4-(cyclohexylamino)butane-1-sulfonic 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:

13 May 2019 - 16 Jul 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

28 July 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Correction factor for purity: not required
- Stability at higher temperatures: stable
- Solubility in water: 110 g/L

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All test concentrations and control
- Sampling method: 2.0 mL from the approximate centre of the test vessels at t=0 h and t=72 h.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the test facility.
- At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at 100 mg/L but without algae and samples for analysis were taken at the start and at the end of the test period.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: the test item was completely soluble in test medium at the concentrations tested. Preparation of test solutions started with the highest concentration of 100 mg/L applying a one-hour period of magnetic stirring to accelerate the dissolution of the test item in test medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
- Controls: Test medium without test item or other additives.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata)
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Age of inoculum at test initiation: 3 days
- Method of cultivation: Algae stock cultures were started by inoculating stock culture medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light (60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm) in a climate room at a temperature of 21-24°C.
- Stock culture medium: M1 medium, according to NPR 6505
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in pre-culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Pre-culture medium and test medium: M2 medium, according to OECD 201

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

22 - 23°C

pH:

Control: 8.0 (t=0 h); 7.9 (t=72 h)
100 mg/L: 8.0 (t=0 h); 7.8 (t=72 h)

Nominal and measured concentrations:

Nominal: 100 mg/L
Measured: 100 mg/L (with algae) at t=0 h 115 mg/L and at t=72h 121 mg/L; 100 mg/L (without algae) at t=0 h 117 mg/L and at t=72 h 121 mg/L
Based on these results, the nominal exposure concentrations were used to determine the effect parameters. See Table 3 in 'Any other information on results incl. tables' for details.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass with aluminium caps, perforated for ventilation, containing 50 mL of test solution
- Aeration: no
- Initial cells density: 10^4 cells/mL
- Control end cells density: 260 x 10^4 cells/mL
- Replicates: 6 replicates of the control and the test concentration, 1 extra replicate of the test concentration without algae.

GROWTH MEDIUM
- Standard medium used: yes, M2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2, formulated using tap-water purified by reverse osmosis.
- Intervals of water quality measurement:
pH: at the beginning and at the end of the test, for the limit concentration and the control.
Temperature of medium: continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod and light intensity: Continuously using TLD-lamps with a light intensity within the range of 85 - 88 µE.m-2.s-1.
- Incubation: Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
- Appearance of the cells: At the end of the limit test, microscopic observations were performed on the 100 mg/L test concentration and the control to observe for any abnormal appearance of the algae.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (performed Apr 2019)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

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

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:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 100 mg/L when compared to the control.
- No significant differences were recorded between the values for growth rate and yield at any of the test concentrations when compared to the control group. No inhibition of growth rate and yield was observed. See Table 1 and Table 2 in 'Any other information on results incl. tables' for details.
- Analytical results: The measured concentrations were at 115 and 121% relative to the nominal concentrations at the start and end of the test, respectively. The concentrations in the samples without algae were comparable, i.e. were 117 and 121% relative to the nominal concentrations at the start and end of the test, respectively. The presence of biomass thus had no effect on the test item concentrations. Based on these results and the minimal exceedance above the 120% limit (121%) given in the study plan, effect parameters were expressed as analytically confirmed nominal concentrations.
- All water quality parameters remained within the requirements as laid down in the study plan throughout the test.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- The EC50 for growth rate inhibition (72h-ErC50) was 1.11 mg/L with a 95% confidence interval ranging from 1.10 - 1.13 mg/L. The historical ranges for growth rate inhibition lie between 0.86 and 2.3 mg/L. Hence, the 72h-ErC50 for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

NOEC and ECx calculations:
An effect was considered to be significant if statistical analysis of the data obtained for the test concentration compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Two-sample Mann-Whitney U-test Procedure, α=0.05, one-sided, smaller).
The ECx-values could not be determined because no algal growth inhibition was obseved.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Table 1: Mean growth rate and yield, inhibition of growth rate and yield

Nominal concentration (mg/L)	Mean growth rate 0-72 h (day^-1, ± SD)	Inhibition of growth rate (%)	Mean yield 0-72 h (x10^4cells/mL, ± SD)	Inhibition of yield (%)
Control	1.854 ± 0.0126	-	259.881 ± 9.8956	-
100	1.861 ± 0.0371	-0.35	266.189 ± 27.2355	-2.4

