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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
The test item was stored in a closed vessel dry at room temperature (20 ± 5 °C).
Vehicle:
no
Remarks:
Due to the poor solubility of the test item, the test item was added directly into the test vessels.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant in D-67435 NW-Lachen-Speyerdorf, Im Altenschemel. Upon arrival in the test facility, the sludge was filtrated, washed with tap water 3 times and re-suspended in tap water. The activated sludge was aerated until usage in the test and fed daily with 50 mL synthetic sewage feed /L. In the first experiment (pre-test), the dry matter was determined as 2.92 g suspended solids/L, giving a concentration of 1.46 g suspended solids/L in the test. In the second experiment (main test), the dry matter of the inoculum was determined as 2.18 g suspended solids/L, giving a concentration of 1.09 g suspended solids/L in the test.
Test type:
not specified
Water media type:
other: tap water
Total exposure duration:
3 h
Post exposure observation period:
After 3 hours, the content of the first vessel was poured in a 250 mL narrow-neck bottle and the respiration rate was determined by measurement of the O2-concentration over a period of max. 5 minutes. The respiration rate was measured over a period of only 5 minutes. In the guideline, a period of 5 to 10 minutes or until the oxygen concentration falls below 2 mg/L is recommended. As the linearity of all regression linear curves (r2 of 0.99 or more) is given, the measurement period can be considered as sufficiently long.
Hardness:
0.88 mmol/L
Test temperature:
18.5 – 20.7 °C (first experiment (pre-test))
19.1 – 21.4 °C (second experiment (main test))
pH:
The pH of the nutrient solutions was 7.0.
The pH of the water used to prepare the solutions was 8.01.
Conductivity:
210 µS/cm
Nominal and measured concentrations:
In the first experiment (pre-test) the test item was tested using 4 concentrations ranging from 1000 to 1 mg/L (1000, 100, 10 and 1 mg/L) nominal concentration.
In the second experiment (main test) the test item was tested using 5 concentrations ranging from 1000 to 10 mg/L (1000, 320, 100, 32 and 10 mg/L) nominal concentration.
Details on test conditions:
In the blank control vessels, 16 mL nutrient solution (synthetic sewage) was mixed with 234 mL water. The positive control vessels and the treatments were prepared by putting the appropriate amount of positive control solution or test item solution into the respective test vessel, adding 16 mL nutrient solution and water to reach a total volume of 250 mL. Then, 250 mL inoculum was added in 5 minutes intervals and the mixtures were aerated.
After 3 hours, the content of the first vessel was poured in a 250 mL narrow-neck bottle and the respiration rate was determined by measurement of the O2-concentration over a period of max. 5 minutes . The following vessels were measured likewise in 5 minutes intervals.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol (CAS-No. 591-35-5) was used as positive control. A stock solution in deionised water containing 501 mg/L of 3,5-Dichlorophenol was freshly prepared for each experiment.
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks on result:
other: confidence interval 350 - 860 mg/L
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
190 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks on result:
other: confidence interval 100 - 270 mg/L
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
320 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Details on results:
For evaluation of the results the nominal concentrations were used because the difference between real concentration and nominal concentration can be stated as not significant.
For the calculation of the EC10 and EC50, the percentage inhibition was plotted versus concentration in a Gauß-logarithmic diagram. EC10 and EC50 were determined from the x values of the regression line at y = 10% and y = 50%.
The coefficient of variation of oxygen uptake rate in blank control replicates was not more than 30% at the end of the test.
The blank controls oxygen uptake rate was more than 20 mg oxygen per gram of activated sludge (dry weight of suspended solids) in 1 hour.
Results with reference substance (positive control):
Concentrations of the positive control were calculated using the concentration of the stock solution and the dilution factor.
Reported statistics and error estimates:
For the treatments with the test item concentrations 1000 and 320 mg/L, it was tested whether the differences between treatment and blank control were significant. For this determination, the values of the O2 consumption were used. The difference between treatment 320 mg/L and the blank control was considered as not significant as the calculated t-value laid below the tabulated t-value and O2 consumption in the treatment was higher than in the blank control. Therefore, the concentration 320 mg/L is stated as NOEC.

Content of Test Vessels in the second experiment (main test):

Vessel No.

