(benzothiazol-2-ylthio)succinic acid

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:

2019-10-30 to 2020-01-29

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:

(2011)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

(2016)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Five concentrations were tested in a geometrical series with a dilution factor of √10: 1.00 – 3.16 – 10.0 – 31.6 – 100 mg/L. The cocentrations are based on the results of a preliminary range finding test (non-GLP).
- Sampling method: At the start, samples were taken from one additional replicate of the test item concentration and the control and at the end samples were taken from pooled replicates.
- Sample storage conditions before analysis: All original samples were stored at room temperature before preparation. Prepared samples were stored in an autosampler at room temperature until analysis.

Vehicle:

no

Details on test solutions:

Stock solution: 100 mg test item/L was freshly prepared with dilution water
Dispersion treatment: The dispersion was treated with ultrasound for 40 minutes at room temperature. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). The test item solution was checked via laser beam (Tyndall effect) for undissolved test item.The Tyndall effect was negative.
Test concentrations: Out of the stock solution 5 concentrations were tested in a geometric series with a dilution factor of √10: 1.00 – 3.16 – 10.0 – 31.6 – 100 mg/L. The test concentrations are based on the results of a preliminary range finding test (non-GLP). Three replicates per concentration level were tested.
Control: Six replicates (without test item) were tested under the same test conditions as the test item replicates.

Test organisms (species):

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

Details on test organisms:

Test organism Pseudokirchneriella subcapitata HINDÁK CCAP 278/4 (axenic)

Synonyms Selenastrum capricornutum; Ankistrodesmus subcapitata; Raphidocelis subcapitata; Ankistrodesmus bibraianus (Experimental Phycology and Culture Collection of Algae at the University of Goettingen 2014)

Reason for the selection Pseudokirchneriella subcapitata is a suitable green alga species
of the test organism according to the guideline.

Origin Culture Collection of Algae and Protozoa (CCAP)
SAMS Research Services Ltd
Dunstaffnage Marine Laboratory
Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK

Cultivation at test facility Fresh stocks are prepared every month on Z-Agar. Light intensity amounts 2567 – 5130 lux for 24 hours per day.

Culture medium Nutrient medium Z according to LÜTTGE et al. (1994)
Acclimation period: No
Culturing media and conditions (same as test or not): Nutrient medium Z for culture and test medium according to OECDTG201

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

32 mg CaCO3/L

Test temperature:

21 - 24 °C controlled at ± 2°C

pH:

Control:
Start (0h): 7.96
End (72h): 8.96

Conductivity:

137 µS/cm

Nominal and measured concentrations:

Nominal concentration measured concentration

0 h:
Control < LOQ < LOQ (10 µg/L)
100 mg/L 94.7 mg/L
31.6 mg/L 31.0 mg/L
10 mg/L 10.4 mg/L
72 h:
Control < LOQ < LOQ
100 mg/L 95.3 mg/L
31.6 mg/LL 28.3 mg/L
10 mg/L 9.98 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile glass Erlenmeyer flasks, volume: 250 mL
- Type (delete if not applicable): sealed with cotton wool plugs
- Material, size, headspace, fill volume: 100 mL
- Aeration: no
- Initial cells density: 5416 cells/mL
- Control end cells density: 1397236 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
GROWTH MEDIUM
- Standard medium used: Nutrient medium Z according to LÜTTGE et al. (1994)
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to OECDTG201
- Total organic carbon: < 2 mgC/L
- Alkalinity: 0.8 mmol/L
- Conductivity: 143µS/cm
- Culture medium different from test medium: yes
- Intervals of water quality measurement: at start(0h) and at the end (72h) of study
OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No.
- Photoperiod: 24 hours/day light
- Light homogeneity: within ± 15% over incubation area
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1
EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
- Chlorophyll measurement: The cell density was measured daily via Chlorophyll a- fluorescence, excitation at 436 nm, emission at 685 nm.
TEST CONCENTRATIONS
- Spacing factor for test concentrations: √10
- Justification for using less concentrations than requested by guideline:
- Range finding study: yes
- Test concentrations: 1; 10; 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

19.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

8.45 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.21 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

< 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No morphological abnormalities observed.
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other:
- 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: No

Results with reference substance (positive control):

- Results with reference substance valid: Yes
- ErC50: 0.940 mg/L (CI; 0.732 – 1.33)
- Other: EyC50 = 0.660 mg/l (CI; 0.393 – 0.700)
based on nominal conccentrations.

