2,2-dimethylthiazolidine

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

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 March 23, 2006, updated Annex 5, 28 July 2011

Deviations:

no

Principles of method if other than guideline:

Deviations from the Study Plan and/or the Guideline
Deviating from the study plan the water samples were missed to stabilize by addition of acetonitrile directly after sampling. Based on the information given by the principal investigator this deviation should have had no influence on the stability of the test item under the recommended storage condition. This deviation has no impact on the integrity of the study.
Deviating from the study plan the analytical replicated samples were combined for the additional determination of the dissolved organic carbon content (DOC), to provide sufficient volume for this analytical method. A further analytical method had to be used, since the test item was not stable in aqueous solution within 24 hours. This deviation has no impact on the integrity of the study.
Otherwise, the study was performed according to the guideline, the study plan and its amendment(s).

GLP compliance:

yes

Specific details on test material used for the study:

Density: 1.02 kg/L (20°C)

Analytical monitoring:

yes

Remarks:

Gas Chromatographic (GC/FID) and DOC Determination

Details on sampling:

- Sampling schedule: Test solutions were analysed at start of exposure period (time 0 hours) and at the end of the exposure at 72 hours
- Storage conditions: Cool, dry, well ventilated, protected from light; in tightly closed container; not above 8 °C (refrigerator)
- Stability under correct storage conditions (expiry date): March 15, 2018

Vehicle:

no

Details on test solutions:

Preparation of Stock, Application and Test Solutions
A stock solution at a nominal concentration of 50 mg test item/L was prepared by directly adding 100.4 mg test item to 2000 mL test medium. This stock solution (S1) was shaken three times over head and thereafter stirred using a magnetic stirrer at average speed for ca. 20 min at ambient temperature. Thereafter this stock solution was left to settle for ca. 10 minutes. Visual examination showed a clear stock solution without undissolved/particulate matter.
The stock solution (S1) was prepared once to prepare the test solutions.
The volume of the stock solution was large enough to prepare the test solutions and all analytical samples and solutions used for conditioning.
The vessels containing the test solutions were stirred for ca. 5 minutes on a magnetic stirrer at room temperature and placed into a temperature controlled room for temperature adaptation.
After temperature adaptation of the test solutions, 1.639 or 3.279 mL of the algal pre-culture (Pseudokirchneriella subcapitata) were added to each of the volumetric flasks containing 500 or 1000 mL of the test solutions, respectively to achieve an algal cell concentration of approximately 0.3×10E4 cells/mL. These manipulations were performed under a laminar flow box. The test solutions were homogenised by manual shaking 3 times overhead shortly after addition of the algal pre-culture and again 15 times overhead, before adding 100±5 mL of the test media to each test vessel to ensure a homogeneous distribution of the algal cells.
The test vessels were placed onto a shaker under light and temperature controlled conditions.
The medium was sterilised by sterile filtration (pore size 0.2 µm) before use. All glassware and materials used for testing purposes were sterilised for at least 3 hours at ≥150 °C using a heating furnace or autoclaved for 20 minutes at 120°C, except preparation flask (volumetric flask) used for preparation of test solution.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

Pre-Culture Conditions
To adapt the algae to the test conditions a pre-culture was inoculated by a liquid algal culture and incubated under test conditions.
Species: Pseudokirchneriella subcapitata (SAG 61.81), currently valid species name: Raphidocelis subcapitata
Supplier: Sammlung von Algenkulturen, Albrecht-von-Haller-Institut, Universität Göttingen, Germany
Date of receipt: February 24, 2016
Charge No. (ECT): A/240216/A
pH-value of the algal medium: 8.2
Culture medium: Mod. OECD medium (OECD 201, EN ISO 8692)
Volume of liquid stock culture per pre-culture vessel: 100±5 mL
Pre-culture vessels: 300 mL Erlenmeyer flasks
Number of replicates: 2
Light: Permanent illumination (24/0 h light/dark); Econlux LED Sunstrip “daylight”
Light intensity: 60–120 µE m-2s-1; (measured: 63.2–70.0 µE m-2s-1)
Shaker: 100±5 oscillations/min
Temperature in the test room: 21–24 C; (measured: 22.0–22.5 °C)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

Chemical analysis of test concentrations: At start (0 hours) and at the end of exposure (72 hours)

Hardness:

No data

Test temperature:

21–24 °C, controlled at ±2 °C

pH:

pH of test medium: 8.0
pH in test solutions: 7.7–8.4

Salinity:

n.a.

