2-(cyclohexylamino)ethanesulphonic 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:

2019-01-09 - 2017-09-21

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)

Version / remarks:

adopted 23rd March 2006, Annex 5 corrected: 28th July 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

March 01, 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/L

Six replicate samples were taken from the test solution and one from the control solution both at the start and at the end of the experiment. The samples were diluted with the eluent in two consecutive steps. In the second step 0.5 mL of Internal Standard Work Solution was added before the solution was filled up to volume. Then samples were analysed with LC/MS/MS applying MRM assay.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The test solution used in the test was prepared by mechanical dispersion. An amount of 0.07 g test item was dissolved in 700 mL dilution water (OECD medium) in order to obtain the concentration of 100 mg/L. The test solution was freshly prepared in the testing laboratory just before introduction of algae (start of the experiment).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: 61.81 SAG
- Source: SAG: Collection of Algal Cultures, 37073 Göttingen, Germany
- Method of cultivation: algae were kept as stock culture on solid agar

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

not specified

Test temperature:

22.0 - 22.2 °C

pH:

8.18– 8.96 (in the control group);
7.78-8.78 (in the treatment group)

Dissolved oxygen:

not specified

Salinity:

not applicable

Conductivity:

not specified

Nominal and measured concentrations:

Nominal concentration: 100 mg/L
Measured concentration: The measured concentrations showed very good correlation with the nominal concentrations. The recovery rate after 72 hours was 94 %.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks 250 mL
- Type: Closed (air-permeable stoppers)
- Initial cells density: 10000 cells/mL
- Control end cells density: approx. 1000000 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted algal growth medium (acc. to OECD TG 201)

OTHER TEST CONDITIONS
- Light intensity and quality: 7915 Lux

EFFECT PARAMETERS MEASURED: The algae
- Determination of cell concentrations: manual counting at 24, 48 and 72 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable as limit test
- Range finding study: yes
- Test concentrations: 100 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: area under the growth curve

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: area under the growth curve

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: none reported
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 0.71 mg/L (ErC50)

Reported statistics and error estimates:

Statistical comparisons of average specific growth rates and yield in control and in treated group were carried out using Student’s t-test (α = 0.05, TOXSTAT software).

Validity of the Test

- The cell density in the control cultures increased by a factor of 85.00 within 72 hours. This corresponds to a specific growth rate of 1.48 /day.

- The mean coefficient of variation (CV) for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72 h-tests) in the control cultures did not exceed 35 %

- CV for section-by-section growth rate day 0-1: 6.40 %

 - CV for section-by-section growth rate day 1-2: 5.70 %

 - CV for section-by-section growth rate day 2-3: 5.61 %

The mean coefficient of variation for section-by-section specific growth rates: 5.90 %.

- The coefficient of variation of average specific growth rates during the whole test period (day 0 - 3) in the control cultures was 1.21 %.

All validity criteria were met. Therefore, the study was considered as valid.

Analytical Results

A single concentration of 100 mg/L (limit test) and a concurrent control were tested in the main test. The concentration of the test item was analytically determined at the start and at the end of the experiment. In the untreated control group the test item was not detected. The measured concentration of the test item was 95 % of the nominal at the start and 94 % at the end of the test. The measured test item concentration remained within ± 20 % of the nominal during the test period of 72 hours. Therefore the biological results were based on the nominal concentration.

Table 1: Test Item concentrations measured in the test solutions

Nominal Concentration (mg/L)	Measured concentrations (mg/L)	In percentage of the nominal	Measured concentrations (mg/L)	In percentage of the nominal
	89.9	90%	97.1	97%
100	94.9	95%	93.9	94%
	93.9	94%	92.3	92%
	95.0	95%	93.3	93%
	92.8	93%	92.6	93%
	101.0	101%	92.7	93%
Mean with 95% confidence interval, (mg/L)	94.6 ± 3.8	95%	93.7 ± 1.9	94%

 Biological Results

In the treated group no inhibition was observed after 72 hours of exposure compared to the control group. The control group was considered to be within the normal biological range.

The results of the statistical evaluation showed that the 0 – 72-h average specific growth rate and yield at the treatment group (100 mg/L) were not statistically significantly different from the untreated control. Because the test item had no significant effect on the growth of algae, no statistical analysis was performed to determine EC values.

