(benzothiazol-2-ylthio)acetic acid

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-06-09 to 2015-06-12

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(benzothiazol-2-ylthio)acetic acid

EC Number:

228-565-0

EC Name:

(benzothiazol-2-ylthio)acetic acid

Cas Number:

6295-57-4

Molecular formula:

C9H7NO2S2

IUPAC Name:

2-(1,3-benzothiazol-2-ylsulfanyl)acetic acid

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

-Test item (Benzothiazol-2-ylthio)acetic acid
-Batch number 41273
-CAS No. 6295-57-4
-EC No. 228-565-0
-Molecular formula C9H7NO2S2
-Molecular weight 225.29
-Active content (certified) 97.58 [GC area-%]

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
7 concentrations were tested in a geometrical series with a dilution factor of 3.16: 0.12 - 0.38 - 1.20 - 3.80 - 12.0 - 38.0 - 120 mg/L.

- Sampling method:
Analytical evaluation of the test concentrations of (Benzothiazol-2-ylthio)acetic acid and the control were carried out via HPLC-DAD from freshly prepared media after 0 hours and old test media after 72 hours. Separate replicates for the test item analysis at the beginning of the exposure were prepared without algae. For the test item analysis after 72 hours, samples were taken out of the test replicates.

Test solutions

Vehicle:

no

Details on test solutions:

Stock solution
A stock solution of 120 mg/L was freshly prepared with dilution water.

Media Preparation
Direct addition of the test item and shaking until the test item is completely dissolved.

Test concentrations (nominal)
7 concentrations were tested in a geometrical series with a dilution factor of 3.16:
0.12 - 0.38 - 1.20 - 3.80 - 12.0 - 38.0 - 120 mg/L.

Control
Six replicates (without test item) were incubated under the same test conditions as the test replicates.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.


ACCLIMATION
- 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

Post exposure observation period:

None

Test conditions

Hardness:

Not measured

Test temperature:

Mean: 23.5 °C, Min: 22.2 °C, Max: 24.0 °C

pH:

Nominal test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
120 7.01 7.04
38.0 7.05 7.68
12.0 7.11 7.78
3.80 7.35 7.89
1.20 7.42 7.95
0.38 7.67 8.07
0.12 7.75 8.17
Control 8.02 8.34

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to information on materials and methods

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks (sterile)
- Type (delete if not applicable): sealed with cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Agitation: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: 4847 cells/mL
- Control end cells density: Mean 754649 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None

GROWTH MEDIUM
- Standard medium used: Yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to the guideline (OECD)

Composition of Dilution water (OECD)

Component Concentration [mg/L]
NH4Cl 15
MgCl2  6 H2O 12
CaCl2  2 H2O 18
MgSO4  7 H2O 15
KH2PO4 1.6
FeCl3  6 H2O 0.064
Na2EDTA  2 H2O 0.1
H3BO3 0.185
MnCl2  4 H2O 0.415
ZnCl2 3 x 10-3
Na2MoO4  2 H2O 7 x 10-3
CoCl2  6 H2O 1.5 x 10-3
CuCl2  2 H2O 1 x 10-5
NaHCO3 50
pH-value 8.1  0.2

This medium had a nominal hardness of 0.24 mmol Ca+Mg/L.


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1 (nominal), 70.0 µE x m-2 x s-1 (measured, n = 24, CV = 5.07%)



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via
Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.



TEST CONCENTRATIONS
- Range finding study: Yes
The results of the preliminary range finding test are presented below:

Results of the Range Finding Test (0-72 h)
Nominal test item
concentration
[mg/L] Growth Rate Inhibition
[%] Yield Inhibition
[%]
120 85 99
12.0 10 41
1.2 6 26
0.12 3 14

Negative Inhibitions = Stimulation of growth


Measured concentrations of the Test Item in the Range Finding Test

The measured concentrations of the test item in the preliminary test are presented below:

Measured Concentrations of the Test Item (Benzothiazol-2-ylthio)acetic acid during the non-GLP Preliminary Range Finding Test
Sampling date 0 hours
Start of the
exposure 72 hours
End of the
exposure
Aqueous phase Aqueous Phase
Nominal
test item
concentration
[mg/L] (Benzothiazol-2-ylthio)acetic acid
Meas. conc.
[mg/L] % Meas. conc.
[mg/L] %
120 121 101 120 100
12.0 12.8 107 12.2 102
1.20 1.42 118 1.21 101
0.12 0.121 101 0.117 98
Control < LOQ < LOQ











Meas. conc. = measured concentration of the test item, single injection, dilution factor taken into account
% = percent of the nominal concentration of the test item

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

38 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

15.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 13.3 - 18.6

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

20.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 18.3 - 23.3

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

34.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 32.6 - 35.9

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 7

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 7.94 - 11.8

Effect concentrations 9

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

11.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 10.2 - 14.1

Effect concentrations 10

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

16.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 14.4 - 22.9

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: Microscopic evaluation of the cells at the start and the end of the exposure period revealed no morphological abnormalities.
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.586 (CI 0.522 - 0.818) mg/L
Yield Inhibition: 0.401 (CI 0.285 - 0.469) mg/L

Reported statistics and error estimates:

EC-values and statistical analysis:
EC-values of the growth rate and yield inhibition were calculated by non-linear sigmoidal dose-response regression.

NOEC, LOEC and statistical analyses
The values for NOEC/LOEC were determined by calculation of statistical significance of inhibition of growth rate and yield. As standard methods, One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is  = 0.05.