Conductivity:

No data

Nominal and measured concentrations:

0.44, 0.97, 2.14, 4.70, 10.3, 22.7 und 50.0 mg test item/L

Details on test conditions:

Material and methods:
Test organism: Pseudokirchneriella subcapitata
Test medium: OECD-Medium (modified)
Endpoints: ECx (e.g. EC50, EC20, EC10), NOEC/LOEC
Biological parameters: Inhibition of growth in relation to control (growth rate & yield)
Test duration: 72 hours
Temperature (target): 21–24 °C, controlled at ±2 °C
Light regime: Permanent illumination
Light intensity (target): 60–120 µE m-2s-1, within ±15% over the incubation area
Test units: Glass Erlenmeyer flasks (300 mL) covered by air-permeable lids
Test concentrations (nominal): 0.44, 0.97, 2.14, 4.70, 10.3, 22.7 und 50.0 mg test item/L and a control
No. of replicates in the control: 6
No. of replicates per test concentration: 3
Renewal of test solution during exposure: None (static system)
Chemical analysis of test concentrations: At start (0 hours) and at the end of exposure (72 hours)
Data evaluation: Shapiro-Wilk’s Test on Normal Distribution; Levene’s Test on Variance Homogeneity; Williams Multiple Sequential t-test and Welsh-t test after Bonferroni-Holm for homogeneous variances; Trend analysis by contrasts for monotonicity 3-parametric cumulative distribution function (CDF)

Exposure Conditions
Test vessels: Glass Erlenmeyer flasks (300 mL) covered by air-permeable lids
Volume of test solution per test vessel: 100±5 mL
Age of the pre-culture: 3 days
Number of cells per mL in the pre-culture before inoculating the test solutions: 91.5×10E4
Number of cells per mL test solution at the beginning of the test: 0.3×10E4
Number of replicates per test item concentration: 3
Number of replicates in the control: 6
Number of replicates for stability check (highest concentration level without algae): 2 (without algae)
Test medium: OECD medium (modified)
pH of test medium: 8.0
pH in test solutions: 7.7–8.4
Light regime: 24 h light/0 h dark
Type of light: Econlux LED Sunstrip “daylight”
Light intensity: 72.45–77.6 µE m–2s–1; mean 75.3 µE m–2s–1;
Temperature: 21.9–22.2, mean 22.1 °C;
Shaker: 100±5 oscillations/min; on a horizontal shaker
Test duration (exposure): 72 hours
Counting of algae: Daily

Method for Counting Cell Numbers
At start of the test, the cell numbers were determined microscopically using a counting chamber (Thoma chamber). Occurrence of morphological deviations of cells was checked.
After 24, 48 and 72 hours, the cell numbers were determined by measuring the fluorescence intensity in 4 samples of 200 µL of test solution per replicate using a fluorometer (Multiple Reader Tecan ULTRA). The results [RFU, relative fluorescence units] were converted into biomass concentration [cells/mL] using a calibration curve which was generated by the data analysis software TIBCO Statistica (TIBCO Software Inc. 2017). A dilution series of the pre-culture was used to determine the calibration line by comparison of cell numbers (counted using a microscope) and fluorescence intensity (measured using a fluorometer) in consideration of background fluorescence of the blank algal medium.

Method for Calculating Growth Inhibition
Growth inhibition was expressed as the effect of the test item concentration that reduced the cell number compared to the control by a certain percentage. The biological results, i.e. growth rate (µ), and yield (Y), were evaluated statistically.

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

23.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limit: 22.1-25.1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.97 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2.14 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

10.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limit: 8.88-11.4.

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.84 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limit: 4.79-6.83

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: 95% confidence limit: 3.69-4.62

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.97 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2.14 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

1.83 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: 95% confidence limit: 1.52-2.11

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.13 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: 95% confidence limit: 0.87-1.38

Details on results:

- Concentrations below 4.70 mg test item/L was not measured, since the expected DOC concentration were below the limit of detection for this analytical method. The results of the DOC analyses could show that at start of the test the test item was applied at an acceptable level based on the nominal concentrations, and that the degradation products were still present at the end of the exposure period. Therefore, the biological results were reported based on nominal test item concentrations.

- In the nominal concentration levels 0 and 0.44–2.14 mg test item/L no deformed and/or damaged algal cells were observed during microscopic inspection. In the 4.70 mg test item/L treatment around 50 % of the observed cells showed deformed (thickened) and/or damaged algal cells. Almost all cells were deformed and/or damaged in the 10.3 and 22.7 mg test item/L concentration levels. In the highest concentration level 50.0 mg test item/L no alga cells were recorded.

