Sodium 3-[[(dimethylamino)thioxomethyl]thio]propanesulphonate

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:

2016-04-11 - 2016-04-14 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD 201 guideline study under GLP on the registered substance itself without deviations.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

OECD Guideline for the Testing of Chemicals No. 201, adopted 23. Mar. 2006, An-nex 5 corrected: 28 July 2011 “Freshwater Alga and Cyanobacteria, Growth Inhibition Test”

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Regulation EC No. 761/2009 amending Regulation EC No. 440/2008, Annex IV, Method C.3: “FRESHWATER ALGAE AND CYANOBACTERIA, GROWTH INHIBITION TEST,” adopted 23. July 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, 55116 Mainz, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 3-[[(dimethylamino)thioxomethyl]thio]propanesulphonate

EC Number:

242-644-7

EC Name:

Sodium 3-[[(dimethylamino)thioxomethyl]thio]propanesulphonate

Cas Number:

18880-36-9

Molecular formula:

C6H13NO3S3.Na

IUPAC Name:

sodium 3-[(dimethylcarbamothioyl)sulfanyl]propane-1-sulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): sodium 3-[[(dimethylamino)thioxomethyl]thio]propanesulphonate, DPS
- Substance type: pure substance
- Storage condition of test material: Room Temperature (20 ± 5°C)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 10, 18, 32, 56, 100 mg/L

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution containing 100.2 mg/L in algal medium (demineralised water enriched with minerals but without algae) was prepared. The lower treatments were prepared by dilution of this stock solution with nutrient medium.
- Controls: Blank control was deionised water with nutrient medium and algae. Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a separate reference test.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): none stated

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular freshwater green alga, Desmodesmus subspicatus
- Strain: SAG Strain Number 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).
- Method of cultivation: 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.

ACCLIMATION
- Culturing media and conditions (same as test or not): same
- Any deformed or abnormal cells observed: none stated

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

none

Test conditions

Test temperature:

22.1 - 23.4 °C

pH:

7.7 (0h) - 7.5 (72h)

Nominal and measured concentrations:

0, 10, 18, 32, 56, 100 mg/L (nominal)
Measured concentrations were withing 91-98%.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks total volume 65 mL
- Type (delete if not applicable): open (covered with perforated plastic foil)
- Material, size, headspace, fill volume: 20 ml headspace
- Initial cells density: 3.2 *10exp3 cells/mL
- Control end cells density: 159313 ± 36644 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
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: All solutions were sterilised before use.
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: 5300 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : daily
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Test concentrations: 0, 10, 18, 32, 56, 100 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9)

Results and discussion

Effect concentrations

Effect concentrations 1

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

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: yield

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 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.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: yield

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: yield

Effect concentrations 7

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

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none observed
- Unusual cell shape: none observed
- Colour differences: none observed
- Flocculation: none observed
- Adherence to test vessels: none observed
- Aggregation of algal cells: none observed
- Any stimulation of growth found in any treatment: yes
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none observed
- Effect concentrations exceeding solubility of substance in test medium: none

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50:
Growth rate: 0.80 mg/L, 95% confidence interval: 0.76 – 0.85 mg/L
Yield: 0.36 mg/L, 95% confidence interval: 0.35 – 0.38 mg/L

Any other information on results incl. tables

Findings

 

Cell Numbers

The cell numbers were determined by determination of the cell numbers via an electronic particle counter. The means and standard deviations of the cell numbers of the control and the treatments are presented in the following table:

Table Cell Number/mL

Nominal Concentration in mg/L	Para-meter	Cell Number/mL
		0 h	24 h	48 h	72 h
Blank control	Mean	3297	12213	63070	162610
Blank control	SD	196	444	6381	36694
10	Mean	3827	13193	61367	176840
10	SD	461	913	8722	38118
18	Mean	3973	14547	72847	194113
18	SD	31	867	5576	25373
32	Mean	3847	17747	86020	275060
32	SD	356	889	4011	9342
56	Mean	4500	15493	67053	227793
56	SD	1214	1962	8380	31490
100	Mean	4060	15027	68933	226647
100	SD	322	1910	13673	36490

SD = Standard Deviation

 

Light Intensity

In the following table,the light intensity during the test is stated:

 

Table Light Intensity

	0 h	24 h	48 h	72 h
Light intensity [lux]	5300	5300	5300	5300

 

pH values

In the following table,the pH values measured at the start and the end of the test are stated:

 

Table pH values

Nominal Concentrationin mg/L	0 h	72 h
Blank control	7.7	7.5
10	7.7	7.5
18	7.7	7.5
32	7.7	7.5
56	7.7	7.5
100	7.7	7.5

 

Microscopical Observations

In the following table, the appearance of the algae at the end of the test is stated:

 

Table Microscopical Observations

Nominal Concentrationin mg/L	Normal and Healthy Appearance of the Algae
Blank control	Yes
10	Yes
18	Yes
32	Yes
56	Yes
100	Yes

 

Analytical Determination

The measured concentrations after 72 hours were in a range between 91 % and 98 % of the nominal concentration. Therefore, the determination of the biological results was based on the nominal concentration.