 

Table 2: Individual cell densities (x10⁴ cells/mL)

		0 h	24 h	48 h	72 h
Control	Replicate 1	1.000	9.804	50.189	259.470
	Replicate 2	1.000	11.572	56.371	272.964
	Replicate 3	1.000	10.257	53.249	248.909
	Replicate 4	1.000	8.828	51.245	259.304
	Replicate 5	1.000	9.987	54.247	272.109
	Replicate 6	1.000	9.235	52.548	252.530
	Mean	1.000	9.947	52.975	260.881
	St. dev	0.000	0.951	2.198	9.896
	CV	0.000	9.556	4.150	3.793
Test item	Replicate 1	1.000	10.005	52.221	275.542
	Replicate 2	1.000	9.310	48.289	212.472
	Replicate 3	1.000	10.194	56.865	277.057
	Replicate 4	1.000	10.688	58.805	285.466
	Replicate 5	1.000	10.447	56.038	281.184
	Replicate 6	1.000	10.297	54.695	271.415
	Mean	1.000	10.157	54.486	267.189
	St. dev	0.000	0.475	3.750	27.236
	CV	0.000	4.675	6.882	10.193

 

Table 3: Analytical results dilution factor into account. Samples might be contaminated in course of sampling. #: sample without algae

Time of sampling (h)	Nominal concentration (mg/L)	Analyzed concentration (mg/L)	Relative to nominal (%)	Relative to initial (%)
0	0	n.d.	n.a.	-
	100	115	115	-
	100#	117	117	-
72	0	0.0074*	n.a.	n.a.
	100	121	121	105
	100#	121	121	103

n.d.: Not detected; n.a.: Not applicable; *: The maximum contribution to the 100 mg/L samples was 0.0066% taking the dilution factor into account. Samples might be contaminated in course of sampling. #: sample without algae

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' for details on validity criteria

Conclusions:

No biologically relevant inhibition of growth rate or yield was recorded at the limit concentration tested (100 mg/L). The 72h-ErC50 and 72h-EyC50 values were determined to be >100 mg/L (analytically confirmed nominal concentration).
The 72h-NOEC for both growth rate inhibition and yield inhibition was 100 mg/L.

Executive summary:

In a 72h toxicity study conducted in accordance with OECD guideline 201, freshwater algae (Pseudokirchneriella subcapitata) were exposed to 100 mg/L and an untreated control (both 6 replicates).

The exposure concentration was, both with and without algae, analytically confirmed to be 115 -121% of nominal throughout the test. Therefore, nominal exposure concentrations were used to determine the effect parameters. No inhibition of growth rate or yield was recorded at the tested limit concentration of 100 mg/L. The 72h-ErC50 and 72h-EyC50 values were beyond the range tested, i.e. exceeded an analytically confirmed nominal concentration of 100 mg/L. The 72h-NOEC for both growth rate inhibition and yield inhibition was 100 mg/L. The study met all validity criteria, and is considered reliable without restriction.

Description of key information

No biologically relevant inhibition of growth rate or yield was recorded at the limit concentration tested (100 mg/L). The 72h-ErC50 and 72h-EyC50 values were determined to be >100 mg/L (analytically confirmed nominal concentration).
The 72h-NOEC for both growth rate inhibition and yield inhibition was 100 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

In a 72h toxicity study conducted in accordance with OECD guideline 201, freshwater algae (Pseudokirchneriella subcapitata) were exposed to 100 mg/L and an untreated control (both 6 replicates).

The exposure concentration was, both with and without algae, analytically confirmed to be 115 -121% of nominal throughout the test. Therefore, nominal exposure concentrations were used to determine the effect parameters. No inhibition of growth rate or yield was recorded at the tested limit concentration of 100 mg/L. The 72h-ErC50 and 72h-EyC50 values were beyond the range tested, i.e. exceeded an analytically confirmed nominal concentration of 100 mg/L. The 72h-NOEC for both growth rate inhibition and yield inhibition was 100 mg/L. The study met all validity criteria, and is considered reliable without restriction.