2	0.0064*	89.1
100	79.9	0.0000*	100.0	0.0000*	100.0	0.0000*	100.0

*statistically significantly different compared to the control values (Bonferroni t-Test; α = 0.05).

Table 5. Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

Concentration		Growth ratemand % inhibition ofm
Nominal (% saturated)	Geometric Mean Measured (mg/L)	0–24 h		0–48 h		0–72 h
		m	%	m	%	m	%
Control	n.d	38.0	0.0	290.0	0.0	1320.0	0.0
6.25	4.59	36.0	5.3	276.0	4.8	1284.0	2.7
12.5	9.75	24.0	36.8	128.0*	55.9	504.0*	61.8
25	19.6	8.0*	78.9	28.0*	90.3	52.0*	96.1
50	42.1	4.0*	89.5	16.0*	94.5	32.0*	97.6
100	79.9	0.0*	100.0	0.0*	100.0	0.0*	100.0

*statistically significantly different compared to the control values (Bonferroni t-Test;a= 0.05).

Table 6. Yield (Y) and Percentage Inhibition of Y during the Test Period

Concentration				Yield (Y) and Percentage Inhibition
Nominal (% saturated)		Geometric Mean Measured (mg/L)	0–72 h
			Y		%
Control	n.d		68.0		0.0
6.25	4.59		67.0		1.5
12.5	9.75		24.7*		63.7
25	19.6		1.0*		98.5
50	42.1		0.7*		99.0
100	79.9		0.0*		100.0

*statistically significantly different compared to the control values (Bonferroni t-Test;a= 0.05).

Table 7. Influence of Test Item on the Growth ofPseudokirchneriella subcapitata

Parameter (0-72 h)	Growth rate (r) [mg/L]	Yield (y) [mg/L]	Biomass (b) [mg/L]
EC10	6.70	5.38	4.96
95 % conf. limits	5.73 – 7.85	4.75 –6.09	4.30 – 5.73
EC20	8.74	6.39	6.14
95 % conf. limits	7.68 – 9.94	5.76 – 7.08	5.45 – 6.90
EC50	14.51	8.87	9.21
95 % conf. limits	13.16 – 15.99	8.19 – 9.60	8.43 –10.06
NOEC	4.59	4.59	4.59
LOEC	9.75	9.75	9.75

Table 8. pH-Values in the Test Media at the Start and End of the Test

Concentration		pH-values
Nominal % saturated	Measured mg/L	Start	End
Control	n.d.	7.38	8.71
			8.64
			8.65
			8.70
			8.63
			8.67
6.25	4.59	7.38	8.52
			8.50
			8.62
12.5	9.75	7.37	8.07
			8.05
			7.95
25	19.6	7.34	7.85
			7.86
			7.87
50	42.1	7.32	7.86
			7.85
			7.88
100	79.9	7.34	7.82
			7.84
			7.82

Table 9. Temperature in the Climate Chamber and in the Test Media during the Test

Parameter		Exposure Time
		Day 0	Day 1	Day 2	Day 3
temperature (ºC) measured in flask		22.7	22.8	22.6	22.5
min / max temperature (ºC) of the climate chamber	min	22.0	22.1	22.0	22.1
	max	23.1	23.3	22.9	23.0

 

Table 10. Cell Number (x 10⁴cell/mL) determined in the Main Experiment

Concentration			Number of cells
Nominal	Measured		0 h	24 h	48 h	72 h
mg/L	mg/L
Control	n.d.		1	5	22	73
			1	4	19	63
			1	5	18	72
			1	4	19	71
			1	3	16	68
			1	4	19	67
		Mean	1.00	4.17	18.83	69.00
		SD	0.0	0.8	1.9	3.7
6.25	4.59		1	4	17	70
			1	3	16	66
			1	5	21	68
		Mean	1.00	4.00	18.00	68.00
		SD	0.0	1.0	2.6	2.0
12.5	9.75		1	3	7	22
			1	3	8	26
			1	3	8	29
		Mean	1.00	3.00	7.67	25.67
		SD	0.0	0.0	0.6	3.5
25	19.6		1	2	2	2
			1	1	2	2
			1	2	2	2
		Mean	1.00	1.67	2.00	2.00
		SD	0.0	0.6	0.0	0.0
50	42.1		1	2	2	2
			1	1	2	2
			1	1	1	1
		Mean	1.00	1.33	1.67	1.67
		SD	0.0	0.6	0.6	0.6
100	79.9		1	1	1	1
			1	1	1	1
			1	1	1	1
		Mean	1.00	1.00	1.00	1.00
		SD	0.0	0.0	0.0	0.0