Content

Concentration (mg/L)

nominal

Nutrient Solution (mL)

Tap Water (mL)

Stock Solution added (mL)

Concen­tration of Stock Solution (mg/L)

Inoculum (mL)

1

Blank Control

0

16

234

0

--

250

2

Blank Control

0

16

234

0

--

250

3

Positive Control

5

16

229

5

501

250

4

Positive Control

10

16

224

10

501

250

5

Positive Control

20

16

214

20

501

250

6

Positive Control

40

16

194

40

501

250

7

Blank Control

0

16

234

0

--

250

8

Blank Control

0

16

234

0

--

250

Vessel No.

Content

Concentration (mg/L) nominal

Nutrient Solution (mL)

Tap Water (mL)

Amount added (mg)

Calculated amount (mg)

Inoculum (mL)

9

Test Item

1000

16

234

500.1

1000.2

250

10

Test Item

1000

16

234

499.8

999.6

250

11

Test Item

1000

16

234

500.3

1000.6

250

12

Test Item

1000

16

234

500.6

1001.2

250

13

Test Item

1000

16

234

499.8

999.6

250

14

Test Item

320

16

234

159.6

319.2

250

15

Test Item

320

16

234

160.4

320.8

250

16

Test Item

320

16

234

160.9

321.8

250

17

Test Item

320

16

234

159.8

319.6

250

18

Test Item

320

16

234

159.6

319.2

250

19

Test Item

100

16

234

50.2

100.4

250

20

Test Item

100

16

234

50.1

100.2

250

21

Test Item

100

16

234

50.2

100.4

250

22

Test Item

100

16

234

50.3

100.6

250

23

Test Item

100

16

234

49.7

99.4

250

Vessel No.

Content

Concentration (mg/L)

nominal

Nutrient Solution (mL)

Tap Water (mL)

Amount added (mg)

Calculated amount (mg)

Inoculum (mL)

24

Test Item

32

16

234

15.9

31.8

250

25

Test Item

32

16

234

15.9

31.8

250

26

Test Item

32

16

234

16.1

32.2

250

27

Test Item

32

16

234

16.1

32.2

250

28

Test Item

32

16

234

16.1

32.2

250

29

Test Item

10

16

234

4.9

9.8

250

30

Test Item

10

16

234

5.1

10.2

250

31

Test Item

10

16

234

4.9

9.8

250

32

Test Item

10

16

234

5.1

10.2

250

33

Test Item

10

16

234

4.9

9.8

250

34

Blank Control

0

16

234

--

--

250

35

Blank Control

0

16

234

--

--

250

Results of the second experiment (main test):

Vessel No.

Content

Concentration in mg/L

nominal

O2consumption in mg/(L*min)

O2consumption in mg/(L*h)

