1-Butoxy-4-[4-(trans-4-ethylcyclohexyl)-1-cyclohexen-1-yl]-2,3-difluorobenzene

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:

Jun 04,2020 - Aug 20, 2020

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

All loading rates and the control were analytically verified via GC MS at the start (0 hours) and at the end of the exposure (72 hours) with algae.
At the start, samples were taken from one additional replicate of the test item loading rate and the control and at the end samples were taken from pooled replicates.

Vehicle:

yes

Remarks:

water

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
A saturated solution with a nominal loading rate of 10.0 mg test item/L was prepared once at room temperature 96 ± 1 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out. The test item was applied onto a glass slide. The glass slide with the test item was inserted into a glass bottle with an appropriate amount of dilution water (see Table 2). The saturated solution was stirred for 96 ± 1 hour (1100 rpm, room temperature). After a separation phase of 1 hour (at room temperature), the saturated solution was removed by siphoning (from the approximate middle of the glass flask). The saturated solution was be checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative.

Dilution levels of the saturated solution were tested in a geometric series with a dilution factor of 1.78: 9.96 - 17.7 - 31.6 - 56.2 - 100% of the saturated solution, corresponding to the geometric mean measured test item concentration of 11.9 – 22.6 – 39.7 – 70.6 – 119 µg/L. The dilution levels are based on the results of a preliminary range finding test (non-GLP).

The control solution was prepared without test item following the same method as specified for the saturated solution. Six replicates (without test item) were tested under the same test conditions as the test item replicates. Potassium dichromate was tested as a reference item

Test organisms (species):

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

Details on test organisms:

Test organism: Pseudokirchneriella subcapitata HINDÁK CCAP 278/4 (axenic)
Origin:
Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Göttingen, Germany

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

46 mg CaCO3/L

Test temperature:

21 - 24 °C

pH:

7.4 - 8.1

Nominal and measured concentrations:

geometric mean measured test item concentration of 11.9 – 22.6 – 39.7 – 70.6 – 119 µg/L
nominal: 9.96 - 17.7 - 31.6 - 56.2 - 100% of the saturated solution

Details on test conditions:

The study was performed as a static test in open vessels under culture conditions. The flasks contained 100 mL either reconstituted water or test medium (test item group).
The duration of exposure was 72 hours. The algal biomass in each flask was determined daily during the exposure period via Chlorophyll a-fluorescence.
Microscopic evaluation of the cells at the start and at the end of the incubation period was carried out.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 119 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: above the water solubility limit of the pure substance

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 119 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: above the water solubility limit of the pure substance

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 119 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: above the water solubility limit of the pure substance

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 2020-04-20 to 2020-04-23. The reference item toxicity is in the valid range which was established by calculation of the average of the historic reference data since 2006, and the limits were set using the threefold standard deviation of these values.
ErC50 = 1.12 mg/L, EyC50 = 0.59 mg/L

Reported statistics and error estimates:

The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours above 119 μg/L and thus above the water saturation limit concentration of the pure test material, respectively.

Cell Densities

Geometric mean measured test item concentration	Replicate	Cell density [cells/mL]
[µg/L]	No.	0 hours	24 hours	48 hours	72 hours
119	1	5375	17001	123342	804243
	2	5375	2692	32510	795251
	3	5375	18318	143572	1040334
	Mean	5375	12670	99808	879943
70.6	1	5375	18069	130663	955496
	2	5375	19779	136723	974946
	3	5375	19562	125869	1001873
	Mean	5375	19137	131085	977438
39.7	1	5375	19547	114257	863229
	2	5375	19867	124804	1092039
	3	5375	20588	146355	1237964
	Mean	5375	20001	128472	1064411
22.6	1	5375	19366	137813	1034225
	2	5375	17658	124813	908629
	3	5375	19230	123210	869680
	Mean	5375	18751	128612	937511
11.9	1	5375	19503	144039	1125221
	2	5375	20855	144576	1053040
	3	5375	18853	146961	1230585
	Mean	5375	19737	145192	1136282
Control	1	5375	20156	134665	1016534
	2	5375	19699	121021	961409
	3	5375	19853	128596	990145
	4	5375	20280	134294	852869
	5	5375	23425	138360	1274910
	6	5375	22098	165385	1177463
	Mean	5375	20919	137054	1045555

Evaluation after 72 hours

Geometric mean measured test item concentration	Replicate	Growth rate		Inhibition of growth rate	Yield		Inhibition of yield
[mg/L]	No.	[d-1]		[%]	[cells/mL]		[%]
119	1		1.67	5		798868	23
	2		1.67	5		789876	24
	3		1.76	0		1034959	1
	Mean	(-)	1.70	3	(-)	874568	16
70.6	1		1.73	2		950121	9
	2		1.73	1		969571	7
	3		1.74	1		996498	4
	Mean	(-)	1.73	1	(-)	972063	7
39.7	1		1.69	3		857854	18
	2		1.77	-1		1086664	-4
	3		1.81	-3		1232589	-19
	Mean	(-)	1.76	0	(-)	1059036	-2
22.6	1		1.75	0		1028850	1
	2		1.71	3		903254	13
	3		1.70	3		864305	17
	Mean	(-)	1.72	2	(-)	932136	10
11.9	1		1.78	-2		1119846	-8
	2		1.76	0		1047665	-1
	3		1.81	-3		1225210	-18
	Mean	(-)	1.78	-2	(-)	1130907	-9
Control	1		1.75			1011159
	2		1.73			956034
	3		1.74			984770
	4		1.69			847494
	5		1.82			1269535
	6		1.80			1172088
	Mean		1.75			1040180