 

Validity criteria fulfilled:

yes

Remarks:

(cell density in control increased by the factor of 69.0>16, mean coefficient of variation for section-by-section specific growth rates in control was 13.24%<35%, coefficient of variation of average specific growth rates in control was 1.3%<7%)

Conclusions:

The 72h-EC50 in green algae Pseudokirchneriella subcapitata was found to be 14.51 mg/L (measured, geometric mean; 95% CL: 13.16 – 15.99 mg/L), the EC10 was 6.70 mg/L (measured, geometric mean; 95% CL: 5.73 – 7.85 mg/L) and the NOEC was determined to be 4.59 mg/L, based on growth rate.

Executive summary:

A Freshwater Alga Growth Inhibition Test was performed in accordance with OECD Guideline 201 and EU Method C.3, following GLP. The effect of test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata, over an exposure period of 72 hours in a static system. Based on the results of a preliminary range-finding test, algae cultures were exposed to 6.25, 12.5, 25, 50 and 100% saturated solutions of the test item (due to low water solubility) or a control. The test design included three replicates per test concentration and six replicates for the untreated control. Test concentrations were analytically determined at the beginning and at the end of the test by an HPLC-UV method. As the deviation from the initial values was greater than 20% from the nominal, measured (geometric mean) concentrations are reported: 4.59, 9.75, 19.6, 42.1 and 79.9 mg/L test item. Positive controls (postassium dichromate) are performed at least twice a year to ensure the quality of the results. All validity criteria were met. The 72h-EC50 in green algae Pseudokirchneriella subcapitata was found to be 14.51 mg/L (measured, geometric mean; 95% CL: 13.16 – 15.99 mg/L), the EC10 was 6.70 mg/L (measured, geometric mean; 95% CL: 5.73 – 7.85 mg/L) and the NOEC was determined to be 4.59 mg/L, both based on growth rate.

Description of key information

Key study. Method according to OECD 201 and EU Method C.3. GLP study. The 72h-EC50 in green algae Pseudokirchneriella subcapitata was found to be 14.51 mg/L (measured, geometric mean; 95% CL: 13.16 – 15.99 mg/L), the EC10 was 6.70 mg/L (measured, geometric mean; 95% CL: 5.73 – 7.85 mg/L) and the NOEC was determined to be 4.59 mg/L, based on growth rate.

Key value for chemical safety assessment

EC50 for freshwater algae:

14.51 mg/L

EC10 or NOEC for freshwater algae:

4.59 mg/L

Additional information

A Freshwater Alga Growth Inhibition Test was performed in accordance with OECD Guideline 201 and EU Method C.3, following GLP. Based on the results of a preliminary range-finding test, algae cultures of Pseudokirchneriella subcapitata were exposed to 6.25, 12.5, 25, 50 and 100% saturated solutions of the test item or a control, over an exposure period of 72 hours in a static system. The corresponding measured (geometric mean) concentrations were 4.59, 9.75, 19.6, 42.1 and 79.9 mg/L, analytically determined at the beginning and at the end of the test. Positive controls (postassium dichromate) are performed at least twice a year to ensure the quality of the results. All validity criteria were met. The 72h-EC50 in green algae was found to be 14.51 mg/L (measured, geometric mean; 95% CL: 13.16 – 15.99 mg/L), the EC10 was 6.70 mg/L (measured, geometric mean; 95% CL: 5.73 – 7.85 mg/L) and the NOEC was determined to be 4.59 mg/L, based on growth rate.