Reaction mass of morpholine and 6-[(p-tosyl)amino]hexanoic acid, compound with morpholine (1:1)

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 06 May 2008 to 09 May 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A nominal 1,000 mg/L solution was prepared in dilulation water, shaken vigorously and allowed to stand for one hour.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0, 1.6, 3.2, 6.3, 12.5, 25 and 50 mg/L.
- Evidence of undissolved material: A clear colourless solution was obtained.

Test organisms (species):

Skeletonema costatum

Details on test organisms:

TEST ORGANISM
- Common name: Skeletonema costatum
- Strain: 1077/5
- Source: Dunstaffnage Marine Laboratory, Scotland
- Method of cultivation: In natural seawater with added nutrients

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

Observations of algal growth rate were noted at 24, 48, and 72 hours of exposure

Test temperature:

20 +/- 1°C

pH:

Between 7.9 and 8.8

Nominal and measured concentrations:

Nominal concentrations 0, 1.6, 3.2, 6.3, 12.5, 25 and 50 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL conical flask
- Initial cells density:
- Control end cells density: 1x10^4 cells/mL
- No. of vessels per concentration (replicates): three
- No. of vessels per control (replicates): six

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: Continuous illumination
- Light intensity and quality: 6000 - 10000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: haemocytometer and microscope
- Chlorophyll measurement: no

TEST CONCENTRATIONS
- Range finding study: yes
- Results used to determine the conditions for the definitive study: 72h EC50 was identified to be between 10 and 32 mg/L

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.37 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: none reported
- Colour differences: none reported
- Flocculation: none reported
- Adherence to test vessels: none reported
- Aggregation of algal cells: none reported
- Any stimulation of growth found in any treatment: none reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none reported
- Effect concentrations exceeding solubility of substance in test medium: none reported

Results with reference substance (positive control):

The algae were exposed to a 1.5 mg/L concentration of 3,5-dichlorophenol for 72 hours under test incubation conditions and the 64% inhibition by growth rate represents an acceptable level of sensitivity (within the range of 20 to 80%).

Validity criteria fulfilled:

yes

Conclusions:

The toxicity of p-toluene sulphonamidocaproic acid morpholine salt to the marine algae Skeletonema costatum was determined in an alga growth inhibition test. Increasing concentrations of p-toluene sulphonamido caproic acid morpholine salt inhibited cell growth relative to the control. At 72 hours, the EC50 was determined to be 15.0 mg/L.

Executive summary:

A guideline study based on BS EN ISO 10253 - Water Quality: Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum (1998) was performed to determine the toxicity of p-toluene sulphonamidocaproic acid morpholine salt to aquatic algae Skeletonema costatum.

Nominal loading rates of 0, 1.6, 3.2, 6.3, 12.5, 25 and 50 mg/L were prepared in seawater following an initial range-finder study. The marine algae were exposed to test concentrations and monitored at 0, 24, 48, and 72 hours to determine the (inhibition) effect on cell density and the EC50.

Algal growth rates were reduced over time relative to the control, particularly at higher concentrations of p-toluene sulphonamido caproic acid morpholine salt, and the EC50 at 72 hours was reported as 15.0 mg/L.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From January 18, 2007 to December 05, 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: p-Toluene sulphonamido caproic acid morpholine salt was described as soluble in seawater, and therefore a 1.0 g/L stock solution was prepared in Guillard's f/2 medium + Si test media. Aliquots of the 1.0 g/L stock were diluted (i.e. dilution series) to achieve nominal concentrations over a definitive range of 0, 1.78, 3.16, 5.62, 10.0, 17.8, and 31.6 mg/L
- Controls: A control (0 mg/L) was used in a range-finding study to enable a suitable range of p-toluene sulphonamido caproic acid morpholine salt to be set. A control was incorporated into the main test at 0 mg/L, and 3,5-dichlorophenol was also used as a positive control (reference substance) at 1.5 mg/L
- Evidence of undissolved material: No

Test organisms (species):

Skeletonema costatum

Details on test organisms:

