2,2'-[1,2-ethanediylbis(oxy)]bis(ethanethiol)

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:

11-23-2016 to 07-03-2017

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Chemical name: 2-[2-(2-sulfanylethoxy)ethoxy]ethanethiol
CAS number: 14970-87-7
Batch number: 471389
Purity: 99.099%
Date of reception
in the test facility: 14 September 2015
Expiration date: 15 July 2017
Internal identification: 0057-15
Appearance: colourless liquid
Water solubility: 15 - 20 g/L at 20°C (according to the SDS)
Relative density: 1.140 kg/m3 at 25°C (according to the SDS)
Storage conditions: well-ventilated place, ambient temperature and obscurity

Analytical monitoring:

yes

Details on sampling:

After 24 and 48h of incubation, aliquot of 200 µL was sampled from each inoculated test flask, pipetted into a microplate (after 72 h, the samples with high cell density were diluted).
Time between sampling and measurement is approximately 15 – 30 min.

Vehicle:

no

Details on test solutions:

For both range-finding test and definitive tests, tests solutions were prepared from dilutions of a stock solution: a known volume of test item (43.86 µL) was poured into a volumetric flask, the volume was then made up to 500 mL with test medium (corresponding to 100 mg/L of nominal final concentration). The solution was kept under high speed stirring at ambient temperature during approximately 30 minutes with a magnetic stir bar.

Test organisms (species):

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

Details on test organisms:

The test organism used for the study was Pseudokirchneriella subcapitata, Strain No. CCAP 278/4, supplied by the Culture Centre of Algae and Protozoa (Ambleside, UK).

Transfers of P. subcapitata were made regularly to provide suitable subcultures. The algae were cultivated under standardized conditions as described in Annex 2 of the OECD 201 guideline.

The absence of micro-organisms and deformed or abnormal cells under microscopic observation was checked to ensure the quality of the stock culture before use.

Three or four days before the start of the exposure, two pre-cultures were prepared by inoculating sufficient cells from the algal stock culture to the growth medium to obtain a low cell density, e.g. 5.10^3 cells/mL to 10^4 cells/mL for pre-culturing, in order to maintain exponential growth until the start of the test. The pre-cultures were incubated under the same conditions as those used for the test cultures. Only one of the two pre-cultures was used to inoculate the testflasks for the study. The second one was only used if the first one was damaged.

At the beginning of the test, the cell density of the pre-culture was determined. The result was used to calculate the volume to be introduced into each test flask in order to obtain an initial cell concentration of 5.10^3 or 10^4 cells/mL as recommended in the OECD 201 guideline.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

Range from 23.0 to 23.5°C (mean: 23.2°C)

pH:

Range from 7.9 to 8.4

Dissolved oxygen:

Range from 8.0 to 10.1 mg/L

Nominal and measured concentrations:

Nominal concentrations of test item: 0.10, 0.32, 1.00, 3.20 and 10.0. mg/L
Measured concentrations of test item (used for statistical analysis) : 0.07, 0.17, 0.53, 1.99, 7.41 mg/L

Details on test conditions:

The incubation was conducted in a phytoculture cabinet that allows test flasks to be incubated under precise conditions: temperature was set to 23 ± 1°C; flasks were continuously shaken with a rotation speed at around 100 rpm and constantly illuminated by fluorescent tubes between 6,000 and 10,000 lux.
Test vessels were made of glass bottles (120 mL capacity) capped with cellulose bungs. They were filled with a volume of 50 mL.

Reference substance (positive control):

yes

Key result

Duration:

96 h

Dose descriptor:

EC10

Effect conc.:

0.51 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

3.11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

96 h

Dose descriptor:

EC10

Effect conc.:

0.44 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

The appearance of the test solutions was visually checked at the beginning and at the end of the test. Solutions were found to be clear, no precipitation was observed at the end of the test.
Microscopic observation confirmed that the algae appeared "normal" at the end of the test: the normal shape of P. subcapitata algae is a crescent shaped cell with an average length of 5-10 µm.

Chemical analysis of test samples taken at 0 and 72 h indicated that concentrations of the test item were not maintained over the 72h test period (i.e. deviation > 20%). As a consequence, the stability of the substance was not confirmed over the test period; geometric means of measured concentrations were thus used to determine EC10 and EC50. The percentages of inhibition are presented in Table 1. The results (growth rate) demonstrated that algal growth was inhibited from 0.4% at the lowest concentration (i.e. 0.10 mg/L nominal concentration) to 68.9% at the highest tested concentration (10.00 mg/L nominal concentration).

