Reaction mass of 2,2'-thiodiethanethiol and 1095071-01-4 and α-sulfanyl-ω-{2-[(2-sulfanylethyl)sulfanyl]ethyl}bis[ethane- 1,2-diylsulfanediylethane- 1,2-diylsulfanediyl(3-hydroxypropane-1,1-diyl) sulfanediyl]

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

28 July 2011

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2,2'-thiodiethanethiol and 1095071-01-4 and α-sulfanyl-ω-{2-[(2-sulfanylethyl)sulfanyl]ethyl}bis[ethane- 1,2-diylsulfanediylethane- 1,2-diylsulfanediyl(3-hydroxypropane-1,1-diyl) sulfanediyl]

EC Number:

947-988-8

Molecular formula:

not applicable

IUPAC Name:

Reaction mass of 2,2'-thiodiethanethiol and 1095071-01-4 and α-sulfanyl-ω-{2-[(2-sulfanylethyl)sulfanyl]ethyl}bis[ethane- 1,2-diylsulfanediylethane- 1,2-diylsulfanediyl(3-hydroxypropane-1,1-diyl) sulfanediyl]

Test material form:

liquid

Sampling and analysis

Analytical monitoring:

yes

Remarks:

HPLC-UV

Details on sampling:

- Concentrations: The concentrations of the test item HIDT were analysed in the duplicate test media samples from all test concentrations, and in the duplicate control samples, from both sampling times (0 and 72 hours).
- Sampling method: The samples were taken from the biological phase of the study.
Collecting, storage and handing over of the samples were the Study Director’s responsibility. The information concerning the samples was provided by the Study Director. Duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from the control were taken at the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the control (containing algae) were taken at the end of the test (after the 72 hours test period) by pouring together the contents of the test beakers of each treatment.
All samples were diluted by a factor of two with acetonitrile.
Additional samples of the control and of the dilution solvent were taken at each sampling without any sample treatment.
- Sample storage conditions before analysis: All samples were stored in a freezer (≤ - 20 °C), protected from light
until analysis was performed. Afterwards the samples were again stored deep frozen (≤ -20 °C) and will be kept stored up to the date of the final report.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low limit of water solubility of the test item a Water
Accommodated Fraction (WAF) of 15.0 mg test item/L and dilutions thereof of 1:3.16, 1:10, 1.31.25 and 1:100 equivalent to nominal concentrations of 15.0, 4.75, 1.50, 0.48 and 0.15 mg test item/L (factor 3.16) and a control were tested. The nominal concentrations of 15.0 and 4.75 mg test item/L correspond to mean measured
concentrations of 7.47 and 2.42 mg test item/L. The nominal concentrations of 1.50 and 0.48 were between LOD and LOQ and per definition would therefore be considered as half the LOQ (i.e. 0.600 mg test item/L). The nominal concentration of 0.15 mg test item/L was below LOD and per definition would therefore be considered as half the LOD (i.e. 0.200 mg test item/L). However, in order to allow a
more realistic estimation of the relevant effect concentrations, the mean recovery which was found in the two highest concentrations (50.5 %) was also considered for the lower concentrations. Thus, mean measured concentrations of 7.58, 2.40, 0.758, 0.242 and 0.076 mg test item/L (i.e. 50.5 % of the nominal values) were used for the determination of the endpoints.

- Evidence of undissolved material (e.g. precipitate, surface film, etc.): There were no remarkable observations except for a slight turbidity at the nominal concentration of 15 mg test item/L

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: Pseudokirchneriella subcapitata (KORSHIKOV), Strain No. 61.81 SAG
- Source (laboratory, culture collection): „Sammlung von Algenkulturen,
Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
- Method of cultivation: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.

ACCLIMATION
- Acclimation period: No
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: No

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3 .

