2,2'-(cyclohexylimino)bisethanol

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-08-09 to 2011-08-12

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-(cyclohexylimino)bisethanol

EC Number:

224-809-5

EC Name:

2,2'-(cyclohexylimino)bisethanol

Cas Number:

4500-29-2

Molecular formula:

C10H21NO2

IUPAC Name:

2,2'-(cyclohexylimino)bisethanol

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The concentrations of the test item were analytically determined from all test concentrations and the control after 0 hours (test start) and 72 hours (test end).
- Sampling method: Samples at test start were taken from an additional replicate without algae. At end of the test samples were taken directly from the pooled test replicates.
- Sample storage conditions before analysis:All samples were stored at room temperature until start of analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 100 mg/L was freshly prepared with dilution water and agitated.
- Eluate: Dilution water
- Differential loading: 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 100 mg/L was freshly prepared and agitated.
- Eluate: Dilution water
- Differential loading: 0.320 - 1.00 - 3.20 - 10.0 - 32.0 - 100 mg/L
- Controls: Six replicated without test item were tested under the same test conditions as the test replicates.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.


ACCLIMATION
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: No

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Test conditions

Hardness:

Not measured

Test temperature:

Mean: 22.5 °C, Min: 22 °C, Max: 23 °C

pH:

Nominal test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
100 9.11 8.10
32.0 8.72 8.06
10.0 8.44 8.49
3.20 8.11 8.79
1.00 8.01 8.82
0.320 7.98 8.72
Control 7.91 8.63

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to Any other information on materials and methods inc. tables

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: 4910 cells/mL
- Control end cells density: Mean 683511 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None


GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.



TEST CONCENTRATIONS
- Range finding study: Yes
Results of the Range Finding Test (0-72 hours)

Nominal
Test Item Concentration [mg/L] Specific Growth Rate Inhibition [%] Yield Inhibition [%]
100 92 100
10.0 8 32
1.00 2 11
0.100 - 1 - 4
100 (neutralized) 95 100

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10.8 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: (8,8-21)

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

8.1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: (5-19 mg/L)

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- 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: Yes

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.560 (CI 0.531 - 0.593) mg/L
Yield Inhibition: 0.260 (CI 0.234 - 0.289) mg/L

Reported statistics and error estimates:

NOEC/LOEC were determined by calculation of statistical
significance of growth rate and yield. 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. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.
Normality test and Equal Variance Test failed for growth rate calculation. The growth rate values were transformed using Graph Pad Prism function (Y = Y squared).
Detection of statistically significant differences were considered negligible when inhibition values were ≤ 10 %. These findings were marked “biologically not significant”. Following the recommendations of the UBA (HEGER ET AL. 1998), inhibitions
> 10 % are significant.

Any other information on results incl. tables

NOEC, LOEC, EC - values and 95 % Confidence Intervals of

                   2,2’-(Cyclohexylimino)diethanol(0-72 hours)

                   based on nominal test item concentrations [mg/L]

                               

	Rate-related Inhibition
NOEC	3.20
LOEC	10.0
ErC10	9.40 (5.37 – 22.4)
ErC20	10.6 (6.25 – 22.7)
ErC50	12.5 (10.2 – 24.1)
	Inhibition of Yield
NOEC	0.320
LOEC	0.320
EyC10	0.320
EyC20	1.98 (1.21 – 3.10)
EyC50	9.89 (9.04 – 11.2)

Cell Densities

	Nominal test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 hours		24 hours	48 hours	72 hours
100		1		4910		8900	6673	5691
		2		4910		10793	7167	7360
		3		4910		9361	5536	8795
		Mean		4910		9685	6459	7282
32.0		1		4910		4910	4910	4910
		2		4910		4910	4910	4910
		3		4910		4910	4910	4910
		Mean		4910		4910	4910	4910
10.0		1		4910		19089	91291	347803
		2		4910		17295	78824	350985
		3		4910		18795	81214	322687
		Mean		4910		18393	83776	340492
3.20		1		4910		19495	114150	509437
		2		4910		19611	119959	559264
		3		4910		21435	106260	512416
		Mean		4910		20180	113456	527039
1.00		1		4910		18791	123340	550531
		2		4910		22143	128557	522909
		3		4910		19336	118744	575817
		Mean		4910		20090	123547	549752
0.320		1		4910		17739	128797	605368
		2		4910		18781	114448	565763
		3		4910		19485	123127	607839
		Mean		4910		18668	122124	592990
Control		1		4910		19208	122068	644702
		2		4910		22288	122423	696255
		3		4910		25142	141115	703533
		4		4910		18778	126357	691685
		5		4910		21506	120917	654653
		6		4910		20768	121262	710235
		Mean		4910		21282	125690	683511

