Tris(4-chlorophenyl)sulfonium 5-benzoyl-4-hydroxy- 2-methoxybenzenesulfonate

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:

03 - 06 Aug 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

The temperature in the experiment was in a range of 20.9 – 25.7 °C and therefore higher than stated in the study plan. Because normal growth was observed, this can be stated as uncritical.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): PM-5927
- Substance type: Whitish/Crystalline powder
- Physical state: solid
- Lot/batch No.: 14-121
- Expiration date of the lot/batch: 01 June 2016
- Storage condition of test material: Room Temperature (20 ± 5 °C); Keep away from light

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The concentration of the test item in the test vessels was measured at the start and at the end of the test.

Test solutions

Vehicle:

no

Details on test solutions:

Preparation:
A stock solution containing 100.4 mg/L test item in nutrient medium (demineralised water enriched with minerals but without algae) was prepared. The test concentrations were prepared by dilution of the stock solution in nutrient medium. For this the volumes 0.44 mL, 0.92 mL, 2.0 mL, 4.4 mL, 9.2 mL and 20 mL of the stock solution were added into a 200 mL volumetric flask and filled up with nutrient medium.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: SAG 86.81
- Source (laboratory, culture collection): The culture of Desmodesmus subspicatus was obtained in February 2014 by MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 2-8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

20.9 – 25.7 °C

pH:

7.4 - 8.0

Nominal and measured concentrations:

control, 0.22, 0.46, 1.0, 2.2, 4.6, and 10 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: glass flasks, total volume 65 mL
- Initial cells density: 7546 cells/mL
- Control end cells density: 432458 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 5300 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer; photometric measurement of optical density at every 24 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 2.2
- Range finding study: yes
Test concentrations: a) 1, 10, and 100 mg/L; b) 0.05, 0.1, 0.5, and 1 mg/L
Results used to determine the conditions for the definitive study: In a first pre-test at the concentrations 1 / 10 / 100 mg/L in all treatments significant inhibition of algal growth could be observed. Therefore a second pre-test using the concentrations 0.05 mg/L, 0.1 mg/L, 0.5 mg/L and 1 mg/L was performed. In the treatments below 1 mg/L no inhibition of algal growth was observed.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.02 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 1.20 - 3.40 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 0.16 - 0.39 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.52 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 0.31 - 0.87 mg/L

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 0.16 - 0.39 mg/L

Effect concentrations 7

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 8

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Results with reference substance (positive control):

72h ErC50: 1.1 mg/L (95% CL: 0.95 – 1.3 mg/L)
72h EyC50: 0.39 mg/L (95% CL: 0.33 – 0.44 mg/L)

Reported statistics and error estimates:

Calculation of results was performed with the help of validated software (Microsoft Excel®). The estimation of the biological data was accomplished using the software ToxRat® Professional, version 3.1.

Any other information on results incl. tables

Analytical Determination:

The determined concentrations of the cation at the start and the end of the test were in the range of 35% - 99%. The determined concentrations of the anion at the start and the end of the test were in the range of 97% - 113%. The values found in the two lowest treatments after 72 hours for the cation where significantly lower than at the beginning. Nevertheless, the determination of the biological results was based on the nominal concentrations as the values for the anion were very close to the nominal concentrations.

Table 1: Measured concentrations (cation)

Nominal Concentration test item	Measured Concentration (t = 0 h)	Measured Concentration (t = 72 h)	% of Nominal Concentration (t = 0h)	% of Nominal Concentration (t = 72h)
mg/L	mg/L	mg/L	mg/L	mg/L
Blank control	n.d.	n.d.	n.d.	n.d.
0.22	0.18	0.08	81%	35%
0.46	0.40	0.31	87%	66%
1.0	0.98	0.90	98%	90%
2.2	2.10	1.90	95%	86%
4.6	4.50	4.51	98%	98%
10	9.59	9.86	96%	99%

Table 2: Measured concentrations (anion)