Results with reference substance (positive control):

A reference tests using potassium dichromate (K2Cr2O7) as reference item was performed in October 2017 in separate study.
Standard: Growth rate ErC50 (0–72 h): 0.952 mg/L (0.916 – 0.990 mg/L; 95%-CL).
Based on an international ring test (ISO (2004). Water Quality – Freshwater algal growth inhibition test with unicellular green algae. European Standard EN ISO 8692, October 2004.) mentioned in OECD guideline 201, the ErC50 (72h)-values for potassium dichromate obtained from different laboratories was 1.19 mg/L with a standard deviation of 0.27 mg/L.
The ErC50 value for the toxic reference item, potassium dichromate, was determined as 0.952 mg/L. This value is within the range of the international ring test (ISO (2004). Therefore the results of this reference test are acceptable and the test conditions are reliable.

Reported statistics and error estimates:

Statistics
The data were evaluated by the Shapiro-Wilk’s Test for normal distribution, and by Levene's Test for homogeneity of variances. The Williams Multiple Sequential t-test and Welsh-t test were applied to find out whether there were significant differences between the growth of algae in the controls and the algae exposed to the test item concentration.
The 3-parametr. Logistic/ weibull CDF (cumulative distribution function) were used to determine of the concentration-response function.
The statistical software package ToxRat 3.2 Professional (ToxRat Solutions GmbH, Naheweg 15, D-52477 Alsdorf) was used for these calculations.
The statistical analysis was performed using nominal concentrations as well as mean measured concentrations. For details see section 23.

The test item showed fast degradation (hydrolysis) in aqueous solution within a short exposure period, thus a detection of the test item wasn’t necessary. Because of this instability of 2,2-Dimethylthiazolidine there was no chance to detect the test item within 24 hours.  Therefore, an additional analytical method was used to detect the degradation products (acetone and aminoethylthiol) of 2,2-Dimethylthiazolidine by measuring the dissolved organic carbon content (DOC).

In the following table the results from the dissolved organic carbon (DOC) analyses were shown. Concentrations below 4.70 mg test item/L wasn’t measured, since the expected DOC concentration were below the limit of detection for this analytical method.

Summary of measured concentrations using DOC analyses at the beginning and at the end of the test.

Conc. Level	Test period	Nominal concentration	Measured concentration	Calculated concentration*	Recovery**
	[d]	[mg test item/L]	[mg DOC/ L]	[mg test item/L]	[%]
Control	0	0	<2	-	-
C4	0	4.70	3.04	5.94	126
C5	0	10.3	5.67	11.06	107
C6	0	22.7	11.5	22.50	99
C7	0	50.0	24.1	47.01	94
Control	3	-	<2	-	-
C4	3	4.70	3.29	6.43	137
C5	3	10.3	5.96	11.62	113
C6	3	22.7	10.8	21.05	93
C7	3	50.0	21.3	21.54	83

Limit of Detection (LOD): (2 mg/L).

*DOC values were recalculated with a factor of 1.95 representing the molar mass of carbon in the test molecule.

**Recovery based on nominal concentration, assuming that the measured DOC is equivalent with the content of carbon in the test item.

The results of the DOC analyses could show that at start of the test the test item was applied at an acceptable level based on the nominal concentrations, and that the degradation products were still present at the end of the exposure period. Therefore, the biological results were reported based on nominal test item concentrations.

Validity criteria fulfilled:

yes

Remarks:

All validity criteria were fulfilled as required by the study plan.

Conclusions:

2,2-Dimethylthiazolidine shows an algae toxicity of EC50 (cell number) = 4.15 mg/L and EC50 (growth rate) = 23.6 mg/L respectively a NOEC of 0.97 and a LOEC of 2.14 after 72 hours according to OECD guideline No. 201.

Executive summary:

The aim of the study is to determine the toxicity of the 2,2-Dimethylthiazolidine towards the green alga, Pseudokirchneriella subcapitata. To achieve this, a series of concentrations of 2,2-Dimethylthiazolidine in aqueous solution were prepared. The test organisms were exposed to these concentrations ( 0.44, 0.97, 2.14, 4.70, 10.3, 22.7 und 50.0 mg), as well as to controls without the test item, for a test period of 72 hours. This study was conducted under static conditions; i.e. the algae will be exposed to a test solution which was not be renewed during the test. The data obtained were analysed in order to estimate the concentration that would cause a x% growth inhibition, i.e. ECx (e.g. EC10, EC20 or EC50). As additional endpoints the No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) were calculated. Growth was determined daily based on determination of the cell number per volume test solution (cell concentration). To verify the nominally applied concentration, samples were taken from the test solution and analytically measured. The samples taken from the test vessels were transferred to the analytical test site and analysed under the responsibility of the Principal Investigator for analysis.

Analytical findings

Samples from the test solutions were analysed to determine actual levels of the test item in solution at start of exposure period (time 0 hours) and at the end of the exposure at 72 hours.

