4-trans-(4-trans-Propylcyclohexyl)-cyclohexancarbonic acid

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:

2018-04-17 to 2018-06-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

All concentration levels and the control were analytically verified via LC-MS/MS after 0 (start of exposure) and 72 hours (end of the exposure) with algae. The samples and the control were diluted (factor 2) with acetonitrile directly after sampling. Further dilutions were made prior to analysis with dilution medium. All original samples were stored at room temperature before preparation. Prepared samples were stored in an auto-sampler at room temperature until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

A saturated solution (100 mg test item/L were weighed out) was freshly prepared with dilution water one day prior to the start of the exposure (day -1). The stock solution was stirred for 24 hours at approx. 1100 rpm at room temperature. After a separation phase of 1 hour at room temperature the test solution was removed from the bottom of the flask. The test item solution was checked via laser beam (Tyndall effect) for undissolved test item. A filtration step was added, because undissolved test item was confirmed by Tyndall effect. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate were discarded. The filtration was interrupted for 10 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. During filtration, the filter will always be kept covered. This saturated solution was used for the test. The saturated solution and four concentration levels were tested in a geometrical series with a dilution factor of 2: 6.25, 12.5, 25.0, 50.0 and 100 %. The concentration levels are based on the results of a preliminary range finding test (non-GLP).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Dunstaffnage Marine Laboratory, Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK
- Cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2590 - 5180 lux corresponding to 35 - 70 µE • m-2 • s-1 for 24 hours per day.
- Culture medium: Nutrient medium Z according to LÜTTGE et al. (1994)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21 - 24 °C controlled at ± 2°C

pH:

7.64 - 9.65

Nominal and measured concentrations:

Geometric mean measured: 1.41, 0.808, 0.385, 0.204 and 0.117 mg/L; Nominal conc.: 100, 50, 25, 12.5 and 6.25 % of the saturated solution

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile glass Erlenmeyer flasks, volume: 250 mL, sealed with cotton wool plugs
- Type: closed
- Initial cells density: Nominal: approximately 5 x 103 - 104 cells/mL Actual: 6442 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: medium according to OECD TG 201
- Intervals of water quality measurement: 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.
- Photoperiod: 24 hours/day light
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: fluorometer
- Chlorophyll measurement: 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 preliminary range finding test at the saturated solution.
- 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
- Range finding study: yes
- Test concentrations: 1.0 – 100 % of saturated solution
- 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.:

> 1.41 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

1.29 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence intervals (1.15 - > 1.41) mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.785 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence intervals (0.697 - 0.898) mg/L

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.385 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.204 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.:

0.87 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95 % confidence intervals (0.764 - 0.999) mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.462 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95 % confidence intervals (0.366 - 0.555) mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.308 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95 % confidence intervals (0.200 - 0.424) mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.204 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.385 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Results with reference substance (positive control):

Regularly, a positive control test to check the test system is carried out with potassium dichromate.
Growth rate: ErC50 = 1.09 mg/L (95% confidence interval: 0.842 - 1.31 mg/L)
Yield inhibition: EyC50 = 0.548 mg/L (95% confidence interval: 0.439 - 0.660 mg/L)

Biological Data

The growth inhibition effects of the test item are summarized in Table 1. Cell densities are listed in Table 2. Evaluations of growth rate inhibition and yield inhibition are given in Table 3 (calculated based on cell density values). Section-by-section and average specific growth rate are given in Table 4.

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.

 

Table 1: NOEC, LOEC, EC_x-values of the test item (0 - 72 hours): (based on the geometric mean measured test item concentrations, with 95% confidence intervals)

 

	Growth Rate Inhibition [mg/L]
NOEC	0.204
LOEC	0.385
ErC10	0.785 (0.697 - 0.898)
ErC20	1.29 (1.15 - > 1.41)
ErC50	> 1.41
	Yield Inhibition [mg/L]
NOEC	0.204
LOEC	0.385
ErC10	0.308 (0.200 - 0.424)
ErC20	0.462 (0.366 - 0.555)
ErC50	0.870 (0.764 - 0.999)

 

Table 2: Cell Densities

Geometric mean measured test item concentration [mg/L]	Replicate	Cell density [cells/mL]
1.41	No.	0 hours	24 hours	48 hours	72 hours
	1	6442	20543	136719	638015
	2	6442	19214	145556	692531
	3	6442	12787	110076	521710
	Mean	6442	17515	130784	617419
0.808	1	6442	21483	174165	1249355
	2	6442	26084	189397	1067487
	3	6442	29370	207450	1277110
	Mean	6442	25646	190337	1197984
0.385	1	6442	34030	238461	1790590
	2	6442	31979	281011	1716165
	3	6442	29849	239852	2020916
	Mean	6442	31953	253108	1842557
0.204	1	6442	38342	325955	2055175
	2	6442	38521	317117	2163362
	3	6442	33543	279730	2267726
	Mean	6442	36802	307601	2162088
0.117	1	6442	37369	253972	2141615
	2	6442	38283	293057	1954038
	3	6442	38134	320731	2072552
	Mean	6442	37929	289253	2056068
Control	1	6442	40611	297317	2133324
	2	6442	39263	312152	2222495
	3	6442	36289	296314	2335350
	4	6442	38039	284437	2161525
	5	6442	41204	314853	2127416
	6	644	33841	286255	2196975
	Mean	6442	38208	29855	2196181

 