Any other information on results incl. tables

Cell Densities

 

Nominal test item concentration	Replicate		Cell density [cells/mL]
[mg/L]	No.		0 hours	24 hours		48 hours		72 hours
120	1		4847	<LOQ		<LOQ		<LOQ
	2		4847	<LOQ		<LOQ		<LOQ
	3		4847	<LOQ		<LOQ		<LOQ
	Mean		4847	n.a.		n.a.		n.a.
38.0	1		4847	13443		29775		60591
	2		4847	12928		51273		37775
	3		4847	12851		32911		32546
	Mean		4847	13074		37986		43637
12.0	1		4847	20851		107570		555149
	2		4847	24459		100874		569580
	3		4847	24425		115459		674545
	Mean		4847	23245		107968		599758
3.80	1		4847	25955		110601		674497
	2		4847	25243		124335		832089
	3		4847	29044		121242		893231
	Mean		4847	26747		118726		799939
1.20	1		4847	22674		128217		847050
	2		4847	28827		129439		775951
	3		4847	23665		149051		840892
	Mean		4847	25055		135569		821298
0.38	1		4847	27124		119173		738525
	2		4847	28447		131627		821699
	3		4847	27033		131267		883032
	Mean		4847	27535		127356		814419
0.12	1		4847	24555		117980		716012
	2		4847	23487		109134		761952
	3		4847	21558		130074		820448
	Mean		4847	23200		119063		766137
Control		1	4847		21424		121877		769072
		2	4847		22131		136404		708556
		3	4847		17219		121896		713174
		4	4847		18504		125177		762145
		5	4847		22198		148070		829107
		6	4847		23131		149465		745837
		Mean	4847		20768		133815		754649

< LOQ     = below the limit of quantification

n.a.            = data not determinable

 Evaluation after 72 hours

Statistically significant differences of growth rates and yield compar control values are marked (+), not significant differences are marked (-).

Nominal test item concentration [mg/L]	Replicate	Growth rate		Growth rate inhibition	Yield		Inhibition of yield
	No.	[d^-1]		[%]	[cells/mL]		[%]
120	1		<LOQ	100		<LOQ	100
	2		<LOQ	100		<LOQ	100
	3		<LOQ	100		<LOQ	100
	Mean	(+)*	n.a.	100	(+)*	n.a.	100
38.0	1		0.84	50		55744	93
	2		0.68	59		32928	96
	3		0.64	62		27699	96
	Mean	(+)	0.72	57	(+)	38790	95
12.0	1		1.58	6		550302	27
	2		1.59	6		564733	25
	3		1.65	2		669698	11
	Mean	(-)	1.61	5	(+)	594911	21
3.80	1		1.65	2		669650	11
	2		1.72	-2		827242	-10
	3		1.74	-3		888384	-18
	Mean	(-)	1.70	-1	(-)	795092	-6
1.20	1		1.72	-2		842203	-12
	2		1.69	-1		771104	-3
	3		1.72	-2		836045	-12
	Mean	(-)	1.71	-2	(-)	816451	-9
0.38	1		1.68	0		733678	2
	2		1.71	-2		816852	-9
	3		1.74	-3		878185	-17
	Mean	(-)	1.71	-1	(-)	809572	-8
0.12	1		1.67	1		711165	5
	2		1.69	0		757105	-1
	3		1.71	-2		815601	-9
	Mean	(-)	1.69	0	(-)	761290	-2
Control	1		1.69			764225
	2		1.66			703709
	3		1.66			708327
	4		1.69			757298
	5		1.71			824260
	6		1.68			740990
	Mean		1.68			749802

negative inhibitions = increase of growth

* = not included in statistical analyses due to values < LOQ
 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, (Benzothiazol-2-ylthio)acetic acid was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours with the following effect values: The NOEC-value after 72 hours of the test item for inhibition of growth rate and yield was 12.0 mg/L and 3.80 mg/L, the LOEC for inhibition of growth rate and yield was 38.0 mg/L and 12.0 mg/L. The EC50-values with 95 % confidence intervals of the test item for inhibition for growth rate (ErC50) and yield (EyC50) after 72 hours were determined to be 34.3 (32.6 – 35.9) mg/L and 16.9 (14.4 – 22.9) mg/L.

Executive summary:

The toxicity of (Benzothiazol-2-ylthio)acetic acid (batch number: 41273) to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 (2011) and Council Regulation (EC) 761/2009 Method C.3 at Dr.U.Noack-Laboratorien in 31157 Sarstedt, Germany from 2015-06-09 to 2015-06-12.The aim of the study was to assess 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 4847 cells/mL. A stock solution with a nominal concentration of 120 mg/L was tested. Seven concentrations were tested in a geometrical series with a dilution factor of 3.16:0.12 - 0.38 - 1.20 - 3.20 - 12.0 - 38.0 - 120 mg/L. Three replicates were tested for the test item concentrations and six for the control. Environmental conditions were determined to be within the acceptable limits.

The test item concentration in the test and control solutions were analysed by HPLC-DAD at the start and the end of exposure. The recovery rate of the test item was in the range of 100 – 108 % of the nominal concentration at the test start and between 99 and 104 % at the test end.
All effect values of (Benzothiazol-2-ylthio)acetic acid are given based on the nominal test item concentrations.

 

 NOEC, LOEC, EC-values and Confidence Intervals of(Benzothiazol-2-ylthio)acetic acid (0-72 hours)

                  based on the nominal test item concentrations [mg/L]

                               

	Growth Rate Inhibition Nominaltest item concentration [mg/L]
NOEC	12.0
LOEC	38.0
ErC10	15.7 (13.3 – 18.6)
ErC20	20.8 (18.3 – 23.3)
ErC50	34.3 (32.6 – 35.9)
	Inhibition of Yield Nominaltest item concentration [mg/L]
NOEC	3.80
LOEC	12.0
EyC10	10.0 (7.94 – 11.8)
EyC20	11.9 (10.2 – 14.1)
EyC50	16.9 (14.4 – 22.9)