The No Observed Effect Concentration (NOEC) was determined to be 100 mg/L and the Lowest Observed Effect Concentration (LOEC) was determined to be >100 mg/L. The 72-h EC₅₀ values based on growth rate and yield were determined to be > 100 mg/L. No abnormal appearance of the algal cells was observed during the experiment.

Table 2: Summary of the Biological Results

Parameter (0-72 -h)	Growth rate (µ) [mg/L]	Yield (y) [mg/L]
EC50 95 % conf. limits	> 100 not calculated	> 100 not calculated
NOEC	100	100
LOEC	> 100	> 100

Table 3: Results of the Preliminary Range-finding Test

Nominal concentrations [mg/L]	Untreated control	0.01	0.1	1.0	10.0	100.0
Average cell number at 72 hours (x 10^4cells/mL)	67.67	70.50	71.00	70.50	71.00	69.50
Growth Rates (µ) [0 – 72-h]	0.0585	0.0591	0.0592	0.0591	0.0592	0.0589
% Inhibition of µ [0 – 72-h]	-	-0.98	-1.14	-0.98	-1.15	-0.64
Yield (Y) [0 – 72-h]	66.67	69.50	70.00	69.50	70.00	68.50
% Inhibition of Y [0 – 72-h]	-	-4.25	-5.00	-4.25	-5.00	-2.75

Table 4: Algal Cell Concentrations (10 cell/ mL) during the Test Period of 72 Hours and Yield

Test Group		0-h	24-h	48-h	72-h	Yield
Control		1	4	15	89	88
		1	4	16	85	84
		1	5	17	89	88
		1	4	16	80	79
		1	4	17	79	78
		1	4	15	88	87
	Mean	1.00	4.17	16.00	85.00	84.0
	SD	0.0	0.4	0.9	4.5	4.5
100 mg/L		1	4	16	84	83
		1	4	18	76	75
		1	4	18	83	82
		1	3	18	87	86
		1	4	17	88	87
		1	4	18	86	85
	Mean	1.00	3.83	17.50	84.00	83.0
	SD	0.0	0.4	0.8	4.3	4.3

Table 5: The Specific Growth Rates during the Test Period of 72 Hours

Test Group		Growth Rates (µ)
		0 – 24-h	0 – 48-h	0 – 72-h
Control		0.0578	0.0564	0.0623
		0.0578	0.0578	0.0617
		0.0671	0.0590	0.0623
		0.0578	0.0578	0.0609
		0.0578	0.0590	0.0607
		0.0578	0.0564	0.0622
	Mean	0.0593	0.0577	0.0617
	SD	0.0038	0.0012	0.0007
100 mg/L		0.0578	0.0578	0.0615
		0.0578	0.0602	0.0601
		0.0578	0.0602	0.0614
		0.0458	0.0602	0.0620
		0.0578	0.0590	0.0622
		0.0578	0.0602	0.0619
	Mean	0.0558	0.0596	0.0615
	SD	0.0049	0.0010	0.0007

Table 6: Growth Rates (µ) and Percentage Inhibition of µ during the Test Period

Test Group	Growth Rates (µ) and % Inhibition of µ
	0 – 24-h		0 – 48-h		0 – 72-h
	µ	%	µ	%	µ	%
Control	0.0593	0.0	0.0577	0.0	0.0617	0.0
100 mg/L*	0.0558	6.0	0.0596	-3.2	0.0615	0.3

Remark: negative inhibition means growth stimulation

*Statistically not significant compared to the control group (α =0.05; p-value: 0.1762)

Table 7: Yield (y) and Percentage Inhibition of y during the Test Period

Test Group	Yield y and % Inhibition of y
	0-72 h
	y	%
Control	84.0	0.0
100 mg/L*	83.0	1.2

*Statistically not significant compared to the control group (α =0.05; p-value: 0.1760)

Table 8: The section-by-section specific growth rates in the control cultures

Section-by-Section Growth Rate
Expressed in hours	0 – 24-h	24 – 48-h	48 – 72-h
	0.0593	0.0562	0.0696
Expressed in days	0 - 1 day	1 - 2 days	2 - 3 days
	1.4235	1.3478	1.6702

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions, the No Observed Effect Concentration (NOEC) was determined to be 100 mg/L and the 72-h EC50 values based on growth rate and yield were also > 100 mg/L.

