Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-12-16 to 2014-12-17
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration 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:
- Name of test material (as cited in study report): (Benzothiazol-2-ylthio) acetic acid
- Molecular formula: C9H7NO2S2
- Molecular weight: 225.29 g/mol
- Physical state: Light yellow white powder
- Analytical purity: 97.58 %
- EC number: 228-565-0

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 10 - 32 - 100 - 320 - 1000 mg/L, each concentration with pH adjustment if necessary, were weighed out directly in Erlenmeyer flasks and stirred over night
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loadings: 10 - 32 - 100 - 320 - 1000 mg/L
- Controls: Test medium without test or reference item. Triplicates at the beginning of the test + triplicates at the end of the test

Test organisms

Test organisms (species):
activated sludge
Details on inoculum:
- Preparation of inoculum for exposure: Non-adapted activated sludge from the sewage plant at Hildesheim (Municipal sewage treatment plant of 31137 Hildesheim, Germany)
- Pretreatment: The sludge was washed twice with chlorine free tap water and adjusted to a dry sludge concentration of 3.0 g/L ± 10 %. The sludge was used within 24 h after sampling.
- Dry sludge concentration: 3.05 g/L, corresponding to 1.53g/L in the test vessel

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h

Test conditions

Hardness:
Not determined
Test temperature:
20.7 °C
pH:
pH-value of the activated sludge 7.57
pH-value of the synthetic waste water 7.43
Salinity:
Not determined
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flask, DIN 12380
- Aeration: Permanent, to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flakes in suspension.



- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6 (Triplicates at the beginning of the test + triplicates at the end of the test.)
- Composition of test medium:

Table for the Test Item Concentrations
Test Item Test concentration [mg/L]
10 32 100 320 1000 1000 pH
Test item [mg] 5 16 50 160 500 500
Inoculum [mL] 250
Synthetic waste water [mL] 16
Demineralised water [mL] filled up to 250 mL



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
After 3 h incubation, the oxygen concentration of each control, reference item and test item replicate was determined by means of oximeter immediately after transferring the solution to the measuring cell and the oxygen depletion was recorded for at least 3 minutes on a flat bed recorder.

TEST CONCENTRATIONS
Inhibition of Respiration (Preliminary Tests)

Inhibitions in the Preliminary Tests

Nominal Test Item Concentration [mg/L] pH(test item in demin. water) pH (adjusted to) pH (test item in medium) Inhibition [%]
Preliminary test – with pH adjustment
10 4.04 7.05* 7.29 4
4.44 - 7.29 -1
100 3.24 - 7.07 4
3.29 - 7.05 14 1000 3.21 - 4.35 93
3.24 7.30* 7.63 38

*)adjusted with 0.5 - -3.5 - 7 mol/L NaOH


Additional replicates were prepared to determine the inhibition of nitrification. Two replicates 1000 mg/L were measured with addition of Allylthiourea ATU (Final concentration: 11.6 mg/L). No indication for the requirement to measure the inhibition of nitrification in the definitive study was observed.

Inhibitions in the Preliminary Test (with / without ATU)

Nominal Test Item Concentration Inhibition of total respiration Inhibition of heterotrophic respiration Inhibition of nitrification
[mg/L] [%] [%] [%]
1000 93 89 97









Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate p.a.

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
227 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 142 - 278 mg/L
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
336 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 295 - 477 mg/L
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: EC50: 97.1 mg/L
Reported statistics and error estimates:
The NOEC was determined by calculation of statistical significance of the inhibition of respiration in comparison to the control. One Way Analysis of Variance (ANOVA) and DUNNETT’s test was used for NOEC 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 test are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

The EC-values of the test item and the reference item were calculated by sigmoidal dose-response regression and linear regression, respectively, using software GraphPadPrism. Calculations of the confidence intervals for the EC-values were carried out using standard procedures.


Any other information on results incl. tables

From the oxygen depletion the oxygen uptake rates were calculated in mg O2/L×h, additionally the specific respiration rates based on the dry sludge concentration were calculated in mg O2/g×h.

The inhibition of the oxygen uptake rates of the test and reference item concentrations in % was calculated in comparison with the oxygen uptake rates of the control.

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration of the Control and Test Item Concentrations

 

Test
Concentration
[mg/L]

Repl.

Oxygen Uptake Rate R
[mg O2/L
×h]

Inhibition

[%]

Mean Inhibition
[%]

 

Control

1

27.2

2

29.2

3

28.8

4

28.4

5

28.8

6

28.0

Test Item

10

1

29.2

-3

-1

2

28.4

0

3

28.0

1

4

29.2

-3

5

28.8

-1

32

1

26.8

6

4

2

28.0

1

3

27.6

3

4

25.2

11

5

29.2

-3

100

1

27.2

4

2

2

28.8

-1

3

28.4

0

4

27.2

4

5

27.6

3

320

1

23.6

17

19

2

22.0

23

3

24.0

15

4

23.2

18

5

22.8

20

1000

1

16.4

42

44

2

17.2

39

3

15.6

45

4

15.2

46

5

15.2

46

Repl. = Replicate

Coefficient of variation of oxygen uptake rates of the control: 2.5%

Suspended solids in the test vessel: 1.53 g/L

Specific Respiration Rates of the Control Replicates

 

 

Repl.

Specific Respiration Rate Rs
[mg O2/g
×h]

 

Control

1

18

2

19

3

19

4

19

5

18

6

19

Mean value

19

Repl. = Replicate

Suspended solids in the test vessel: 1.53 g/L

 

Oxygen Uptake Rates and Inhibition of the Respiration of the Reference Item Concentrations

 

Test Concentration
[mg/L]

Repl.

Oxygen Uptake

Rate R
[mg O2/L
×h]

Inhibition

 

[%]

Mean Inhibition

[%]

Reference Item

58

1

21.2

25

28

2

20.0

30

3

20.4

28

100

1

14.0

51

51

2

14.0

51

3

13.6

52

180

1

6.4

77

77

2

6.4

77

3

6.8

76

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The NOEC of (Benzothiazol-2-ylthio) acetic acid is 100 mg/L. The test item is not toxic up to the concentration of 100 mg/L to activated sludge of a municipal sewage treatment plant. The EC10 is 227 mg/L, the EC20 is 336 mg/L and the EC50 and EC80 of the test item are > 1000 mg/L.

Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item(Benzothiazol-2-ylthio) acetic acid(Batch number:41273) from 2014-12-16 to 2014-12-17 at the Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany. The test item concentrations were pH-measured, pH-adjusted (if necessary) and stirred overnight. The test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the test item concentrations10 - 32 - 100 - 320 - 1000 mg/L. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The mean inhibition of respiration for the test item replicates ranged from 1 % to 44 %.

 

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC50-value for the reference item was 97.1 mg/L.

NOEC and EC-Values with Confidence Interval of(Benzothiazol-2-ylthio) acetic acidafter 3 h exposure time, Inhibition of Total Respiration

 

EC-values

[mg/L]

Confidence interval
P = 95 [%]
[mg/L]

NOEC*

100

EC10

227

142 – 278

EC20

336

295 – 477

EC50

>1000

EC80

>1000

*) No statistically significant inhibition (P < 0.05), ANOVA, Dunnett`s Method

 

 

The NOEC of(Benzothiazol-2-ylthio) acetic acidis100mg/L.

The test item is not toxic up to the concentration of 100 mg/L to activated sludge of a municipal sewage treatment plant.

The EC10is 227 mg/L, the EC20is 336 mg/L and the EC50and EC80of the test item are >1000 mg/L.