TEST ORGANISM
- Common name: Skeletonema costatum
- Source: Culture Collection of Algae and Protozoa, Dunstaffnage (CCAP1077/5)
- Method of cultivation: Stock cultures were maintained in Guillard's f/2 medium + Si in accordance with STL Runcorn SOP I.18

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

Observations of algal growth rate were noted at 24, 48, and 72 hours of exposure

Test temperature:

Temperature of the control and treated media was taken at 0, 24, 48, and 72 hours according to STL Runcorn SOP III.19, with mean temperature calculated as 20.5 ± 1.6 °C

pH:

In control and treated flasks, media pH was obtained at the beginning and end of the study according to STL Runcorn SOP III.19, with a mean pH of 8.4 ± 0.3. The pH at 0 (control) and 1.78 mg/L after 72 hours was noted as high due to high cell growth: 9.09 and 9.11, respectively

Nominal and measured concentrations:

Nominal: 0, 1.78, 3.16, 5.62, 10.0, 17.8, and 31.6 mg/L

Details on test conditions:

TEST SYSTEM
- Initial cell density: An initial algal cell concentration of 1112 cells per mL was counted microscopically, which equated to a mean cell density of 2511 cells per mL when determined using a Particle Count Analyser
- Control end cells density: Mean cell numbers per ml was 124321 at 72 hours, however, mean cell density was not specified
- No. of organisms per vessel: Initially, 2511 cells per mL
- No. of vessels per concentration: Three replicates
- No. of vessels per control: Six replicates

GROWTH MEDIUM
- Standard medium used: Yes, Guillard's f/2 medium + Si

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: p-Toluene sulphonamido caproic acid morpholine salt is soluble in seawater and, therefore, a stock solution of 1.0 g/L was prepared in test media, with aliquots taken from the main stock serially diluted to obtain a test range over 1.78 to 31.6 mg/L
- Alkalinity (pH): 8.44
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No
- Photoperiod: Continuous light for 72 hours
- Light intensity and quality: 6000-10000 lux
- Salinity: Not specified
- Other: Culture flasks were incubated in a rotary incubator at a target temperature of 20 ± 2 °C

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: To determine (mean) cell density, sub-samples of 5 - 10 ml of each vessel were obtained following incubation at 0, 24, 48, and 72 hours and measured with an electronic particle counter in accordance with STL Runcorn SOP III.19
- Chlorophyll measurement: No
- Other: Growth rate and percentage inhibition of growth (EC50) at 24, 48, and 72 hours were calculated for each test media

TEST CONCENTRATIONS
- Range-finding study: Yes, measuring the response of cell numbers (per ml) after a 72 hour exposure period
- Test concentrations: 0 (control), 1.0, 10.0, and 100.0 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

2.67 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% Fiducial Limits (mg/L): 2.04 - 2.97

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

4.45 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% Fiducial Limits (mg/L): 3.07 - 7.46

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.43 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.51 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% Fiducial Limits (mg/L): 5.41 - 10.4

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

16.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% Fiducial Limits (mg/L): 11.4 - 58.4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

5.62 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): No unusual cell growth or deformity
- Other: Clear evidence of a dose response in this study, with all parameters measured within the acceptable limits of tolerance. The lowest test concentration producing 100% inhibition was 17.8 mg/L after 72 hours
- Any stimulation of growth found in any treatment: Mean cell number (per mL) grew from 0 to 72 hours over the range of 1.78 - 10.0 mg/L of test material, although the stimulation of growth within this range became less evident as p-toluene sulphonamido caproic acid morpholine salt concentration increased. At 17.8 and 31.6 mg/L, cell number at the end of the study was less than the initial population of 1112 cells per mL. Overall, cell number after 72 hours of exposure was less in treated media than that in the control, highlighting an inhibitory affect

Reported statistics and error estimates:

The statistical computer programme Toxcalc (Tidepool Scientific, STL Runcorn SOP V.4) was used to determine the concentration of p-toluene sulponamido caproic acid morpholine salt required to elicit 50% inhibition of growth rate

