Sodium 3-morpholin-4-ylpropane-1-sulfonate

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:

2012-01-09 to 2012-03-08

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 23 March 2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

adopted 31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For the measurement of the samples of the study, a fresh calibration was performed and used for quantification. The stability was determined by measuring a Standard Solution containing 100 mg/L in algal test medium at the time levels 0 h and 72 h at room temperature.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
A stock solution containing 125 mg/L test item in deionised water was prepared.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: CHODAT
- Source: Institut für Pflanzenphysiologie of Universität Göttingen
- Method of cultivation: The algae are kept as stock culture on solid agar at 8 °C. From an aliquot of this stock culture the pre-culture was prepared.

ACCLIMATION
Four days before the start of each test, an aliquot of the stock culture containing a few cells was brought into pre-culture medium and incubated for 96 hours. The resulting culture is grown exponentially. Before usage, the culture was checked on the absence of cell aggregates.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

26 °C

pH:

8.1 – 10.2

Nominal and measured concentrations:

Nominal concentration: 100 mg/L;
Measured concentration: recovery of the test item was between 99 – 100 %

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks 50 mL volume
- Type: closed
- Initial cells density: 3.5 E4 cells /mL
- No. of vessels per concentration: six replicates
- No. of vessels per control: six replicates

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: before and after the test

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: continuous illumination
- Light intensity and quality: 4800 Lux

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Photometric measurement

TEST CONCENTRATIONS
- Spacing factor for test concentrations: limit test

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:

LOEC

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:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

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 curve (AUC)

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Area under the curve (AUC)

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 curve (AUC)

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- 72-h ErC50: 1.2 mg/L (95 %-CI: 1.1-1.4 mg/L)
- 72-h EbC50: 0.61 mg/L (95 %-CI: 0.57-0.67 mg/L)
- 72-h EyC50: 0.48 mg/L (95 %-CI: 0.43-0.53 mg/L)

FINDINGS

Cell Numbers

Table 1: Cell Number/mL Main Study

 

Nominal Conc. in mg/L	Parameter	Cell Number/mL
		0 h	24 h	48 h	72 h
0	Mean	18102	170159	859845	3088201
0	SD	0	65393	112684	974621
100	Mean	18102	214207	864371	3311158
100	SD	0	81894	73028	587978

 

Microscopical Observation

In the following table the appearance of the alga at the end of the test were stated:

Table 2: Microscopical Observation Main Study

 

Nom. Conc. in mg/L	Observation
0	Normal growth
100	Normal growth

 

Analytical Determinations

The measured concentrations showed very good correlation with the nominal concentrations. The recovery rate after 72 hours was 96 %. Therefore the nominal concentration was used for the determination of the results.

Table 3: Measured Concentrations in mg/L and corresponding Recovery Rates in %

 

Nom. Conc. in mg/L	Conc. Test Item mg/L 0h	Conc. Test Item mg/L 72h	Corr. Nom. 0h in %	Corr. Nom. 72h in %	Recovery in %
0	< LOQ	<LOQ	< LOQ	< LOQ	< LOQ
100	99.50	98.95	99.51%	98.95%	99.44%

 

Table 4: Area under the Curve, Growth Rate, Yield

 

Nom. Conc. in mg/L		Growth Rate [day^1]	AUC [Cell Conc./mL*day]	Yield [Cell Conc./mL]
0	Mean	1.70	2528849	3070099
	SD	0.11	627483	974621
100	Mean	1.73	2688901	3293056
	SD	0.06	317126	587978

SD = Standard Deviation

 

Table 5: Inhibition Values

 

Nom. Concentration in mg/L	% Inhibition
	Growth Rate µ	Area under the Curve AUC	Yield
0	0	0	0
100	-1.91	-6.33	-7.26

 

Table 6: Biological Results of the Test Item

 

Parameter	Value	95%-confidence-interval
NOEC (Growth Rate) 72 h	100 mg/L	not determinable
NOEC (AUC) 72 h	100 mg/L	not determinable
NOEC (Yield) 72 h	100 mg/L	not determinable
LOEC (Growth Rate) 72 h	> 100 mg/L	not determinable
LOEC (AUC) 72 h	> 100 mg/L	not determinable
LOEC (Yield) 72 h	> 100 mg/L	not determinable
72h ErC50	> 100 mg/L	not determinable
72h EbC50	> 100 mg/L	not determinable
72h EyC50	> 100 mg/L	not determinable
72h-ErC100	> 100 mg/L	not determinable
72h-EbC100	> 100 mg/L	not determinable
72h-EyC100	> 100 mg/L	not determinable

 

VALIDITY

Table 7: Growth Rate: Increase Factor: The cell concentration in the control should increase by a factor of at least 16 within 72h. The following increase factors were reached:

 

