Cyclohexanepentanol, .beta.-methyl-

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:

2010-11-12 to 2010-11-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

(2006)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Council Regulation (EC) No. 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2010-10-20

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all test item concentrations and control; additionally dilution levels of 0.320, 3.20 and 100 % of the saturated solution were analysed after 24 and 48 hours (high, medium and low); to check the stability of the test item under test conditions the lowest and highest dilution level after 24, 48 and 72 hours were analysed without algae
- Sampling method: Separate samples were prepared for each test item concentration and control.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until start of analysis, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A solution with a nominal loading of 10 mL test item/L was prepared with dilution water three days prior to application. The test item was placed on the surface of the dilution water. A slow stirring procedure in closed bottles was applied. Gentle stirring (to avoid the formation of an emulsion) was carried out for 72 hours. After a separation phase of 1 hour the saturated solution was taken from the homogeneous liquid phase.
- Loading levels: The saturated solution and further 5 dilutions of it were tested in a geometrical series with a dilution factor of √10: 0.32 - 1.00 - 3.20 - 10.0 - 32.0 - 100 %

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: CHODAT SAG 86.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, 37073 Göttingen, Germany
- Method of cultivation(at test facility): fresh stocks were prepared every month on Z-Agar; light intensity amounted to 35 - 70 µE•m-2•s-1 for 24 h per day.
- Preculture: A three day old preculture was used as inoculum. Incubation was performed in 500 mL Erlenmeyer flasks with dilution water. For the start of the test the preculture was directly pipetted into each test concentration and the control.
- Culturing media and conditions (same as test or not): Nutrient medium Z according to LÜTTGE et al. (1994).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

no data

Hardness:

no data

Test temperature:

Nominal range: 21 – 24 °C, controlled at +/- 2 °C
room temperature (measured): min.: 21 °C, max.: 22 °C, mean value: 21.5 °C

pH:

measured values: between pH 8.00 - 8.15 (at start of the test), between 8.14 - 9.44 (at 72 hr)

Dissolved oxygen:

not applicable

Salinity:

The test medium had a nominal hardness of 0.24 mmol Ca+Mg/L

Nominal and measured concentrations:

geometric mean measured exposure: 0.0321, 0.0838, 0.328, 0.986, 3.12, 12.8 mg/L

Details on test conditions:

TEST SYSTEM
- Type: closed, with regard to the volatility of the test item glass flasks without headspace were used to reduce losses of test item.
- Test vessel, material, size, headspace, fill volume: Sterile headspace flasks, (fill) volume: 119 mL, with aluminium tops with PTFE seals.
- Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
- Initial cells density: approximately 2 - 5 x 10E+03 cells/mL (nominal); approx. 4 x 10E+03 cells/mL (actual)
- Control end cells density: 3.7 x 10E+05 (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, according to the guideline (OECD 201 medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to the guideline (OECD 201 medium)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no data
- Photoperiod: 24 hours/day light
- Light intensity and quality: 60 - 120 µE • m-2 • s-1; measured values (n=18): mean: 101 µE•m-2•s-1, RSD: 3.48 %

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: after 0, 24, 48 and 72 h, measured daily via Chlorophyll-a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.
- Other: pH-value (at the beginning of the test was measured in one additional replicate each of dilution level and control; at the end it was measured from one pool per dilution level and control, respectively); room temperature; light intensity (prior to test start); microscopic evaluation of the cells (at the start and end of the incubation); 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: 3 - 4 (for geometric mean measured concentrations)

PRELIMINARY RANGE FINDING TEST
A non GLP preliminary range finding test was conducted with the saturated solution of the test item (100 %) and further 3 dilutions of the saturated solution (0.1, 1 and 10 %) over a period of 72 h. The specific algal growth rate inhibitions were 100 % at 100 % saturated solution, 19 % at 10 % saturated solution and 1 % at 0.1 and 1 % saturated solution. The recovery rates were between 77 % at 100 % saturated solution and 25 % at 0.1 % saturated solution.
- 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.:

0.328 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Basis for effect:

growth rate

Remarks:

(rate-related inhibition); same for inhibition of yield

Remarks on result:

other: -

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.986 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Basis for effect:

growth rate

Remarks:

(rate-related inhibition); same for inhibition of yield

Remarks on result:

other: -

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.03 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Basis for effect:

growth rate

Remarks:

(rate-related inhibition)

Remarks on result:

other: 1.01-1.06

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.564 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Basis for effect:

growth rate

Remarks:

(rate-related inhibition)

Remarks on result:

other: 0.497-0.635

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.557 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Basis for effect:

biomass

Remarks:

(inhibition of yield)

Remarks on result:

other: 0.507-0.618

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.327 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Basis for effect:

biomass

Remarks:

(inhibition of yield)

Remarks on result:

other: 0.292-0.368

Details on results:

BIOLOGICAL DATA
- Exponential growth in the control: yes
- Observation of abnormalities: Microscopic evaluation of the cells at start and end of the incubation period revealed no morphological abnormalities.