Nominal Concentration test item	Measured Concentration (t = 0 h)	Measured Concentration (t = 72 h)	% of Nominal Concentration (t = 0h)	% of Nominal Concentration (t = 72h)
mg/L	mg/L	mg/L	mg/L	mg/L
Blank control	n.d.	n.d.	n.d.	n.d.
0.22	0.22	0.22	99%	101%
0.46	0.52	0.45	113%	97%
1.0	1.07	1.03	107%	103%
2.2	2.16	2.13	98%	97%
4.6	4.62	4.55	100%	99%
10	9.65	9.82	97%	98%

Table 3: Growth rate and yield

Nominal Concentration in mg/L		Growth Rate (0-72h) [day^-1]	Yield (0-72h) [Cell Concentration/mL]
Blank control	Mean	1.35	424912
	SD	0.03	36099
0.22	Mean	1.40	500551
	SD	0.01	19414
0.46	Mean	1.11	205539
	SD	0.02	15485
1.0	Mean	0.88	102051
	SD	0.09	31784
2.2	Mean	0.60	38089
	SD	0.02	2713
4.6	Mean	0.43	20123
	SD	0.01	1245
10	Mean	0.31	11499
	SD	0.03	1647

SD = Standard deviation

Table 4: Inhibition values

Nominal concentration in mg/L	% Inhibition
	Growth Rate (0-72h)	Yield (0-72h)
Blankcontrol	0	0
0.22	-4.04	-17.80
0.46	17.47	51.63
1.0	34.51	75.98
2.2	55.54	91.04
4.6	67.90	95.26
10	77.18	97.29

Table 5: Cell number/mL

Nominal Concentration in mg/L	Parameter	Cell Number/mL
		0 h	24 h	48 h	72 h
Blank control	Mean	7546	24075	107081	432458
Blank control	SD	0	2783	18086	36099
0.22	Mean	7546	24794	140499	508097
0.22	SD	0	1078	3293	19414
0.46	Mean	7546	23716	90911	213085
0.46	SD	0	2156	3465	15485
1.0	Mean	7546	26591	64680	109597
1.0	SD	0	1245	3887	31784
2.2	Mean	7546	22997	37371	45635
2.2	SD	0	622	1245	2713
4.6	Mean	7546	20123	24075	27669
4.6	SD	0	622	1647	1245
10	Mean	7546	18685	15811	19045
10	SD	0	1245	1647	1647

Table 6: Daily growth rates of the controls

Growth rates	days 0 – 1	days 1 – 2	days 2 – 3	CV of sectional daily growth rates	days 0 – 3
Replicate 1	0.999	1.425	1.563	22%	1.33
Replicate 2	1.050	1.361	1.562	19%	1.32
Replicate 3	1.312	1.386	1.275	4%	1.32
Replicate 4	1.145	1.671	1.261	20%	1.36
Replicate 5	1.190	1.528	1.372	12%	1.36
Replicate 6	1.232	1.540	1.402	11%	1.39
Mean	1.155	1.485	1.406	15%	1.349
Standard deviation	0.116	0.117	0.133		0.027
CV	10%	8%	9%		2%

CV = Coefficient of variation

Validity:

Increase Factor:

The cell concentration in the blank control should increase by a factor of at least 16 within 72h.

Daily Growth Rates:

Mean coefficient of variation of daily growth rates in the blank controls should be 35% at the most. Coefficient of variation of average growth rate during the whole test period should be 7% at the most.

Values and assessment:

The daily growth rates of the blank controls were calculated. Means, standard deviations and coefficients of variation were determined.

Change of pH:

The pH of the blank control should not fluctuate by more than 1.5 units. The pH changed by 0.5 units in the blank control.

All validity criteria were met. The result of the test can be considered valid.

Applicant's summary and conclusion

Conclusions:

CLP: Aquatic chronic 2, H411 (basis for classification: not rapidly biodegradable and a NOEC (growth rate) of 0.22 mg/L for algae)