Registration Dossier

Ecotoxicological information

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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
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 concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
12 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
38 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
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
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
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
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
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
12 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
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
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
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)