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 [mg/L]	Replicate No.	Growth Rate [d^-1]	Inhibition of Growth Rate [%]	Yield [cells/mL]	Inhibition of yield [%]
1.41	1	1.53	21	631573	71
	2	1.56	20	686089	69
	3	1.47	25	515268	76
	Mean	(+) 1.52	22	(+) 610977	72
0.808	1	1.76	10	1242913	43
	2	1.70	12	1061045	52
	3	1.76	9	1270668	42
	Mean	(+) 1.74	10	(+) 1191542	46
0.385	1	1.88	3	1784148	19
	2	1.86	4	1709723	22
	3	1.92	1	2014474	8
	Mean	(+) 1.89	3	(+) 1836115	16
0.204	1	1.92	1	2048733	6
	2	1.94	0	2156920	2
	3	1.96	-1	2261284	-3
	Mean	(-) 1.94	0	(-) 2155646	2
0.117	1	1.94	0	2135173	2
	2	1.91	2	1947596	11
	3	1.93	1	2066110	6
	Mean	(-) 1.92	1	(-) 2049626	6
Control	1	1.93		2126882
	2	1.95		2216053
	3	1.96		2328908
	4	1.94		2155083
	5	1.93		2120974
	6	1.94		2190533
	Mean	1.94		2189739

 

Table 4: Section-by-Section and Average Specific Growth Rates of the Control Group (0 - 72 hours)

	Replicate No.	Specific growth rate [d^_1] section-by-section			Mean (0 – 72 hours)	SD ±	CV [%]	Mean CV [%]
		0 - 24 hours	24-48 hours	48 - 72 hours
Control	1	1.84	1.99	1.97	1.93	0.081	4.2	7.74
	2	1.81	2.07	1.96	1.95	0.134	6.9
	3	1.73	2.10	2.06	1.96	0.205	10.4
	4	1.78	2.01	2.03	1.94	0.141	7.3
	5	1.86	2.03	1.91	1.93	0.091	4.7
	6	1.66	2.14	2.04	1.94	0.252	12.9
				Mean	1.94
				SD ±	0.01
				CV [%]	0.60

 

Table 5: Environmental Conditions

Geometric mean measured test item concentration [mg/L]		pH-values
		Start; 0 hours	End; 72 hours
1.14		7.64	8.73
0.808		7.68	9.17
0.385		7.72	9.47
0.204		7.77	9.63
0.117		7.86	9.65
Control		7.93	9.38
Room temperature [°C]:	min.: 22.0	max.: 24.0	mean value: 23.0
Light intensity [lux]	n = 24	mean value: 5778 range of the measured values: 5040 to 6172 equalling -12.8 to 6.82%	CV [%]: 5.21%

CV = Coefficient of variation              

n = number of measuring points

 

Table 6: Measured Concentration of the test item in the Definitive Test with Algae

Sampling date	Fresh Media, 0 hours		Old Media, 72 hours
Nominal test item concentration [mg/L]	Test Item					Geometric mean measured concentration of the test item [mg/L]
	Meas. cone. [mg/L]	%		Meas. cone. [mg/L]	%
10.0	1.57	2		1.27	1	1.41
3.16	0.756	2		0.864	2	0.808
1.00	0.388	2		0.382	2	0.385
0.316	0.196	2		0.212	2	0.204
0.100	0.121	2		0.113	2	0.117
Control	< LOQ			< LOQ

Meas. conc. = measured concentration of the test item, dilution factors taken into account

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

LOQ = limit of quantification (0.0700 mg/L of the test item)

 

Validity Criteria

The study meets the validity criteria of the guideline:

 

Table 7: 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	341-fold (specific growth rate 1.94 /day)
Mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures	< 35 %	7.74 %
Coefficient of variation of average specific growth rates during the whole test period in replicate control cultures	< 7 %	0.60 %

 

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions, the EC50-vaiues for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 1.41 mg/L and 0.870 (C.I: 0.764 - 0.999) mg/L, respectively. The EC10 for growth rate was 0.785 mg/L.

Executive summary:

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. The aim of the study was the determination of the effects on the 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 concentration of 100 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 2: 6.25 - 12-5 - 25.0 - 50.0 - 100 % (1.41, 0.808, 0.385, 0.204 and 0.117 mg/L). 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 measured concentrations in the test solutions at the start of the exposure were 2 % of the nominal concentrations. At the end of exposure after 72 hours, the measured concentrations were in the range of 1 to 2 % of the nominal concentrations. All validity criteria were met. Under the test conditions, the EC₅₀-values for inhibition of growth rate (ErC₅₀) and yield (EyC₅₀) after 72 hours were > 1.41 mg/L and 0.870 (C.I: 0.764 - 0.999) mg/L, respectively. The EC10 for growth rate was 0.785 mg/L.

Description of key information

The test item was tested to Pseudokirchneriella subcapitata in an open static test system according to OECD 201 (2011). Under the test conditions, the EC₅₀-vaiues for inhibition of growth rate (ErC₅₀) and yield (EyC₅₀) after 72 hours were > 1.41 mg/L and 0.870 (C.I: 0.764 - 0.999) mg/L, respectively. The EC10 value for growth rate was 0.785 mg/L (reference 6.1.5-1).

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.785 mg/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. 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 concentration of 100 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 2: 6.25 - 12-5 - 25.0 - 50.0 - 100 % (1.41, 0.808, 0.385, 0.204 and 0.117 mg/L). 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 measured concentrations in the test solutions at the start of the exposure were 2% of the nominal concentrations. At the end of exposure after 72 hours, the measured concentrations were in the range of 1 to 2% of the nominal concentrations. All validity criteria were met. Under the test conditions, the EC₅₀-values for inhibition of growth rate (ErC₅₀) and yield (EyC₅₀) after 72 hours were > 1.41 mg/L and 0.870 (C.I: 0.764 - 0.999) mg/L, respectively. The EC10 value for growth rate was 0.785 mg/L.