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

Nominal test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
100	1		0.049	97.0		781	99.9
	2		0.135	91.8		2450	99.6
	3		0.194	88.2		3885	99.4
	Mean	(+)	0.126	92.3	(+)	2372	99.7
32.0	1		0.000	100		0	100
	2		0.000	100		0	100
	3		0.000	100		0	100
	Mean	(+)	0.000	100	(+)	0	100
10.0	1		1.42	13.7		342893	49.5
	2		1.42	13.5		346075	49.0
	3		1.40	15.2		317777	53.2
	Mean	(+)	1.41	14.1	(+)	335582	50.6
3.20	1		1.55	5.94		504527	25.7
	2		1.58	4.05		554354	18.3
	3		1.55	5.82		507506	25.2
	Mean	(+)*	1.56	5.27	(+)	522129	23.1
1.00	1		1.57	4.37		545621	19.6
	2		1.56	5.41		517999	23.7
	3		1.59	3.46		570907	15.9
	Mean	(+)*	1.57	4.41	(+)	544842	19.7
0.320	1		1.61	2.45		600458	11.5
	2		1.58	3.82		560853	17.4
	3		1.61	2.36		602929	11.2
	Mean	(+)*	1.60	2.88	(+)	588080	13.3
Control	1		1.63			639792
	2		1.65			691345
	3		1.66			698623
	4		1.65			686775
	5		1.63			649743
	6		1.66			705325
	Mean		1.65			678601

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 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.36	1.85	1.66	1.63	0.245	15.1	10.5
	2	1.51	1.70	1.74	1.65	0.121	7.35
	3	1.63	1.73	1.61	1.66	0.062	3.76
	4	1.34	1.91	1.70	1.65	0.286	17.3
	5	1.48	1.73	1.69	1.63	0.135	8.25
	6	1.44	1.77	1.77	1.66	0.187	11.3
				Mean	1.65
				SD ±	0.01
				CV [%]	0.81

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study 2,2’-(Cyclohexylimino)diethanol was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours. The NOEC-values for inhibition of specific growth rate and inhibition of yield after 72 hours were 3.20 mg/L and < 0.320 mg/L, respectively. The LOEC-values for inhibition of specific growth rate and inhibition of yield after 72 hours were 10.0 mg/L and 0.320 mg/L, respectively. The EC50-values for inhibition of specific growth rate (ErC50) and yield (EyC50) with 95 % Confidence Intervals after 72 hours were 12.5 (10.2 – 24.1) and 9.89 (9.04 – 11.2) mg/L, respectively.
All effect levels are given based on nominal concentrations of 2,2’-(Cyclohexylimino)diethanol.

Executive summary:

The toxicity of 2,2’-(Cyclohexylimino)diethanol(batch number: ESD0011452) to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany from August 09 to 12, 2011. The aim of the study was to assess 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 5 x 10³ cells/mL. Based on a preliminary test 6 concentration levels were tested in a geometrical series with a dilution factor of √10, nominal: 0.320 - 1.00 - 3.20 - 10.0 - 32.0 - 100 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of 2,2’-(Cyclohexylimino)diethanolwere analytically verified by LC-MS/MS at test start and test end of all test concentrations and the control.
The measured concentrations of 2,2’-(Cyclohexylimino)diethanolat test start were in the range of 101 to 105 % and after 72 hours in the range of 110 to 115% of the nominal values. Therefore, all effect values given are based on the nominal concentrations of the test item 2,2’-(Cyclohexylimino)diethanol.

NOEC, LOEC, EC - values and 95 % Confidence Intervals of

                   2,2’-(Cyclohexylimino)diethanol(0-72 hours)

                   based on nominal test item concentrations [mg/L]

                               

	Rate-related Inhibition
NOEC	3.20
LOEC	10.0
ErC10	9.40 (5.37 – 22.4)
ErC20	10.6 (6.25 – 22.7)
ErC50	12.5 (10.2 – 24.1)
	Inhibition of Yield
NOEC	0.320
LOEC	0.320
EyC10	0.320
EyC20	1.98 (1.21 – 3.10)
EyC50	9.89 (9.04 – 11.2)