Evaluation after 72 hours: Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

Nominal test item concentration	Replicate	Growth rate		Inhibition of growth rate	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
100	1		n.a.	100		n.a.	100
	2		n.a.	100		n.a.	100
	3		n.a.	100		n.a.	100
	Mean	(+)	n.a.	100	(+)	n.a.	100
31.6	1		0.272	85		6835	100
	2		0.479	74		17396	99
	3		0.181	90		3897	100
	Mean	(+)	0.311	83	(+)	9376	99
10.0	1		1.61	13		669238	52
	2		1.58	15		609167	56
	3		1.63	12		707403	49
	Mean	(+)	1.60	13	(+)	661936	52
3.16	1		1.75	5		1032488	26
	2		1.69	9		858068	38
	3		1.72	7		945740	32
	Mean	(+)*	1.72	7	(+)	945432	32
1.00	1		1.79	3		1148055	18
	2		1.78	4		1126976	19
	3		1.79	4		1139876	18
	Mean	(+)*	1.78	4	(+)	1138302	18
Control	1		1.85			1395446
	2		1.86			1440475
	3		1.89			1548350
	4		1.84			1334061
	5		1.82			1257731
	6		1.85			1374855
	Mean		1.85			1391820

*      = statistically significant but biologically not relevant (inhibition < 10%)

Validity criteria fulfilled:

yes

Conclusions:

The test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values based on the nominal test item concentrations: The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 19.5 (CI: 17.3 – 21.6) mg/L and 9.21 (CI: 7.57 - 10.7) mg/L, respectively. The NOEC-values for inhibition of growth rate and yield after 72 hours were 3.16 and < 1.00 mg/L respectively.

Executive summary:

The toxicity of the test itemto the unicellular freshwater green alga Pseudokirchneriella subcapitatawas determined according to the principles of OECD 201 andCouncil Regulation (EC) No. 266/2016/Method C.3.The aim of the study was the determination of the effects on growth rate and yield over a period of 72 hours.The study was conducted under static conditions with an initial cell density of 5416 cells/mL. A test item solution with a nominal concentration of 100 mg test item/L was prepared with dilution water. The dispersion was treated with ultrasound for 40 minutes at room temperature.Undissolved particles were removed by membrane filtration (membrane filterµm, RC,Macherey-Nagel).The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate were discarded. The filtration was interrupted for 15 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following filtrate, i.e. the stock solution, was used in the test. During filtration, the filter was always kept covered. The test item solution was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative. Out of the stock solution five concentrations were tested in a geometrical series with a dilution factor of √10:1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L.Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditionswere within the acceptable limits. The test media were visually clear throughout the test period. The concentrations of the test item in all concentration levels and the control were analytically verified by LC-MS at the start and at the end of exposure. The measured concentrations of the test item in the fresh media were in the range of 95 to 104% of the nominal values.At the end of exposure after 72 hours, the measured concentrations were in the range of 90 to 100% of the nominal test item concentrations. All measured test item concentrations were within ± 20% of the nominal at the start and the end of the exposure. This indicates that the test item concentrations were successfully maintained for the duration of the test. Therefore, all affect values are given as nominal concentrations. The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 19.5 (CI: 17.3 – 21.6) mg/L and 9.21 (CI: 7.57 - 10.7) mg/L, respectively. The NOEC-values for inhibition of growth rate and yield after 72 hours were 3.16 and < 1.00 mg/L respectively.

Description of key information

The toxicity of the test itemto the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3.The aim of the study was the determination of the effects on growth rate and yield over a period of 72 hours.The study was conducted under static conditions with an initial cell density of 5416 cells/mL. A test item solution with a nominal concentration of 100 mg test item/L was prepared with dilution water. The dispersion was treated with ultrasound for 40 minutes at room temperature. Undissolved particles were removed by membrane filtration (membrane filterµm, RC,Macherey-Nagel).The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate were discarded. The filtration was interrupted for 15 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following filtrate, i.e. the stock solution, was used in the test. During filtration, the filter was always kept covered. The test item solution was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative. Out of the stock solution five concentrations were tested in a geometrical series with a dilution factor of √10:1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L.Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditionswere within the acceptable limits.The test media were visually clear throughout the test period. The concentrations of the test item in all concentration levels and the control were analytically verified by LC-MS at the start and at the end of exposure. The measured concentrations of the test item in the fresh media were in the range of 95 to 104% of the nominal values.At the end of exposure after 72 hours, the measured concentrations were in the range of 90 to 100% of the nominal test item concentrations. All measured test item concentrations were within ± 20% of the nominal at the start and the end of the exposure. This indicates that the test item concentrations were successfully maintained for the duration of the test. Therefore, all affect values are given as nominal concentrations. The EC50-values for inhibition of growth rate (ErC₅₀) and yield (EyC₅₀) after 72 hours were 19.5 (CI: 17.3 – 21.6) mg/L and 9.21 (CI: 7.57 - 10.7) mg/L, respectively. The NOEC-values for inhibition of growth rate and yield after 72 hours were 3.16 and < 1.00 mg/L respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

19.5 mg/L

EC10 or NOEC for freshwater algae:

8.45 mg/L

Additional information

8.45 is the 72h- EC10 value of growth inhibition.