At time 0 hours the measured concentrations range between <LOD–2.5% of the nominal concentrations. After 72 hours exposure, the test concentrations decrease to levels of <LOD of the nominal concentrations. Therefore, the test item concentration based on the nominal concentration remained not stable within ±20% throughout the exposure period.

Since the test item showed fast degradation (hydrolysis) in aqueous solution within 24 hours, a detection of the test item wasn’t necessary. Because of this instability of 2,2-Dimethylthiazolidine there was no chance to detect the test item using gas chromatography. Therefore, an additional analytical method was used to detect the degradation products of 2,2-Dimethylthiazolidine by measuring the dissolved organic carbon content (DOC).  

The results of the DOC analyses could show that at start of the test the test item was applied at an acceptable level based on the nominal concentrations, and that the test item (degradation product) was still present at the end of the exposure period. Therefore, the biological results were reported based on nominal test item concentrations.

Biological findings

A clear concentration-response relationship was observed for both biological parameters growth rate and yield during the exposure period.

The following ECx, NOEC and LOEC values for the parameters cell number and biomass expressed in yield and growth rate, based on statistical evaluation of biological results and measured geometric mean concentrations of the test item (2,2-Dimethylthiazolidine) are presented in table:

Endpoints [mg test item/L]
Parameter	EC10	EC20	EC50	NOEC	LOEC
Yield	1.13	1.83	4.15	0.97	2.14
(95% confidence limit)	(0.87 -1.38)	(1.52 -2.11)	(3.69 -4.62)
Growth Rate	5.84	10.2	23.6	0.97	2.14
(95% confidence limit)	(4.79 -6.83)	(8.88 -11.4)	(22.1 -25.1)

Description of key information

The aim of the study is to determine the toxicity of the 2,2-Dimethylthiazolidine towards the green alga, Pseudokirchneriella subcapitata. To achieve this, a series of concentrations of 2,2-Dimethylthiazolidine in aqueous solution were prepared. The test organisms were exposed to these concentrations ( 0.44, 0.97, 2.14, 4.70, 10.3, 22.7 und 50.0 mg), as well as to controls without the test item, for a test period of 72 hours. This study was conducted under static conditions; i.e. the algae will be exposed to a test solution which was not be renewed during the test.

The data obtained were analysed in order to estimate the concentration that would cause a x% growth inhibition, i.e. ECx (e.g. EC10, EC20 or EC50). As additional endpoints the No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) were calculated.

Growth was determined daily based on determination of the cell number per volume test solution (cell concentration).

To verify the nominally applied concentration, samples were taken from the test solution and analytically measured. The samples taken from the test vessels were transferred to the analytical test site and analysed under the responsibility of the Principal Investigator for analysis.

Analytical findings

Samples from the test solutions were analysed to determine actual levels of the test item in solution at start of exposure period (time 0 hours) and at the end of the exposure at 72 hours.

At time 0 hours the measured concentrations range between <LOD–2.5% of the nominal concentrations. After 72 hours exposure, the test concentrations decrease to levels of <LOD of the nominal concentrations. Therefore, the test item concentration based on the nominal concentration remained not stable within ±20% throughout the exposure period.

Since the test item showed fast degradation (hydrolysis) in aqueous solution within 24 hours, a detection of the test item wasn’t necessary. Because of this instability of 2,2-Dimethylthiazolidine there was no chance to detect the test item using gas chromatography. Therefore, an additional analytical method was used to detect the degradation products of 2,2-Dimethylthiazolidine by measuring the dissolved organic carbon content (DOC).  

The results of the DOC analyses could show that at start of the test the test item was applied at an acceptable level based on the nominal concentrations, and that the test item (degradation product) was still present at the end of the exposure period. Therefore, the biological results were reported based on nominal test item concentrations.

Biological findings

A clear concentration-response relationship was observed for both biological parameters growth rate and yield during the exposure period.

The following ECx, NOEC and LOEC values for the parameters cell number and biomass expressed in yield and growth rate, based on statistical evaluation of biological results and measured geometric mean concentrations of the test item (2,2-Dimethylthiazolidine) are presented in table:

Endpoints [mg test item/L]
Parameter	EC10	EC20	EC50	NOEC	LOEC
Yield	1.13	1.83	4.15	0.97	2.14
(95% confidence limit)	(0.87 -1.38)	(1.52 -2.11)	(3.69 -4.62)
Growth Rate	5.84	10.2	23.6	0.97	2.14
(95% confidence limit)	(4.79 -6.83)	(8.88 -11.4)	(22.1 -25.1)

Key value for chemical safety assessment

EC50 for freshwater algae:

23.6 mg/L

EC10 or NOEC for freshwater algae:

5.84 mg/L