4-[4-(3,4-Difluorophenyl)-cyclohexyl]-cyclohexanone

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:

2018-08-10 till 2018-12-13

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

At the start of the exposure (0 hours), samples of the fresh media were taken after preparation of each concentration and analyzed. At the end of the exposure (72 hours), samples of the old media were taken from the test vessels for analyses.

Vehicle:

no

Details on test solutions:

A saturated solution with a nominal loading of 10.0 mg test item/L was freshly prepared prior to the start of the exposure. An appropriate amount of the test item was weighed out and transferred with an appropriate amount of the dilution water into the glass flask. This dispersion was treated with ultrasound for 60 minutes at room temperature. Thereafter, the saturated solution was removed by siphoning from the middle of the flask. The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: HINDAK, CCAP 278/4
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Dunstaffnage Marine Laboratory, Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK
- Method of cultivation: On Z-Agar

ACCLIMATION
- Acclimation period: no
- Culturing media and conditions: same as test conditions

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23 - 24°C

pH:

7.82 - 9.38

Nominal and measured concentrations:

Nominal concentrations: 0.1, 0.316, 1, 3.16 and 10 mg/L
Geometric mean measured concentration: 10, 28.4, 113, 417 and 1423 µg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: closed with cotton wool plugs
- Material, fill volume: glass, 250 mL
- Aeration: no
- Initial cells density: 6442 cells/mL
- Control end cells density: 1531776 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: According to the guideline OECD 201

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: 24 hours/day light
- Light intensity: 5286 to 6432 lux

EFFECT PARAMETERS MEASURED
Determination of cell concentrations: Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm measured daily
Microscopic evaluation of the cells at the start and the end of the incubation period was carried out.
The pH-values at the start of exposure were measured in one additional replicate of each concentration level and the control. At the end of exposure, the pH-values were measured from pooled samples of the test item concentrations and the control. The room temperature was measured continuously. Light intensity was measured prior to the start of exposure.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: √10
Range finding study:
- Test concentrations: 0.1, 1, 3, 10 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

250 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

61.6 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

66.1 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

12.6 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Growth rate and yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

28.4 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Growth rate and yield

Details on results:

The study meets the validity criteria of the guideline:
- The required increase of the cell growth in the control cultures was exponentially (> 16-fold corresponding to a specific growth rate of 0.92 day-1), in this study the increase was 238-fold (specific growth rate 1.82 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 should be < 35%, in this study the coefficient was 25.5%.
- Coefficient of variation of average specific growth rates during the whole test period in replicate control cultures should be < 7%, in this study the coefficient was 1.70%.
Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities. All effect values are given based on the nominal test item concentrations.

Results with reference substance (positive control):

The toxicity of potassium dichromate (SIGMA ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2018-03-26 to 2018-03-29. The reference item toxicity is in the valid range following test facility SOPs. The measured EC50 based on growth rate inhibition was 1.09 (95% confidence interval: 0.842-1.31), the EC50 based on yield was 0.548 (95% confidence interval: 0.439 - 0.660).

Reported statistics and error estimates:

EC10-, EC20- and ECso-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression. The NOEC and LOEC were determined by calculation of statistical significant differences of growth rates and yield. As a standard, One Way Analysis of Variance (ANOVA) and DUNNETT'S test were used for NOEL/LOEL calculations. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05. Equal Variance test failed for calculation of growth rate. Therefore, data were transformed (Y=10Y). The data for the tables in this report were computer-generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures. Calculations were carried out using software:
• Excel, MICROSOFT CORPORATION
• SigmaPlot, SPSS INC.
• GraphPad Prism, GRAPHPAD SOFTWARE, INC

Table 1: Measured Concentration of the test item in the definitive test with Algae

Sampling date	Fresh Media, 0 hours	Old Media, 72 hours		Geometric mean measured concentration of the test item [ug/L]
Dilution level of the saturated solution [%]	Test item
	Meas. cone. [µg/L]	Meas. cone. [µg/L]	%
100	1319	1534	116	1423
31.6	476	365	77	417
10.0	128	97.7	77	113
3.16	36.4	21.9	61	28.4
1.00	< LOQ (15.4)	< LOQ (5.77)	37	10.0*)
Control	< LOQ	<LOQ

Meas. cone. = measured concentration of the test item, enrichment and dilution factors taken into account

%               = percent of the initially measured concentration of the test item

LOQ           = limit of quantification (20µg/L of the test item)

*)                = for geometric mean % LOQ (10µg/L of the test item) taken in lo account

   

