3-methylthiazolidine-2-thione

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:

2016-05-30 - 2016-07-11 (experimental phase)

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:

Version dated 23-March-2006

Deviations:

no

Remarks:

The concentration of NaHCO3 was increased to be twice of that indicated in the Guideline 201. This concentration has been found to be optimal.

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Mainzer Straße 80, D65189 Wiesbaden

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 100 mg/L
- Sample storage conditions before analysis: The supporting analyses could not be performed on the same day of collection of the specimens. Therefore, these specimens from freshly prepared test solutions were stored deep-frozen, thawed over-night and then analysed.

Vehicle:

no

Remarks:

medium was used

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: An aliquot of the algal stock culture was diluted with test medium to an absorbance value of E578 nm = 0.015 of the algal suspension giving an initial algal cell concentration of 3*10E3 cells/mL. The determination of cell concentration was performed by photometrical measurement using a calibration curve prepared by using a spectrophotometer at 578 nm. The test item was prepared as follows: Separate solutions were made for each test concentration in ultrapure water. Aliquots of these solutions were made up with „intermediate dilution“ and algal inoculum (or diluted „intermediate dilution“) to give 50 mL of final volume.
The test item was introduced into the test solutions as follows: First, suspensions of the test item were individually prepared for each test concentration in ultrapure water and stirred on a magnetic stirrer for 30 min. at room temperature after 10 minutes of ultrasonication (screening test) or by shaking on a shaking machine for 0.5 hrs at approx. 130 rpm and stirring for 15 min on a magnetic stirrer. Thereafter the suspensions were transferred into 8 test vessels (screening test) or 10 test vessels, respectively (main test). By the addition of the "intermediate dilution" and the algal inoculation the final nominal concentrations (see below) were achieved.
- Controls: medium without any test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none stated

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: Desmodesmus subspicatus CHODAT
- Source (laboratory, culture collection): Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen [SAG], Strain-No. 86.81
- Method of cultivation: The algae were cultivated under aseptic / sterile conditions.

ACCLIMATION
- Acclimation period: Three days before starting of the test (main test), an algal pre-culture was made which was inoculated from an algal stock culture derived from the algal type culture collection Göttingen in order to guarantee sterile conditions at all.
- Culturing media and conditions (same as test or not): The pre-culture was incubated under the same conditions as the test solutions to be tested afterwards.
- Any deformed or abnormal cells observed: none stated

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

24.2 – 25.2 °C (measured by aid of a data logger, system ELPRO)

pH:

7.99 - 9.72

Nominal and measured concentrations:

Nominal: 0, 100 mg/L
Found (t0): Found after “procedural recovery” correction (t0): -, 98.8 mg/L
Found (t48h): Found after “procedural recovery” correction (t48h): -, 107.1 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: cylinders
- Material, size, headspace, fill volume: heat-sterilized glass, 50 mL final volume
- Initial cells density: 3*10E3
- Control end cells density: 7.13*10E5
- No. of vessels per concentration (replicates): 7 (main test) / 5 (screening)
- No. of vessels per control (replicates): 7 (main test) / 5 (screening)

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Ultrapure water generating: Purelab Classic D1, USF Seral; Ser.-No. 1470984-10 01 (ELGA Labwater)
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continuous
- Light intensity and quality: illumination rate of ≥ 120 µE/m²s ([≈8000 Lux]

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer, daily

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0, 1, 10, 100 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: both growth rate and yield

Remarks on result:

other:

Remarks:

Results should be based on the nominal concentrations applied because the necessary stability of ≥ 80% could be shown.

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: both growth rate and yield

Remarks on result:

other:

Remarks:

Results should be based on the nominal concentrations applied because the necessary stability of ≥ 80% could be shown.

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: both growth rate and yield

Remarks on result:

other:

Remarks:

Results should be based on the nominal concentrations applied because the necessary stability of ≥ 80% could be shown.

