2,2-dimethylthiazolidine

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

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 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

Gas Chromatographic (GC/FID) and DOC Determination

Details on sampling:

Sampling for Chemical Analysis:
- Samples of stock solution and test solutions were taken to determine the actual test item concentrations in comparison to the nominally applied concentrations
- Control and test solutions were sampled in duplicate at the start of exposure and after 48 hours; the duplicate samples were kept separately as a reserve; the nominal volume per sample was 80 mL. mixed with 10 mL of acetonitrile; the volume of each sample was recorded
- After sampling and before shipment, all samples were stored in amber glass bottles in the dark at a temperature of ≤ -18°C; a record was kept for each sample

Vehicle:

no

Remarks:

Aetone is one of the hydrolysis products

Details on test solutions:

Preparation of Stock and Test Solutions:
- A stock solution was prepared at a nominal concentration rate of 100 mg/L by adding 100.27 mg of the test item to 1000 mL of the test medium, stirred (500 rpm) for 15 minutes at ambient temperature
- Thereafter this stock solution was left to settle for 20 minutes
- Visual inspection of the flask showed a clear solution
- The volume of the stock solution was large enough to prepare the test concentrations and all analytical samples
- The volumetric flasks containing the test solutions were stirred for ca. 5 minutes on a magnetic stirrer

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM:
- Species: Daphnia magna STRAUS (clone M10), a water flea
- Origin: The organisms were originally supplied by KU Leuven, Belgium and cultured at ECT Oekotoxikologie GmbH since December 22, 2011
- Age of parental daphnids: less than 35 d
- Age of the daphnids at the beginning of the test: less than 24 h

HOLDING CONDITIONS:
- Material of stock vessel: Glass
- Volume of medium per stock vessel: 1.8 L
- Depth of medium in the stock vessel: approximately 14.5 cm
- Number of daphnids kept as stock per culture vessel (batch size): approximately 20
- Separation of adult and young daphnids: 2-5 times per week
- Medium: Elendt medium M4
- Renewal of medium: twice per week
- Temperature: within required ranges (20 +/- 2°C)
- Photoperiod (light/dark) 16/8 h
- Light intensity: within required ranges (50 to 1000 lx)
- Aeration: None
- Food: algae (Desmodesmus subspicatus), instant baker’s yeast suspension, TetraMin® suspension
- Feeding frequency: 4 times per week

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

14.2 °dH (254 CaCO3 mg/L)

Test temperature:

19.7 - 20.7 °C

pH:

7.7 - 8.6

Dissolved oxygen:

5.6 - 8.2 mg/L

Salinity:

n.a.

Conductivity:

no data

Nominal and measured concentrations:

The biological endpoints are expressed based on nominal (100 mg/L) and geometric mean measured concentrations (mg/L).
- After 0 hour: 100 mg/L test concentration (Nominal) equals 81.039 mg/L measured geometric concentration
- After 48 hours: 100 mg/L test concentration (Nominal) equals 51.029 mg/L measured concentration and 64.3 mg/L measured geometric concentration

Details on test conditions:

EXPOSURE CONDITIONS:
- Test units: 100-mL glass beakers, closed by watch glass
- Volume of test solution per test vessel: 50-60 mL
- Test medium: medium M4 (OECD 2004)
- Number of daphnids per test vessel: 5
- Adjustment of pH in the medium: none
- Application of test item: test item dissolved in Medium M4 applied once at the beginning of the test
- Aeration of test vessels: none
- Test concentrations (nominal): Control; 1.94, 4.27, 9.39, 20.7, 45.5 and 100 mg test item/L
- Number of replicates per concentration: 4
- Number of replicates in the controls: 4
- Renewal of the test solution during the test period: static, no renewal
- Feeding during exposure: none
- Temperature (target): 18 to 22°C, constant within a range of 2°C
- Light intensity (target): 50 to 1000 lx
- Light regime: 16 h light; 8 h dark
- Biological parameter: immobility

WATER QUALITY MEASUREMENTS:
At start of the test, after temperature adaptation of the test solutions, and at the end of the test the following parameters were measured and recorded in one replicate test vessel of the control and the highest test concentration:
- Temperature,
- pH,
- Dissolved oxygen content in % of air saturation value (ASV) and in mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

6.03 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limit: 4.16-8.45 mg/L

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

1.55 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limit: 0.728-2.46 mg/L

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

3.53 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limit: 0.331-6.98 mg/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limit: 4.87-24.02 mg/L

Details on results:

The results of the DOC analyses showed that at start of the test the test item was applied at an acceptable level based on the nominal concentrations. The degradation products were also still present at the end of the exposure period, but to a lesser extent than at start of exposure. Therefore, the biological results were reported based on nominal and on geometric mean test item concentrations.

The test item concentrations were not stable during the test period within 80-120% of the nominal concentration.

Results with reference substance (positive control):

The most recent reference test with potassium dichromate performed in a separate study (Study No. IDA1711; November 2017) resulted in an EC50(24 h) of 0.74 mg/L.

According to the OECD guideline No. 202 (OECD, April 2004) EC50(24 h)-values for potassium dichromate obtained from different laboratories should range between 0.6 mg/L and 2.1 mg/L. The toxicity of the reference item is within this range. Therefore, the results of this reference test are acceptable and the test conditions are reliable and the sensitivity of the test system could be demonstrated.