Table 2: Cell densities

Geometric mean measured test item concentration [µg/L]
	Replicate		Cell density [cells/mL]
	No.	0 hours	24 hours	48 hours	72 hours
	1	6442	6924	4513	<LOQ cell density
1423	2	6442	7115	6159	2837
	3	6442	6646	5583	3038
	Mean	6442	6895	5418	n.a.
	1	6442	9731	12864	19994
417	2	6442	10980	13093	26019
	3	6442	9332	17747	55114
	Mean	6442	10014	14568	33709
	1	6442	14987	60903	314987
113	2	6442	14885	87764	538884
	3	6442	15007	92272	641039
	Mean	6442	14960	80313	498303
	1	6442	17114	144236	1009442
28.4	2	6442	19383	143014	1227355
	3	6442	22889	150735	1242648
	Mean	6442	19795	145995	1159815
	1	6442	23177	192749	1512992
10.0	2	6442	18730	193275	1284403
	3	6442	21625	177864	1458129
	Mean	6442	21177	187963	1418508
	1	6442	26170	217981	1482258
	2	6442	22278	225994	1660403
	3	6442	21523	209729	1615420
Control	4	6442	26205	230145	1301086
	5	6442	25105	247294	1482705
	6	6442	24793	234792	1648785
	Mean	6442	24346	227656	1531776

LOQ cell density = 2653 N/mL; n.a. = not available

Table 3: Evaluation after 72 hours: Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

Geometric mean measured test item concentration	Replicate	Growth Rate		Inhibition of Growth Rate	Yield	Inhibition of Yield
[µg/L]	No.		[d-1]	[%]	[cells/mL]	[%]
	1		n.a.	100	n.a.	100
1423	2		-0.27	100	-3605	100
	3		-0.25	100	-3404	100
	Mean		(+) n.a.	100	(+) n.a.	100
	1		0.38	79	13552	99
	2		0.47	74	19577	99
417	3		0.72	61	48672	97
	Mean		(+) 0.52	72	(+) 27267	98
	1		1.30	29	308545	83
	2		1.48	19	532442	65
113	3		1.53	16	634597	58
	Mean		(+) 1.44	21	(+) 491861	68
	1		1.69	8	1003000	34
	2		1.75	4	1220913	20
28.4	3		1.75	4	1236206	19
	Mean		(+) 1.73	5	(+) 1153373	24
	1		1.82	0	1506550	1
	2		1.77	3	1277961	16
10.0	3		1.81	1	1451687	5
	Mean		(-) 1.8	1	(-) 1412066	7
	1		1.81		1475816
	2		1.85		1653961
	3		1.84		1608978
	4		1.77		1294644
	5		1.81		1476263
Control	6		1.85		1642343
	Mean	1.82			1525334

n.a. = not available

Validity criteria fulfilled:

yes

Conclusions:

In this study, the effect values of the test item on the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours were determined as follows (based on geometric mean measured test item concentrations): The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 250 µg/L and 66.1 µg/L, respectively. The EC10 value for inhibition of growth rate was 61.6 µg/L.

Executive summary:

The toxicity of the test item to the unicellular freshwater green algaPseudokirchneriella subcapitatawas determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3 at the test facility. The aim of the study was the determination of the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 6442 cells/mL. A saturated solution with a nominal loading of 10.0 mg/L was prepared with dilution water. Out of the saturated solution five concentration levels were tested in a geometrical series with a dilution factor of: 1.00 – 3.16 - 10.0 - 31.6 - 100%. Three replicates were tested for each test item concentration level and six replicates for the control. The environmental conditions were within the acceptable limits. The initially measured concentrations of the test item were 15.4 - 36.4 - 128 -476 - 1319 µg/L At the end of exposure after 72 hours, the measured concentrations were in the range of < LOQ to 116% of the initially measured concentrations. The geometric mean measured test item concentrationswere calculated to be10.0 - 28.4 - 113 - 417 - 1423 µg/L. The following effect values were calculated ((based on geometric mean measured test item concentrations): The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 250 (231 - 291) µg/L and 66.1 (50.2 - 83.5) µg/L, respectively. The EC10 value for inhibition of growth rate was 61.6 µg/L.

Description of key information

The effect values of the test item on the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours were determined as follows (based on geometric mean measured test item concentrations): The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 250 µg/L and 66.1 µg/L, respectively. The EC10 value for inhibition of growth rate was 61.6 µg/L (reference 6.1.5 -1).

Key value for chemical safety assessment

EC50 for freshwater algae:

0.25 mg/L

EC10 or NOEC for freshwater algae:

61.6 µg/L

Additional information

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3 at the test facility. The aim of the study was the determination of the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 6442 cells/mL. A saturated solution with a nominal loading of 10.0 mg/L was prepared with dilution water. Out of the saturated solution five concentration levels were tested in a geometrical series with a dilution factor of: 1.00 – 3.16 - 10.0 - 31.6 - 100%. Three replicates were tested for each test item concentration level and six replicates for the control. The environmental conditions were within the acceptable limits.The initially measured concentrations ofthe test itemwere 15.4 - 36.4 - 128 -476 - 1319 µg/L At the end of exposure after 72 hours, the measured concentrations were in the range of < LOQ to 116% of the initially measured concentrations. The geometric mean measured test item concentrationswere calculated to be10.0 - 28.4 - 113 - 417 - 1423 µg/L. The following effect values were calculated ((based on geometric mean measured test item concentrations): The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 250 (231 - 291) µg/L and 66.1 (50.2 - 83.5) µg/L, respectively. The NOEC for both inhibition of growth rate and yield were 10.0 µg/L, respectively (reference 6.1.5 -1).