Study	Increase Factor	Assessment
Reference Study	88	ok
Main Study	171	ok

 

Table 8: Assessment Main Study

 

Parameter	Criteria	Value	Assessment
Increase factor of Cell Concentration	factor 16 in 72-h	171	valid
Mean coefficient of Variation of daily growth rates	max. 35%	32%	valid
Coefficient of Variation of average growth rate during the whole test period	max, 7%	6%	valid
Exponential growth during the whole test period given? (graphical assessment)			yes

 

Table 9: Cell Numbers

The cell numbers for each replicate and the corresponding means and standard deviations are presented in the following table. Because of clouded test solution in the fourth replicate of the control, photometric measurement was biased. This replicate was not used for evaluation of the results. No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.

 

Nom. Conc. in mg/L	Repl.	Cell Number/mL
		0 h	24 h	48 h	72 h
0	1	10102	99561	669774	2090781
	2	18102	199122	859845	2253699
	3	18102	117663	886998	2896320
	4	Not used	Not used	Not used	Not used
	5	18102	262479	959406	4208715
	6	18102	171969	923202	3991491
	Mean	18102	170159	859845	3088201
	SD	0	65393	112684	974621
100	1	18102	235326	868896	4272072
	2	18102	126714	841743	3421278
	3	18102	343938	733131	3204054
	4	18102	162918	886998	2588586
	5	18102	153867	941304	2851065
	6	18102	262479	914151	3529890
	Mean	18102	214207	864371	3311158
	SD	0	81894	73028	587978

SD = Standard Deviation

 

Table 10: Growth Rate M

The growth rate u for each replicate and the corresponding means and standard deviations are given in the following table. Because of clouded test solution in the fourth replicate of the control, photometric measurement was biased. This replicate was not used for evaluation of the results. No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.

 

Nom. Conc. in mg/L	Repl.	Growth Rate u	[day^1]
		24 h	48 h	72 h
0	1	1.70	1.81	1.58
	2	2.40	1.93	1.61
	3	1.87	1.95	1.69
	4	Not used	Not used	Not used
	5	2.67	1.99	1.82
	6	2.25	1.97	1.80
	Mean	2.18	1.93	1.70
	SD	0.39	0.07	0.11
100	1	2.56	1.94	1.82
	2	1.95	1.92	1.75
	3	2.94	1.85	1.73
	4	2.20	1.95	1.65
	5	2.14	1.98	1.69
	6	2.67	1.96	1.76
	Mean	2.41	1.93	1.73
	SD	0.38	0.04	0.06

SD = Standard Deviation

 

Table 11: Area under the Curve (Bio Mass Integral)

The Area under the Curve (Bio Mass Integral) AUC for each replicate and the corresponding means and standard deviations are given in the following table. Because of clouded test solution in the fourth replicate of the control, photometric measurement was biased. This replicate was not used for evaluation of the results. No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.

 

Nom. Conc. in mg/L	Repl.	AUC [Cell Concentration/mL*day]
		24 h	48 h	72 h
0	1	40730	407295	1769471
	2	90510	601892	2140562
	3	49781	534009	2407566
	4	Not used	Not used	Not used
	5	122189	715029	3280988
	6	76934	606417	3045662
	Mean	76028	572928	2528849
	SD	32697	113029	627483
100	1	108612	642621	3195003
	2	54306	520433	2633841
	3	162918	683351	2633841
	4	72408	579264	2298954
	5	67883	597366	2475449
	6	122189	692402	2896320
	Mean	98053	619239	2688901
	SD	40947	66104	317126

SD = Standard Deviation

 

Table 12: Yield

The Yield for each replicate and the corresponding means and standard deviations are given in the following table. Because of clouded test solution in the fourth replicate of the control, photometric measurement was biased. This replicate was not used for evaluation of the results. No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.

 

Nom. Conc. in mg/L	Repl.	Yield [Cell Concentration/mL]
		72 h
0	1	2072679
	2	2235597
	3	2878218
	4	Not used
	5	4190613
	6	3973389
	Mean	3070099
	SD	974621
100	1	4253970
	2	3403176
	3	3185952
	4	2570484
	5	2832963
	6	3511788
	Mean	3293056
	SD	587978

SD = Standard Deviation

 

Table 13: Inhibition Values of Single Replicates

The inhibition values of the single replicates are presented in the following table. Because of clouded test solution in the fourth replicate of the control, photometric measurement was biased. This replicate was not used for evaluation of the results. No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.