Reported statistics and error estimates:

Probit analysis using linear max. likelihood regression with immobility at 48 h

Analytical Results:

Due to rapid decomposition of test item in the test solutions, no 2,2-dimethylthiazolidine could be detected by GC/FID in any of the test solution samples at concentrations higher than 0.619 mg/L, which was used as the lowest standard concentration during this study. Therefore, a surrogate quantification of the actual test concentrations was performed by DOC measurement.

Summary of measured concentrations during the test:

Nominal concentration (mg/L)	Test period (d)	Age of test solution (h)	Measured concentration (mg /L)	% of nominal concentration	Geometric mean measured concentration (mg/L)
0 (Control)	0	0	--	--	--
1.94	0	0	0.254	13.1	--
4.27	0	0	2.171	50.8	--
9.39	0	0	6.360	67.7	--
20.7	0	0	16.030	77.4	--
45.5	0	0	36.640	80.5	--
100	0	0	81.039	81.0	--
0 (Control)	48	48	--	--	--
1.94	48	48	0.980	50.5	0.499
4.27	48	48	1.475	34.5	1.79
9.39	48	48	4.753	50.6	5.50
20.7	48	48	11.874	57.4	13.8
45.5	48	48	26.841	59.0	31.4
100	48	48	51.029	51.0	64.3

Validity criteria fulfilled:

yes

Remarks:

immobilisation and other abnormalities in the controls did not exceed 10% by the end of the test; dissolved oxygen concentration remained above 3 mg/L throughout the exposure period

Conclusions:

An EC 50 value of the parameter "immobility of daphnids" based on statistical evaluation of biological results and mean measured concentrations of 6.03 mg/L (95% confidence limit 4.16-8.45 mg/L) was measured. The test was conducted under static conditions for 48 hours.

Executive summary:

The study was performed according to OECD Guideline for Testing of Chemicals No. 202 'Daphnia sp., Acute Immobilization Test'.

A series of concentrations of the test item in aqueous solution (100, 45.5, 20.7, 9.39, 4.27 and 1.94 mg test item /L in geometrical series)

was prepared, and the test organisms were exposed to these concentrations as well as to controls without the test item for a period of 48 hours under static conditions. During the test period the test solutions were not aerated. The daphnids were not fed. Those animals not able to swim within 15 seconds after gentle agitation of the test vessel were considered to be immobile. The data obtained were analysed in order to estimate the concentration that would cause a x% immobilisation, i.e. ECx (e.g. EC50, EC20, EC10). To verify the nominally applied concentrations, samples were taken from the test solutions and analytically measured (DOC determination). The results of the DOC analyses showed that at start of the test the test item was applied at an acceptable level based on the nominal concentrations. The degradation products were also still present at the end of the exposure period, but to a lesser extent than at start of exposure. Therefore, the biological results were reported based on nominal and on geometric mean test item concentrations. The test item concentrations were not stable during the test period within 80-120% of the nominal concentration. An EC 50 value of the parameter "immobility of daphnids" based on statistical evaluation of biological results and mean measured concentrations of 6.03 mg/L (95% confidence limit 4.16-8.45 mg/L) was measured. The test was conducted under static conditions for 48 hours. The toxicity of the reference item (24 h-EC50 0.74 mg/L) was within this range. All validity criteria were fullfilled. This toxicity study is classified as acceptable and satisfies the guideline requirements for the acute Daphnia study.

Description of key information

According to OECD Guideline 202 an EC 50 value of the parameter "immobility of daphnids" based on statistical evaluation of biological results and mean measured concentrations of 6.03 mg/L (95% confidence limit 4.16-8.45 mg/L) was measured. The test was conducted under static conditions for 48 hours.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

6.03 mg/L

Additional information

The samples from the biological test were extracted with dichloromethane after addition of internal standard. The organic extract was dried by sodium sulphate and analysed by gas chromatography and FID-detection. Since the test item showed fast degradation (hydrolysis) in aqueous solution within 24 hours, a detection of the test item was in fact performed but was found to be not expedient. Due to the instability of the test item it was not possible to detect the test item with any analytical instrumentation. Additionally, gas chromatography which was used for the analysis of the test item did not show any peaks of potential metabolites.

Therefore, an additional analytical method was used to detect the degradation products of the test item by measuring the dissolved organic carbon content (DOC).

The results of the DOC analyses show that at start of the test the test item was applied at an acceptable level based on the nominal concentrations. The degradation products were also still present at the end of the exposure period, but to a lesser extent than at start of exposure. Therefore, the biological results were reported based on nominal and on geometric mean test item concentrations. The test item concentrations of 2,2-Dimethylthiazolidinewere not stable during the test period within 80-120% of the nominal concentration. According to information from the sponsor, acetone is one hydrolysis product. As this substance has a high Henry´s law constant, the lower recoveries found in test samples (based on DOC) might reflect evaporation of acetone during preparation of the test solution and during the performance of the test.

Summary of measured concentrations during the test after 48 hours of exposure:

Nominal concentration (mg/L)	Test period (d)	Age of test solution (h)	Measured concentration (mg /L)	% of nominal concentration	Geometric mean measured concentration (mg/L)
0 (Control)	48	48	--	--	--
1.94	48	48	0.980	50.5	0.499
4.27	48	48	1.475	34.5	1.79
9.39	48	48	4.753	50.6	5.50
20.7	48	48	11.874	57.4	13.8
45.5	48	48	26.841	59.0	31.4
100	48	48	51.029	51.0	64.3