Mean skeletonema costatum cell numbers (per mL) determined for each control and test media containing p-toluene sulphonamido caproic acid morpholine salt:

Exposure period (hours)	Test material concentration (mg/L)
	Control	1.78	3.16	5.62	10	17.8	31.6	3,5-dichlorophenol (1.5 mg/L)
0	1112	1112	1112	1112	1112	1112	1112	1112
24	8572	4714	2463	1574	940	651	652	6797
48	45238	29503	9302	6340	1556	597	553	18922
72	124321	109952	68740	35716	6760	1005	401	38628

Percentage inhibition of growth rate (Iµi) values determined for each test media containing p-toluene sulphonamido caproic acid morpholine salt:

Exposure period (hours)	Test material concentration (mg/L)
	Control	1.78	3.16	5.62	10.0	17.8	31.6	3,5-dichlorophenol (1.5 mg/L)
24		29.3	61.1	83.0	>100.0	>100.0	>100.0	11.4
48		11.5	42.7	53.0	90.9	>100.0	>100.0	23.5
72		2.6	12.6	26.4	61.7	>100.0	>100.0	24.8

pH data for media in control and test flasks:

Test material concentration (mg/L)	Hours
	0	24	48	72
0	8.44	-	-	9.09
1.78	8.02	-	-	9.11
3.16	8.13	-	-	8.97
5.62	8.19	-	-	8.35
10.0	8.25	-	-	8.24
17.8	8.25	-	-	8.25
31.6	8.35	-	-	8.27
3,5-dichlorophenol (1.5 mg/L)	8.46	-	-	8.44

Temperature (°C) data for media in control and test flasks:

Test material concentration (mg/L)	Hours
	0	24	48	72
0	18.0	20.0	20.5	20.0
1.78	18.0	21.5	22.0	20.0
3.16	18.0	21.5	22.0	21.5
5.62	18.0	21.0	22.0	22.0
10.0	18.0	21.0	22.0	22.0
17.8	18.0	21.0	22.0	22.0
31.6	18.0	21.0	22.0	22.0
3,5-dichlorophenol (1.5 mg/L)	18.0	21.0	21.0	21.5

Validity criteria fulfilled:

not specified

Conclusions:

The toxicity of p-toluene sulphonamido caproic acid morpholine salt to the marine algae Skeletonema costatum was determined in a short-term static test based on nominal loading rates between 1.78 and 31.6 mg/L. Increasing concentrations of p-toluene sulphonamido caproic acid morpholine salt inhibited cell growth relative to the control (0 mg/L). At 72 hours, the EC50 was determined to be 7.51 mg/L.

Executive summary:

A guideline study based on BS EN ISO 10253 - Water Quality: Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum (1998) was performed to determine the toxicity of p-toluene sulphonamido caproic acid morpholine salt to aquatic algae Skeletonema costatum. Nominal loading rates of 0, 1.78, 3.16, 5.62, 10.0, 17.8, and 31.6 mg/L were prepared in seawater following an initial range-finder study. The marine algae were exposed to test concentrations and monitored at 0, 24, 48, and 72 hours to determine the (inhibition) effect on cell density and the EC50. Algal growth rates were reduced over time relative to the control, particularly at higher concentrations of p-toluene sulphonamido caproic acid morpholine salt, and the EC50 at 72 hours was reported as 7.51 mg/L.

Description of key information

The acute toxicity of p-toluene sulphonamido caproic acid morpholine salt to marine water algae was determined in two studies conducted in accordance with ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum), with no reported deviation.

First study performed on Skeletonema costatum resulted in an EC₅₀ = 7.51 mg/L based on growth inhibition. It was concluded that the salt had not been fully neutralised during the test and therefore a purer sample was tested.

This second study concluded that the EC₅₀ of the test substance was 15 mg/L based on growth inhibition of Skeletonema costatum. This result is considered as more reliable taking into account the purity of the sample used for testing.

Based on these results it is concluded that the EC₅₀ for the registered substance should be reported as 15 mg/L.

Key value for chemical safety assessment

EC50 for marine water algae:

15 mg/L