 

Concentration (mg/L)	Replicate	Inhibition Growth Rate u	Inhibition Area under the Curve AUC	Inhibition Yield
0	1	6.85	30.03	32.49
0	2	5.38	15.35	27.18
0	3	0.46	4.80	6.25
0	4	—	—	~
0	5	-6.87	-29.74	-36.50
0	6	-5.83	-20.44	-29.42
0	Mean	0	0	0
0	SD	6.27	24.81	31.75
100	1	-7.16	-26.34	-38.56
100	2	-2.81	-4.15	-10.85
100	3	-1.52	-4.15	-3.77
100	4	2.66	9.09	16.27
100	5	0.77	2.11	7.72
100	6	-3.42	-14.53	-14.39
100	Mean	-1.91	-6.33	-7.26
100	SD	3.43	12.54	19.15

SD = Standard Deviation

Validity criteria fulfilled:

yes

Conclusions:

The 72-hour NOErC was determined to be 100 mg test item/L and the associated 72-hour LOErC was > 100 mg test item/L.

Executive summary:

The acute toxicity of the test item was investigated in a GLP-study according to OECD test guideline 201 using unicellular freshwater green alga Desmodesmus subspicatus CHODAT. For this purpose, exponentially growing cultures of this unicellular green algal species were exposed to a nominal concentration of 100 mg/L of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus, over several algal generations. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium. This study encompassed six replicates for the test item concentration of 100 mg/L and control. The EC₅₀s of potassium dichromate were tested in a reference test. The cell concentration of each replicate was determined by measuring the absorption at 440 nm every 24 hours with a spectral photometer. With these measured values, the number of cells was calculated (linear correlation between cell concentration and absorption given). Then the growth rate µ, the area under the growth curve (AUC) and the Yield were determined. At the start and at the end of the test, the quantification of the test item in the test samples was performed using high performance liquid chromatography (HPLC) with UV. The recovery after 72 hours was 99 % of the start concentration, the measured concentration at the beginning and at the end of the test between 100 % and 99 % of the nominal concentration, respectively. Therefore, the determination of the results was based on the nominal concentrations. All validity criteria were met. The effect of the test item on freshwater green alga Desmodesmus subspicatus according to OECD TG 201 showed the following results: the 72-hour E_yC₅₀ was calculated to be > 100 mg test item/L, the 72-hour E_bC₅₀ was calculated to be > 100 mg test item/L and the 72-hour E_rC₅₀ value was calculated to be > 100 mg test item/L. The 72-hour NOE_yC was determined to be 100 mg test item/L and the associated 72-hour LOE_yC was > 100 mg test item/L. The 72-hour NOE_bC was determined to be 100 mg test item/L and the associated 72-hour LOE_bC was > 100 mg test item/L. The 72-hour NOE_rC was determined to be 100 mg test item/L and the associated 72-hour LOE_rC was > 100 mg test item/L. No abnormal appearance of the algal cells was observed during the experiment.

Description of key information

The 72-hour E_yC₅₀, E_bC₅₀ and E_rC₅₀ was calculated to be > 100 mg test item/L. The 72-hour NOE_yC, NOE_bC and NOE_rC was determined to be 100 mg test item/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 acute toxicity of the test item was investigated in a GLP-study according to OECD test guideline 201 using unicellular freshwater green alga Desmodesmus subspicatus CHODAT. For this purpose, exponentially growing cultures of this unicellular green algal species were exposed to a nominal concentration of 100 mg/L of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus, over several algal generations. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium. This study encompassed six replicates for the test item concentration of 100 mg/L and control. The EC₅₀s of potassium dichromate were tested in a reference test. The cell concentration of each replicate was determined by measuring the absorption at 440 nm every 24 hours with a spectral photometer. With these measured values, the number of cells was calculated (linear correlation between cell concentration and absorption given). Then the growth rate µ, the area under the growth curve (AUC) and the Yield were determined. At the start and at the end of the test, the quantification of the test item in the test samples was performed using high performance liquid chromatography (HPLC) with UV. The recovery after 72 hours was 99 % of the start concentration, the measured concentration at the beginning and at the end of the test between 100 % and 99 % of the nominal concentration, respectively. Therefore, the determination of the results was based on the nominal concentrations. All validity criteria were met. The effect of the test item on freshwater green alga Desmodesmus subspicatus according to OECD TG 201 showed the following results: the 72-hour E_yC₅₀ was calculated to be > 100 mg test item/L, the 72-hour E_bC₅₀ was calculated to be > 100 mg test item/L and the 72-hour E_rC₅₀ value was calculated to be > 100 mg test item/L. The 72-hour NOE_yC was determined to be 100 mg test item/L and the associated 72-hour LOE_yC was > 100 mg test item/L. The 72-hour NOE_bC was determined to be 100 mg test item/L and the associated 72-hour LOE_bC was > 100 mg test item/L. The 72-hour NOE_rC was determined to be 100 mg test item/L and the associated 72-hour LOE_rC was > 100 mg test item/L. No abnormal appearance of the algal cells was observed during the experiment.