CONCENTRATIONS
The test item was stable under test conditions for 72 hours under absence of algae. With increasing algae cell density the test item concentrations decreased up to 50 % (RR) of the measured initial concentrations. Metabolisation of the test item as well as adsorption to algae are possible reasons for these findings. All effect levels were based on geometric mean measured concentrations of the test item.

SELF FLUORESCENCE
No self-fluorescence has been found in the preliminary range finding test at the saturated solution.

ANALYTICAL METHOD VALIDATION RESULTS
-Specificity:
Values of two blank samples without test item and spiked with 100µL methanol had to be lower than the lowest calibration standard to prove specificity. Specificity of the analytical method was demonstrated because the test item was not detected in the blank samples.
-Linearity:
Linearity of detector response was checked by analysis of standards and plotting a calibration graph of peak area versus concentration. The coefficients of determination were calculated. The analytical system gave linear responses in the range of nominal 1 to 50 µg/L test item. R2 of the calibration curves was ≥ 0.992.
- Repeatability of injections: While working with SPME it is not possible to use a sample vial more than once. Due to this fact repeatability in the common use is not possible.
- Precision:
A relative standard deviation (RSD) < 20 % was necessary to follow guideline requirements.
- Accuracy:
Five replicates of algae dilution water fortified with 5 µg/L test item (1x LOQ) and 1000 µg/L test item (200x LOQ), latter diluted 1:40 with dilution water to the measured concentration and spiked with 100 µL MeOH were prepared and analysed. A mean recovery of 105% with a rel. standard deviation (RSD) of 4.24 % was obtained for the samples series fortified on 1 x LOQ level indicating sufficient accuracy and precision according to SANCO 3029/99 rev.4 (2000). Furthermore a validation series spiked on 200 x LOQ level fulfilled the requirements of the corresponding guideline (SANCO 3029/99 rev.4) with a mean recovery of 99% and a RSD of 3.16%, as well.
- LOQ:
The LOQ of the analytical method was fixed, a minimum signal to noise ratio of 9 was provided.

Results with reference substance (positive control):

- Results with reference substance valid? The toxicity of potassium dichromate to Desmodesmus subspicatus was determined over a period of 72 h.
The reference item toxicity is in the valid range following test facility SOPS.
- EC50 (growth rate): 0.589 mg/L (72 h) with 0.535-0.652 mg/L 95 % confidence interval
- EC50 (biomass yield): 0.263 mg/L (72 h) with 0.252-0.274 mg/L 95 % confidence interval

Reported statistics and error estimates:

As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s Test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO.WILK-Test was used to test for normally distributed populations. Normality test and Equal Variance test failed for growth rate calculation and Normality Test failed for yield calculation. The growth rates were transformed using Graph Pad Prism function (Y = y squared). No possible transformation was found for yield values to pass Normality Test.
EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals of the EC-values was carried from the best fit values, the standard error and the t-distribution with the software Graph Pad Prism.

 Cell Densities 

Dilution level out of the saturated solution	Geometric mean measured exposure test item concentration	Mean Cell density [cells/mL]
[%]	[mg/L]	0 hours	24 hours	48 hours	72 hours
100	12.8	4069	680	78	296
32.0	3.12	4069	7783	1807	898
10.0	0.986	4069	19735	42799	47350
3.20	0.328	4069	21401	107726	333778
1.00	0.0838	4069	21096	119966	389722
0.320	0.0321	4069	18961	115785	396592
Control		4069	21407	105448	371028

 

Evaluation after 72 hours

Dilution level out of the saturated solution	Geometric mean measured exposure test item concen-tration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[%]	[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
100	12.8	1		-0.902	100		-3797	100
		2		-0.815	100		-3716	100
		3		-0.912	100		-3805	100
		Mean	(+)	-0.876	100	(+)	-3773	100
32.0	3.12	1		-0.777	100		-3674	100
		2		-0.483	100		-3114	100
		3		-0.369	100		-2724	100
		Mean	(+)	-0.543	100	(+)	-3171	100
10.0	0.986	1		0.792	47.3		39785	89.2
		2		0.804	46.5		41261	88.8
		3		0.855	43.1		48796	86.7
		Mean	(+)	0.817	45.6	(+)	43281	88.2
3.20	0.328	1		1.49	0.65		354676	3.35
		2		1.46	2.76		322140	12.2
		3		1.45	3.44		312312	14.9
		Mean	(-)	1.47	2.28	(-)	329709	10.2
1.00	0.0838	1		1.53	-1.55		392000	-6.82
		2		1.53	-1.47		390712	-6.47
		3		1.51	-0.53		374248	-1.99
		Mean	(-)	1.52	-1.18	(-)	385653	-5.09
0.320	0.0321	1		1.53	-1.60		392952	-7.08
		2		1.55	-3.38		426160	-16.1
		3		1.50	0.42		358456	2.32
		Mean	(-)	1.53	-1.52	(-)	392523	-6.96
Control		1		1.56			439628
		2		1.50			362544
		3		1.47			330036
		4		1.48			335440
		5		1.51			370412
		6		1.50			363692
		Mean		1.50			366959