Test temperature:

Water temperature was:
23.1 to 23.5 °C

pH:

pH was:
7.9 to 8.0 at test start and
8.7 to 9.9 at test end

Nominal and measured concentrations:

nominal: 15.0, 4.75, 1.50, 0.48 and 0.15 mg test item/L
mean measured: 7.58, 2.40, 0.758, 0.242 and 0.076 mg test item/L (i.e. 50.5 % of the nominal values)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Material, size, headspace, fill volume: glass, fill volume 50 mL
- Aeration: No
- Initial cells density: 5000 cells/mL
- Control end cells density:
- No. of vessels per concentration (replicates): three
- No. of vessels per control (replicates): six

GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the test medium was adjusted with 1 M NaOH to pH 8.1 prior to start of the experiments.
- Photoperiod: continuous
- Light intensity and quality: Mean light intensity: 5965 lux (range: 5710 to 6430 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer
Yield (y):
The yield is defined as the mean cell density at the end of the test minus the mean cell density at the start of the test.
Average specific growth rate (µ) and Percentage reduction of growth rate µ (I R ): y and µ were calculated for each test flask. The means were then determined as arithmetic mean values over all test flasks per treatment.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16

Reference substance (positive control):

yes

Remarks:

For the evaluation of the quality of the algae and the experimental conditions the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.05 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.84 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.48 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks:

and growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks:

and growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.85 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.87 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Effect concentrations 7

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.758 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

yield

Remarks:

and growth rate

Effect concentrations 8

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.242 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

yield

Remarks:

and growth rate

Effect concentrations 9

Duration:

72 h

Dose descriptor:

EC10

Remarks:

growth

Effect conc.:

1.54 mg/L

95% CI:

1.34 - <= 1.76

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 10

Duration:

72 h

Dose descriptor:

EC10

Remarks:

yield

Effect conc.:

0.437 mg/L

95% CI:

0.355 - <= 0.539

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

yield

Effect concentrations 11

Duration:

72 h

Dose descriptor:

EC50

Remarks:

growth

Effect conc.:

4.97 mg/L

95% CI:

4.66 - <= 5.31

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 12

Duration:

72 h

Dose descriptor:

EC50

Remarks:

yield

Effect conc.:

1.45 mg/L

95% CI:

1.29 - <= 1.62

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

yield

Details on results:

- Exponential growth in the control (for algal test): yes

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: yield: 0.442 mg/L; growth rate: 1.11 mg/L; Biomass: 0.487 mg/L

Any other information on results incl. tables

Influence of HIDT on the Growth of Pseudokirchneriella subcapitata
Parameter	Yield [mg test item/L]	Growth rate [mg test item/L]
72 h EC50	1.45	4.97
95% Conf. interval	1.29-1.62	4.66-5.31
72 h EC20	0.658	2.30
95% Conf. interval	0.564-0.774	2.08-2.54
72 h EC10	0.437	1.54
95% Conf. interval	0.355-0.539	1.34-1.76
72 h NOEC	0.242(0.6)	0.242(0.6)
72 h LOEC	0.758(0.6)	0.758(0.6)
Concentrations refer to mean measured test concentrations, considering 50.5 % of the nominal values (mean recovery of the two highest concentrations) for all test concentrations. The values in brackets show the mean measured concentrations which would be considered if the definition according to OECD Guidance Document No. 23 (half the LOQ for values <LOQ, LOD for values <LOD) would be followed

Summary of Analytical Results
Sample Description [mg test item/L]	% of nominal^1	RSD [%]	Mean measured concentration [mg test item/L]^1	n
Control	n.a.	n.a.	n.a.	4
Dilution 1:100	133	0	0.200	4
Dilution 1:31.25	125	0	0.600	4
Dilution 1:10	40	0	0.600	4
Dilution 1:3.16	51	3	2.42	4
WAF of 15 mg/L	50	2	7.47	4
^1  mean value of all measured samples per treatment group RSD: relative standard deviation per treatment group n: number of analysed samples; n.a.: not applicable italic: For values <LOQ, half the LOQ and for values <LOD, the LOD was used for calculating measured concentrations according to OECD Guidance Document No. 23