Section-by-Section and Average Specific Growth Rates of the Control Group (0 - 72 hours)

	Replicate No.	Specific growth rate [d-1]			Mean (0 - 72 hours)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.32	1.90	2.02	1.75	0.374	21.39	20.7
	2	1.30	1.82	2.07	1.73	0.394	22.79
	3	1.31	1.87	2.04	1.74	0.384	22.09
	4	1.33	1.89	1.85	1.69	0.313	18.56
	5	1.47	1.78	2.22	1.82	0.377	20.66
	6	1.41	2.01	1.96	1.80	0.332	18.50
				Mean	1.75
				SD ±	0.05
				CV [%]	2.76

SD = Standard deviation    CV = Coefficient of variation

Environmental conditions

Geometric mean measured test item concentration		pH-values
[mg/L]		Start; 0 hours	End; 72 hours
119		7.65	7.41
70.6		7.66	7.48
39.7		7.79	7.56
22.6		7.88	7.59
11.9		7.91	7.81
Control		7.99	8.07
Room temperature [°C]:	min.: 22	max.: 23		mean value: 22.5
Light intensity [lux]	n = 18	mean value: 5057 range of the measured values: 4512 to 5524 equalling -10.8 to 9.23%		CV [%]: 6.83%

CV = Coefficient of variation                              n = number of measuring points

Water Quality parameters

Parameters of the dilution water (measured on 2020-04-27)
Conductivity [µS/cm]	Total hardness [mg CaCO3/L]	Acidity [mmol/L]	Alkalinity [mmol/L]	Total organic carbon [mg C/L]
154	46	0.2	0.5	< LOQ*

*Limit of quantification = 2.00 mg C/L

Measured Concentrations and Percent of Initial of the Test Item

Sampling date	Fresh media, 0 hours	Old media, 72 hours	Geometric mean measured concentration of the test item [µg/L]
[%] of the saturated solution	Test Item
	Meas. conc. [µg/L]	Meas. conc. [µg/L]
100	172	81.8	119
56.2	98.2	50.8	70.6
31.6	52.4	30.1	39.7
17.7	29.6	17.2	22.6
9.96	16.9	8.33	11.9
Control	< LOQ	< LOQ	< LOQ

Validity criteria fulfilled:

yes

Conclusions:

The EC50 and EC10 value (based on growth rate) of the test material is thus clearly above the water solubility of the pure substance corresponding to a nominal concentration of 100 mg test item/L (cf. OECD 201).

Executive summary:

The toxicity of the test material 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.
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 5375 cells/mL. A saturated solution with a nominal loading rate of 10.0 mg test item/L was prepared once 96 ± 1 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out and applied onto a glass slide. The glass slide with the test item was inserted into a glass bottle with an appropriate amount of dilution water. The saturated solution was stirred for 96 ± 1 hour (1100 rpm, room temperature) with a magnetic stirrer. After a separation phase of 1 hour (at room temperature), the saturated solution was removed by siphoning (from the approximate middle of the glass flask). The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative.

The original saturated solution and further 4 dilution levels in a geometrical series with a dilution factor of 1.78 were tested: 9.96 - 17.7 - 31.6 - 56.2 - 100% of the saturated solution, corresponding to the geometric mean measured test item concentration of 11.9 – 22.6 – 39.7 – 70.6 – 119 µg/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 concentrations of the test item were analytically verified via GC-MS at the start (0 hours) and at the end of the exposure (72 hours) in all loading rates and in the control.

All effect values given are based on the geometric mean measured test item concentrations. For results, please refer to Table below:

	Growth Rate Inhibition [µg/L]
NOEC	≥ 119 (nominal 100 mg/L)
LOEC	> 119 (nominal 100 mg/L)
ErC10	> 119 (nominal 100 mg/L)
ErC20	> 119 (nominal 100 mg/L)
ErC50	> 119 (nominal 100 mg/L)
	Yield Inhibition [µg/L]
NOEC	≥ 119 (nominal 100 mg/L)
LOEC	> 119 (nominal 100 mg/L)
EyC10	78.1 (not applicable)
EyC20	> 119 (nominal 100 mg/L)
EyC50	> 119 (nominal 100 mg/L)

Description of key information

In the OECD 201 assay performed for this compound, no effects on Daphnia magna were observed up to the saturated solution of the test item.

The EC50 and EC10 value for inhibition of growth rate after 72 hours were > 119 mg/L (geometric mean measured) corresponding to a nominal concentration of 100 mg/L.

Additional information

The toxicity of the test material 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.
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 5375 cells/mL. A saturated solution with a nominal loading rate of 10.0 mg test item/L was prepared once 96 ± 1 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out and applied onto a glass slide. The glass slide with the test item was inserted into a glass bottle with an appropriate amount of dilution water. The saturated solution was stirred for 96 ± 1 hour (1100 rpm, room temperature) with a magnetic stirrer. After a separation phase of 1 hour (at room temperature), the saturated solution was removed by siphoning (from the approximate middle of the glass flask). The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative.

The original saturated solution and further 4 dilution levels in a geometrical series with a dilution factor of 1.78 were tested: 9.96 - 17.7 - 31.6 - 56.2 - 100% of the saturated solution, corresponding to the geometric mean measured test item concentration of 11.9 – 22.6 – 39.7 – 70.6 – 119 µg/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 concentrations of the test item were analytically verified via GC-MS at the start (0 hours) and at the end of the exposure (72 hours) in all loading rates and in the control.

No adverse effects were observed up to the saturated solution. The EC50 and EC10 value for inhibition of growth rate after 72 hours were > 119 mg/L (geometric mean measured) corresponding to a nominal concentration of 100 mg/L.