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: both growth rate and yield

Remarks on result:

other:

Remarks:

Results should be based on the nominal concentrations applied because the necessary stability of ≥ 80% could be shown.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: both growth rate and yield

Remarks on result:

other:

Remarks:

Results should be based on the nominal concentrations applied because the necessary stability of ≥ 80% could be shown.

Details on results:

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

Results with reference substance (positive control):

not required

Cell Numbers (Mean Values, Screening Test)

Times	Nominal Concentrations mg test item/L
	Control	1 mg/L	10 mg/L	100 mg/L
t0	3*10^3	3*10^3	3*10^3	3*10^3
t22.75hr	1.8*10^4	2.0*10^4	2.0*10^4	1.9*10^4
t49.25hr	1.51*10^5	1.61*10^5	1.56*10^5	1.62*10^5
t72hr	8.01*10^5	8.24*10^5	8.27*10^5	8.53*10^5

 

Inhibition Values(Mean Values, Screening Test)

Nominal Concentration of the Test Item [mg/L]	Growth Rates (day^-1)	Inhibition of the Growth Rate after 72hr [%]	Yield after 72hr^1)	Inhibition of the Yield after 72hr [%]
Control (0)	1.905	-	7.99*10^5	-
1	1.914	-0.5^2)	8.21*10^5	-2.8^2)
10	1.916	-0.6^2)	8.25*10^5	-3.3^2)
100	1.926	-1.1^2)	8.51*10^5	-6.5^2)

¹⁾Yield = Cell number at time t_72hr– cell number at time t₀;

²⁾negative value indicates an activation

 

Cell Numbers (Mean Values, Main Test [Limit])

Times	Nominal Concentrations mg the test item/L
	Control	100 mg/L
t0	3*10^3	3*10^3
t24hr	2.3*10^4	2.2*10^4
t48.25hr	1.41*10^5	1.31*10^5
t72hr	7.13*10^5	7.00*10^5

 

Inhibition Values (Mean Values, Main Test)

Nominal Concentration of the Test Item [mg/L]	Growth Rates (day^-1)	Inhibition of the Growth Rate after 72hr [%]	Yield after 72hr^1)	Inhibition of the Yield after 72hr [%]
Control (0)	1.866	-	7.10*10^5	-
100	1.859	0.4	6.97*10^5	1.9

1)           Yield = Cell number at time t_72hr– cell number at time t₀;

 

 

pH-Values

pH values increased during the course of the study upon growth of the algae. This increase of pH in the controls was in the required range of ≤ 1.5 units according to the validity criteria given in OECD test guideline 201.

 

 

Results of the Analytical Part (GC-MS)

Sample Designation	mg test item/L found	mg test item/L found after “procedural recovery” correction	% Recovery on Basis of t0
Control time t0	<cal	-	-
100 mg/L time t0	72.2	98.8	-
Control time t72 hr	<cal	-	-
100 mg/L time t72 hr	78.4	107.1	108.6
Spike 100 mg/L (“procedural recovery”)	73.2	(100)	-

 

As a result of the supporting analysis it can be stated that the results of the biological part should be based on the nominal concentrations applied because the necessary stability of ≥ 80% could be shown.

 

 

 

Discussion

The toxicity of the test item towards algae was tested according to OECD-Guideline No. 201, in the Version dated 23-March-2006. The toxic effect was investigated by determination of the inhibition of the growth rate of the algae and the yield during the exposure period of 72 hours. The maximum concentration investigated was 100 mg/L, and the main test was performed as a limit test using 100 mg/L as the sole concentration. The following EC-values are assumed:

 

On the Basis of the nominal Concentrations [mg the test item/ L]

Yield (0 - 72 hr)                                                              EC10           >100            95%-CL     lower           >100                             upper           >100                                           EC20           >100            95%-CL     lower           >100                             upper           >100                                           EC50           >100            95%-CL     lower           >100                             upper           >100                              Yield    LOEC           >100                            NOEC           >100 Section-by-section growth rate (48.25-72hr)                              EC10           >100            95%-CL     lower           >100                             upper           >100                                           EC20           >100            95%-CL     lower           >100                             upper            >100                               EC50          >100            95%-CL     lower           >100                             upper           >100                                                     LOEC           >100                            NOEC           >100

Growth rate (0 - 72 hr)                                                     EC10           >100            95%-CL     lower           >100                             upper           >100                               EC20           >100            95%-CL     lower           >100                             upper           >100                               EC50          >100            95%-CL     lower           >100                             upper            >100       Growth rate    LOEC           >100                            NOEC           >100

As a conclusion of the analytical part of this study, it can be stated that the concentrations of the test item remained sufficiently stable during incubation of 72hr: After 72 hr there was a recovery of 108.6% of the initial concentration measured a time t₀.