Section-by-Section and Average Specific Growth Rates of the Control Group

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 hours)	SD ±	RSD [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.76	1.54	1.39	1.56	0.185	11.8	17.4
	2	1.63	1.48	1.40	1.50	0.117	7.76
	3	1.73	1.58	1.11	1.47	0.323	22.0
	4	1.36	1.88	1.18	1.48	0.365	24.7
	5	1.67	1.74	1.11	1.51	0.346	22.9
	6	1.76	1.37	1.37	1.50	0.227	15.1
				Mean	1.50
				SD ±	0.03
				RSD [%]	2.24

RSD = rel. standard deviation

SD = Standard deviation

Concentrations of the test item

Sampling date	2010-11-15 0 h	2010-11-16 24 h			2010-11-17 48 h		2010-11-18 72 h
Start of analysis	2010-11-16	2010-11-16			2010-11-17		2010-11-18
Dilution Level of the Saturated Solution [%]	Test item								Geometric  Mean  Exposure  Concen-tration  [mg/L]
	Meas. conc. [mg/L]		Meas. conc. [mg/L]	RR [%]	Meas. conc. [mg/L]	RR [%]	Meas. conc. [mg/L]	RR [%]
100	12.2		13.9	114	12.8	105	11.5	94	12.8
100 (without algae)	-		15.2	125[1]	12.1	99	12.0	98	--
32	3.31		-	-	-	-	2.94	89	3.12
10	1.23		-	-	-	-	0.790	64	0.986
3.2	0.375		0.353	94	0.343	91	0.226	60	0.328
1.0	0.118		-	-	-	-	0.0595	50	0.0838
0.32	0.0353		0.0330	93	0.0364	103	0.0213	60	0.0321
0.32 (without algae)	-		0.0367	104[2]	0.0413	117	0.0345	98	--
Control	< LOQ		< LOQ		< LOQ		< LOQ		< LOQ

Meas. conc. = Measured concentration, dilution factor taken into account

RR  = Recovery rate related to the initially measured concentration of the test item in the fresh media

LOQ = Limit of quantification of the analytical method (5 µg test item/L)

- = No samples measured

-- = Not calculated

[1]based on 12.2 mg/L

[2]based on 0.0353 mg/L

Validity criteria fulfilled:

yes

Remarks:

cell growth increased 91-fold after 72 h in the control; mean coeff. of variation for section-by-section specific growth rates in control was 17.4 %; - rel. standard dev. of average specific growth rates during test period in replicate control was 2.24 %

Conclusions:

In this study the test item was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours. The geometric mean measured exposure NOEC-value for both inhibition of specific growth rate and inhibition of yield after 72 hours was 0.328 mg/L. The geometric mean measured exposure LOEC-values for both inhibition of specific growth rate and inhibition of yield after 72 hours was 0.986 mg/L. The geometric mean measured exposure EC50-values for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 1.03 (1.01 – 1.06) and 0.557 (0.507 – 0.618) mg/L, respectively.

Executive summary:

The toxicity test to aquatic algae and cyanobacteria performed according to OECD 201 and GLP was rated with RL=1 (reliable without restrictions) and used as a key study. The results indicate that the test item inhibits the growth of the freshwater green alga Desmodesmus subspicatus at a test item concentration of 0.986 mg/L (LOEC, growth rate) and greater. The NOEC is 0.328 mg/L. The EC50-values with 95 % confidence intervals for the growth rate (EC50) after 72 h were 1.03 (1.01 - 1.06) mg/L. All effect levels are given based on measured geometric mean value of the concentrations of the test item.

Description of key information

ErC10 (72h) 0.56 mg/L  (geom. mean measured, growth rate)
ErC50 (72h)  1.03 mg/L (geom. mean measured, growth rate)

 

Key value for chemical safety assessment

EC50 for freshwater algae:

1.03 mg/L

EC10 or NOEC for freshwater algae:

0.56 mg/L

Additional information

The toxicity test to aquatic algae and cyanobacteria (key_Toxicity to aquatic algae and cyanobacteria_2011_RL1) performed according to OECD 201 and GLP was rated RL=1 (reliable without restrictions) and used as key study. The 72 -h ErC10/ErC50 amount to 0.56 mg/L and 1.03 mg/L (geom. mean measured, growth rate), respectively.