Yield y and Percentage Inhibition of y during the Test Period
	Yields y [10000 cells/mL] and % inhibition of y
Mean measured concentration [mg test item/L]	0-24 h		0-48 h		0-72 h
	y	%	y	%	y	%
Control	5.335	-	39.994	-	157.359	-
0.076 (0.200)	5.295	0.7	40.103	-2.8	162.561	-3.3
0.242 (0.600)	4.741	11.1	36.553	6.3	155.810	1.0
0.758 (0.600)	3.157	40.8*	27.678	29.0*	118.722	24.6*
2.40 (2.42)	2.207	58.6*	11.333	70.9*	48.594	69.1*
7.58 (7.47)	1.256	76.5*	6.747	82.7*	2.690	98.3*
negative values in ‘% inhibition’ indicate an increase in growth relative to that of the control * mean value significantly different from the control (tested with Williams t-test (24h and 48h) and Bonferroni-Welch t-test (72h), α = 0.05, one-sided) Concentrations refer to mean measured test concentrations, considering 50.5 % of the nominal values (mean recovery of the two highest concentrations) for all test concentrations. The values in brackets show the mean measured concentrations which would be considered if the definition according to OECD Guidance Document No. 23 (half the LOQ for values <LOQ, LOD for values <LOD) would be followed.
Growth Rates µ and Percentage Inhibition of µ during the Test Period
	Yields y [10000 cells/mL] and % inhibition of y
Mean measured concentration [mg test item/L]	0-24 h		0-48 h		0-72 h
	µ	%	µ	%	µ	%
Control	2.456	-	2.184	-	1.918	-
0.076 (0.200)	2.450	0.3	2.198	-0.6	1.929	-0.6
0.242 (0.600)	2.348	4.4	2.152	1.5	1.914	0.2
0.758 (0.600)	1.983	19.3	2.014	7.8*	1.825	4.9*
2.40 (2.42)	1.683	31.5	1.568	28.2*	1.520	20.8*
7.58 (7.47)	1.125	54.2	1.329	39.2*	0.611	68.2*
negative values in ‘% inhibition’ indicate an increase in growth relative to that of the control * mean value significantly different from the control (tested with sequentially rejective median test (24h) and Bonferroni-Welch t-test (48 and 72h), α = 0.05, one-sided) Concentrations refer to mean measured test concentrations, considering 50.5 % of the nominal values (mean recovery of the two highest concentrations) for all test concentrations. The values in brackets show the mean measured concentrations which would be considered if the definition according to OECD Guidance Document No. 23 (half the LOQ for values <LOQ, LOD for values <LOD) would be followed.

 

 

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The influence of HIDT on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour E y C 50 was calculated to be 1.45 mg test item/L and the 72-hour E r C 50 value was calculated to be 4.97 mg test item/L. The 72-hour NOE y C was determined to be 0.242 mg test item/L and the associated 72-hour LOE y C was 0.758 mg test item/L. The 72-hour NOE r C was determined to be 0.242 mg test item/L and the associated 72-hour LOE r C was 0.758 mg test item/L.
The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to mean measured values since the concentrations of the test item were not within ± 20 % of the nominal concentrations during the test.

Executive summary:

In a 72 hour acute toxicity study, the cultures of Pseudokirchneriella subcapitata were exposed to HIDT at nominal concentrations of 0, 15.0, 4.75, 1.50, 0.48 and 0.15 mg mg a.i./L} corresponding to mean measured concentrations of 0, 7.58, 2.40, 0.758, 0.242 and 0.076 mg test item/L (i.e. 50.5 % of the nominal values) under static conditions in accordance with the OECD test guideline 201 (2011). The NOEC and EC50 values based on yield and growth rate were 1.45 and 4.97 mg a.i./L and 0.242 mg/L, respectively. The % growth inhibition in the treated algal culture as compared to the control ranged from 0 to 68.2%.

There were no compound related phytotoxic effects.

This toxicity study is classified as acceptable and satisfies the guideline requirements for an acute toxicity to aquatic algae and cyanobacteria study.

Results Synopsis

 

Test Organism: Pseudokirchneriella  subcapitata

 

Test Type (Static):

72 hr EC50yield: 1.45 mg a.i./L                95% C.I.: 1.29 to 1.62 mg a.i./L

72 hr EC50growth rate: 4,97 mg a.i./L                95% C.I.: 4.66 to 5.31 mg a.i./L

72 hr NOEC: 0.242 mg a.i./L                   

Endpoint(s) Effected: Yield and growth rate