The details are given in the following table:

 

Table Calculated Concentrations

Nominal Concentration Test Item	Measured Concentration  Test Item t = 0 h	Measured Concentration Test Item t = 72 h
mg/L	mg/L	mg/L
Blank control	< LOQ	< LOQ
10	9.08	9.50
18	17.12	17.39
32	30.45	31.23
56	53.78	54.68
100	95.51	97.65

LOQ (Limit of quantification) = 0.04 mg/L

 

Table Percentage of nominal Concentrations and Recovery

Nominal Concentration Test Item	% of Nominal concentration t = 0 h	% of Nominal concentration t = 72 h
mg/L	%	%
Blank control	--	--
10	91	95
18	95	97
32	95	98
56	96	98
100	96	98

 

Growth Rate, Yield

From the cell numbers, the Growth Rate µ and the Yield were calculated. The means and standard deviations at the end of the test are given in the following table:

 

Table Growth Rate µ, Yield

Nominal Concentration in mg/L	Parameter	Growth Rate (0-72h) [day^-1]	Yield (0-72h) [Cell Concentration/mL]
Blank control	Mean	1.29	159313
	SD	0.08	36644
10	Mean	1.27	173013
	SD	0.08	38059
18	Mean	1.29	190140
	SD	0.04	25379
32	Mean	1.42	271213
	SD	0.04	9695
56	Mean	1.31	223293
	SD	0.13	32584
100	Mean	1.34	222587
	SD	0.03	36171

SD = Standard Deviation

 

Inhibition

The following mean inhibition values were calculated for the Growth Rate µ and the Yield.

 

Table Inhibition Values

Nominal Concentration in mg/L	% Inhibition
	Growth Rate (0-72h)	Yield (0-72h)
Blank control	0	0
10	1.45	-8.60
18	-0.16	-19.35
32	-10.20	-70.24
56	-1.63	-40.16
100	-3.58	-39.72

Negative inhibition values indicate a stimulation of algal growth compared to the control.

 

Statistics and Results

Calculation of results was performed with the help of validated software (Microsoft Excel®). Biological results are stated using two significant digits.

 

Biological Results

The following values were determined:

 

Table Biological Results of the Test Item

Parameter	Value	95% confidence interval
NOEC (Growth Rate) 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 (Yield) 72 h	> 100 mg/L	not determinable
72h ErC10	> 100 mg/L	not determinable
72h EyC10	> 100 mg/L	not determinable
72h ErC50	> 100 mg/L	not determinable
72h EyC50	> 100 mg/L	not determinable

 

Biological Results of the Reference Study

The results of the last study with the positive control K₂Cr₂O₇ are presented in the following table. The study was performed under GLP conditions in November 2015.

 

Table Biological Results of K₂Cr₂O₇

Parameter	Value	95% confidence interval
72h ErC50	0.80 mg/L	0.76 – 0.85 mg/L
72h EyC50	0.36 mg/L	0.35 – 0.38 mg/L

 

Validity

 

Growth

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. Values and assessment can be found in the following tables.

Table 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	1.422	1.407	0.752	32%	1.194
Replicate 2	1.286	1.591	0.801	33%	1.226
Replicate 3	1.206	1.776	0.794	39%	1.259
Replicate 4	1.396	1.677	1.076	22%	1.383
Replicate 5	1.268	1.697	1.085	23%	1.350
Replicate 6	1.284	1.679	1.063	23%	1.342
Mean	1.311	1.638	0.929	29%	1.194
Standard deviation	0.082	0.127	0.161		0.076
CV	6%	8%	17%		6%

CV = Coefficient of Variation

 

Table Assessment

Parameter	Validity criteria	Observed value	Assessment
Increase factor biomass	factor 16 in 72 h	49	Valid
Mean coefficient of variation of daily growth rates	max. 35%	29%	Valid
Coefficient of variation of average growth rate during the whole test period	max. 7%	6%	Valid

 

Change of pH

The pH of the blank control should not fluctuate by more than 1.5 units. The change was 0.2 units in the blank control.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP according to OECD guideline 201 and EU method C.3 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the toxicity of DPS towards algae.
No significant inhibition of algal growth was observed in all tested concentrations of DPS up to 100 mg/L.
Based on the obtained results, sodium 3-[[(dimethylamino)thioxomethyl]thio]propanesulphonate does not need to be classified as hazardous to the aquatic environment according to the Regulation (EC) No. 1272/2008.

Executive summary:

In a 72 hour acute toxicity study, the cultures of Desmodesmus subspicatus, SAG strain number 86.81, were exposed tosodium 3-[[(dimethylamino)thioxomethyl]thio]propanesulphonate at nominal concentrations of 0, 10, 18, 32, 56, 100 mg/L under static conditions in accordance with the OECD guideline 201 and EU method C.3. Growth rate μ and the yield were determined from the cell densities every 24 hours. The NOEC, LOEC and EC₅₀values based on growth and yield were ≥ 100 mg/L (NOECs) and > 100 mg/L (LOECs, EC₅₀s), respectively. The % growth inhibition in the treated algal culture did not differ from the one of the control.

There were no abnormalities, and no compound related phytotoxic effects noted.

This toxicity study is classified as acceptable and satisfies the guideline requirements for a 72h algae toxicity study.