Results with reference substance (positive control):

The sensitivity of the test system and the methodology are evaluated every two months by performing an algal growth inhibition test on potassium dichromate. The latest value of ErC50 obtained in March 2017 was 1.41 mg/L.
For information, according to ISO 8692 standard, 72h-ErC50 obtained from an inter-laboratory exercise on potassium dichromate was in the range 0.65 to 1.73 mg/L.

Reported statistics and error estimates:

See table 2.

Table 1

Test solution (mg/L)	Geometric means measured conc. (mg/L)	Growth rate Iµi (%)	Yield Iyi (%)
0.10	0.07	0.4	2.4
0.32	0.17	0.8	4.1
1.00	0.53	3.8	15.7
3.20	1.99	44.6	86.3
10.00	7.41	68.9	96.1

Table 2

Growth Function	72h-EC10(mg/L)	95% confidence level		72h-EC50 (mg/L)	95% confidence level
		Lower limit	Upper limit		Lower limit	Upper limit
Growth rate	0.51	0.32	0.70	3.11	2.61	3.74
Yield	0.44	0.36	0.52	1.00	0.89	1.12

Validity criteria fulfilled:

yes

Conclusions:

The concentrations that result in a 10 and 50% reduction in growth rate (72h-ErC10 and 72h-ErC50, respectively) and yield (72h-EyC10 and 72h-EyC50, respectively) were determined.
Chemical analysis of the test item showed the instability of the substance over the 72-hour test period. Based on these results, the exposure concentrations were based on the geometric mean of measured concentrations.

72h-ErC10 = 0.51 mg/L
72h-ErC50 = 3.11 mg/L

Executive summary:

This study was designed to determine the effects of the test item on the growth ofPseudokirchneriella subcapitatain a 72-hour test according to the OECD 201 Guideline.

 Tests solutions were prepared from dilutions of a stock solution prepared at 100 mg/L (nominal concentration).

The concentrations that result in a 10 and 50% reduction in growth rate (72h-ErC10 and 72h-ErC50, respectively) and yield (72h-EyC10 and 72h-EyC50, respectively) were determined.

 Chemical analysis of the test item showed the instability of the substance over the 72-hour test period. Based on these results, the exposure concentrations were based on the geometric mean of measured concentrations.

Growth Function	72h-EC10(mg/L)	95% confidence level		72h-EC50 (mg/L)	95% confidence level
		Lower limit	Upper limit		Lower limit	Upper limit
Growth rate	0.51	0.32	0.70	3.11	2.61	3.74
Yield	0.44	0.36	0.52	1.00	0.89	1.12

 

The definitive test met the validity criteria of the test guideline detailed as follows:

-The biomass in the control cultures increased exponentially by a factor of 71.2 within the 72-hour test period (required: at last 16-fold)

-The mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures were less than or equal to 18.0 (required: < 35%)

-The coefficient of variation of average specific growth rates during the whole test period in the control cultures was 3.5% (required: < 7%)

Description of key information

This study was designed to determine the effects of the test item on the growth ofPseudokirchneriella subcapitatain a 72-hour test according to the OECD 201 Guideline.

Chemical analysis of the test item showed the instability of the substance over the 72-hour test period. Based on these results, the exposure concentrations were based on the geometric mean of measured concentrations.

Growth Function	72h-EC10(mg/L)	95% confidence level		72h-EC50 (mg/L)	95% confidence level
		Lower limit	Upper limit		Lower limit	Upper limit
Growth rate	0.51	0.32	0.70	3.11	2.61	3.74
Yield	0.44	0.36	0.52	1.00	0.89	1.12

 

The definitive test met the validity criteria of the test guideline detailed as follows:

-The biomass in the control cultures increased exponentially by a factor of 71.2 within the 72-hour test period (required: at last 16-fold)

-The mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures were less than or equal to 18.0 (required: < 35%)

-The coefficient of variation of average specific growth rates during the whole test period in the control cultures was 3.5% (required: < 7%)

Key value for chemical safety assessment

EC50 for freshwater algae:

3.11 mg/L

EC10 or NOEC for freshwater algae:

0.51 mg/L