Executive summary:

The toxicity of the test item on algal growth was assessed using the unicellular green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata and Selenastrum capricornutum) in a GLP-study according to OECD 201. For this purpose, exponentially-growing cultures of Raphidocelis subcapitata were exposed to the test item for 72 hours, over several generations under defined conditions (static test). The algal growth was determined over a fixed test period of 72 hours in relation to a concurrent untreated control culture. The reference item potassium dichromate served as the reference control. Per test concentration and control, six replicates were used. The alga cell concentration was approximately 10e4 cells/mL at the start of the test for all test cultures. Glass flasks with a total capacity of 250 mL were used as test vessels. The volume of the test liquid in the vessels was 100 mL. The alga cell concentration was determined by microscope in each testing flask at 24, 48 and 72 hours. Based on the results of a non-GLP preliminary test, in which the test item had no toxic effect on algae up to 100 mg/L (limit concentration), the main test was performed with 100 mg/L as a limit test. As measured test item concentration remained within ± 20 % of the nominal over the test period of 72 hours, the biological results were based on the nominal concentration. The purpose of the analytical part of the study was to verify the concentrations of the test item in the test medium. The measured concentration of the test item was 95 % of the nominal at the start and 94 % at the end. Statistical comparisons of average specific growth rates and yield in control and in treated group were carried out using Student’s t-test (α = 0.05). The results of the statistical evaluation showed that the 0 – 72-h average specific growth rate and yield in the treatment group (100 mg/L) were not statistically significantly different from the untreated concurrent control. The No Observed Effect Concentration (NOEC) was determined to be 100 mg/L and the Lowest Observed Effect Concentration (LOEC) was determined to be >100 mg/L (limit concentration). Consequently, the 72-h EC₅₀ values based on growth rate and yield were also > 100 mg/L. As a limit test was performed and as no effects were observed, no further statistical analysis was performed.

Description of key information

Under the test conditions, the NOEC was determined to be 100 mg/L and the 72-h EC₅₀ values based on growth rate and yield were also > 100 mg/L (reference 6.1.5-1).

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The toxicity of the test item on algal growth was assessed using the unicellular green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata and Selenastrum capricornutum) in a GLP-study according to OECD 201. For this purpose, exponentially-growing cultures of Raphidocelis subcapitata were exposed to the test item for 72 hours, over several generations under defined conditions (static test). The algal growth was determined over a fixed test period of 72 hours in relation to a concurrent untreated control culture. The reference item potassium dichromate served as the reference control. Per test concentration and control, six replicates were used. The alga cell concentration was approximately 10e4 cells/mL at the start of the test for all test cultures. Glass flasks with a total capacity of 250 mL were used as test vessels. The volume of the test liquid in the vessels was 100 mL. The alga cell concentration was determined by microscope in each testing flask at 24, 48 and 72 hours. Based on the results of a non-GLP preliminary test, in which the test item had no toxic effect on algae up to 100 mg/L (limit concentration), the main test was performed with 100 mg/L as a limit test. As measured test item concentration remained within ± 20 % of the nominal over the test period of 72 hours, the biological results were based on the nominal concentration. The purpose of the analytical part of the study was to verify the concentrations of the test item in the test medium. The measured concentration of the test item was 95 % of the nominal at the start and 94 % at the end. Statistical comparisons of average specific growth rates and yield in control and in treated group were carried out using Student’s t-test (α = 0.05). The results of the statistical evaluation showed that the 0 – 72-h average specific growth rate and yield in the treatment group (100 mg/L) were not statistically significantly different from the untreated concurrent control. The No Observed Effect Concentration (NOEC) was determined to be 100 mg/L and the Lowest Observed Effect Concentration (LOEC) was determined to be >100 mg/L (limit concentration). Consequently, the 72-h EC₅₀ values based on growth rate and yield were also > 100 mg/L. As a limit test was performed and as no effects were observed, no further statistical analysis was performed.