 

Based on this, the reported effect concentrations (EC values) should refer to nominal concentrations of the test item.

 

The validity criteria for the study as given by the OECD Guideline 201 were met and the study is therefore considered to be valid. The increase of pH in the control by 1.44 meets the requirements of the Guideline OECD 201.

Validity criteria fulfilled:

yes

Conclusions:

The study was performed according to OECD TG 201 with no relevant deviations, the results were so obtained via a scientifically reasonable method. The validity criteria for the study as given by the OECD Guideline 201 were met and the study is therefore considered to be valid. Hence, there is no doubt that the obtained results are not reliable: The toxic effect was investigated by determination of the inhibition of the growth rate of the algae and the yield during the exposure period of 72 hours. The maximum concentration investigated was 100 mg/L, and the main test was performed as a limit test using 100 mg/L as the sole concentration. Due to the lack of any effects compared to control, all NOEC, LOEC, EC10, EC20, and EC50 are considered to be > 100 mg/l.
Based on these results, the test item does not need to be classified as acute or chronic toxic to the aquatic environment.

Executive summary:

The toxicity of 3-Methyl-thiazolidin-thion-2 towards algae was tested according to OECD-Guideline No. 201 (GLP), in the Version dated 23-March-2006. The toxic effect was investigated by determination of the inhibition of the growth rate of the algae and the yield during the exposure period of 72 hours. The maximum concentration investigated was 100 mg/L, and the main test was performed as a limit test using 100 mg/L as the sole concentration. The following EC-values are assumed:

 

On the Basis of the nominal Concentrations [mg 3-Methyl-thiazolidin-thion-2 / L]
Yield (0 - 72 hr)                                                              EC10           >100            95%-CL     lower           >100                             upper           >100                                           EC20           >100            95%-CL     lower           >100                             upper           >100                                           EC50           >100            95%-CL     lower           >100                             upper           >100                              Yield    LOEC           >100                            NOEC           >100 Section-by-section growth rate (48.25-72hr)                              EC10           >100            95%-CL     lower           >100                             upper           >100                                           EC20           >100            95%-CL     lower           >100                             upper            >100                               EC50          >100            95%-CL     lower           >100                             upper           >100                                                     LOEC           >100                            NOEC           >100		Growth rate (0 - 72 hr)                                                     EC10           >100            95%-CL     lower           >100                             upper           >100                               EC20           >100            95%-CL     lower           >100                             upper           >100                               EC50          >100            95%-CL     lower           >100                             upper            >100       Growth rate    LOEC           >100                            NOEC           >100

 

As a conclusion of the analytical part of this study, it can be stated that the concentrations of 3-Methyl-thiazolidin-thion-2 remained sufficiently stable during incubation of 72hr: After 72 hr there was a recovery of 108.6% of the initial concentration measured a time t₀.

 

Based on this, the reported effect concentrations (EC values) should refer to nominal concentrations of the test item.

 

The validity criteria for the study as given by the OECD Guideline 201 were met and the study is therefore considered to be valid. The increase of pH in the control by 1.44 meets the requirements of the Guideline OECD 201.

Description of key information

Acute toxicity to algae: NOEC, LOEC, EC10, EC20, and EC50 > 100 mg/L for Desmodesmus subspicatus (OECD 201, GLP, static)

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 result EC50 (72h) > 100 mg/L does not trigger classification as harmful for the aquatic environment, neither acute nor chronic, according to Regulation (EC) 1272/2008.