Inhibition in %

pH meeasured at the end of the test

1

Blank Control

0

0.7024

42.142

0

8.1

2

Blank Control

0

0.7713

46.279

0

8.1

3

Positive Control

5

0.4971

29.826

31.7

8.2

4

Positive Control

10

0.3127

18.764

57.0

8.2

5

Positive Control

20

0.1557

9.345

78.6

8.3

6

Positive Control

40

0.0800

4.799

89.0

8.3

7

Blank Control

0

0.6504

39.022

0

8.2

8

Blank Control

0

0.7296

43.773

0

8.1

9

Test Item

1000

0.0278

1.666

96.2

4.6

10

Test Item

1000

0.0583

3.497

92.0

4.5

11

Test Item

1000

0.0550

3.301

92.4

4.5

12

Test Item

1000

0.0232

1.395

96.8

4.5

13

Test Item

1000

0.0294

1.767

96.0

4.5

14

Test Item

320

0.7550

45.299

-3.7

7.7

15

Test Item

320

0.7025

42.148

3.5

7.8

16

Test Item

320

0.7595

45.569

-4.3

7.8

17

Test Item

320

0.7541

45.245

-3.6

7.8

18

Test Item

320

0.7431

44.587

-2.1

7.8

19

Test Item

100

0.7737

46.420

-6.3

8.0

20

Test Item

100

0.7575

45.452

-4.1

8.0

21

Test Item

100

0.6835

41.011

6.1

8.0

22

Test Item

100

0.7034

42.204

3.4

8.1

23

Test Item

100

0.6415

38.488

11.9

8.1

24

Test Item

32

0.7666

45.997

-5.3

8.1

25

Test Item

32

0.7391

44.346

-1.5

8.1

26

Test Item

32

0.7621

45.727

-4.7

8.1

27

Test Item

32

0.7468

44.810

-2.6

8.1

28

Test Item

32

0.7860

47.163

-8.0

8.0

29

Test Item

10

0.7508

45.048

-3.1

8.1

30

Test Item

10

0.7357

44.141

-1.1

8.1

31

Test Item

10

0.7544

45.265

-3.6

8.1

32

Test Item

10

0.7528

45.168

-3.4

8.1

33

Test Item

10

0.7735

46.411

-6.3

8.1

34

Blank Control

0

0.7763

46.578

0

8.1

35

Blank Control

0

0.7378

44.269

0

8.1

Validity criteria fulfilled:
yes
Remarks:
No observations were made which might cause doubts concerning the validity of the study outcome.
Conclusions:
The following results for the test item CDTA HHQ were determined:
3h NOEC = 320 mg/L
3h EC10 = 190 mg/L (confidence interval 100 - 270 mg/L)
3h EC50 = 500 mg/L (confidence interval 350 - 860 mg/L)

Note: According to the guideline, NOEC is determined by comparing the respective treatment with the blank control. Statistically insignificant variation is considered as “no observed effect”, although the EC10 which is read from the graph toxicity vs. concentration may lie lower.
Executive summary:

Two experiments were performed.

In the first experiment (pre-test) the test item was tested using 4 concentrations ranging from 1000 to 1 mg/L nominal concentration. For the treatment 1000 mg/L, 5 replicates were used; for the other treatments, 1 replicate each.

Duration of the test was 3 hours. Activated sludge was used as inoculum. It was taken from a domestic sewage treatment plant and washed before usage. The dry matter was determined as 2.92 g suspended solids/L, giving a concentration of 1.46 g suspended solids/L in the test.3,5-Dichloro-phenol was used as positive control.

Because significant inhibition was observed, an additional experiment had to be performed.

In the second experiment (main test) the test item was tested using 5 concentrations ranging from 1000 to 10 mg/L nominal concentration. For all treatments 5 replicates were used. The dry matter of the inoculum was determined as 2.18 g suspended solids/L, giving a concentration of 1.09 g suspended solids/L in the test.

All validity criteria were met. For the estimation of the EC50 of the positive control, the fits showed good statistical correspondence of the data with the dose-response-equation. The positive control gave an EC50 of 8.8 mg/L (first experiment pre-test) and 8.4 mg/L (second experiment main test) which lie within the recommended range of 2 – 25 mg/L stated in the OECD guideline. The coefficient of variation of oxygen uptake rate in control replicates was below 30% at the end of the test. The oxygen uptake rate of the blank controls was above 20 mg O2per gram activated sludge in 1 hour.

For evaluation of the results the nominal concentrations were used because the difference between real concentration and nominal concentration can be stated as not significant.

Because no inhibition was observed in the 3 lowest concentrated treatments, the 2 lowest concentrated treatments were not used for evaluation to receive a linear regression.

No observations were made which might cause doubts concerning the validity of the study outcome.

The result of the test can be considered valid.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
January 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
It is reasonable that the toxicity to microorganisms of tripotassium hydrogen ethylenediaminetetraacetate is caused by the ammonium groups of the substance. In a read-across approach, a guideline study on CDTA is used to evaluated the toxicity as these substance has the same moiety. Furthermore, with 4 ammonium groups (in CDTA) compared to only 2 (in tripotassium hydrogen ethylenediaminetetraacetate) it can be assume that the toxicity of the latter is not higher than the one of the first. Accordingly, the toxicity might be overestimated but not underestimated.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
The test item was stored in a closed vessel dry at room temperature (20 ± 5 °C).
Vehicle:
no
Remarks:
Due to the poor solubility of the test item, the test item was added directly into the test vessels.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant in D-67435 NW-Lachen-Speyerdorf, Im Altenschemel. Upon arrival in the test facility, the sludge was filtrated, washed with tap water 3 times and re-suspended in tap water. The activated sludge was aerated until usage in the test and fed daily with 50 mL synthetic sewage feed /L. In the first experiment (pre-test), the dry matter was determined as 2.92 g suspended solids/L, giving a concentration of 1.46 g suspended solids/L in the test. In the second experiment (main test), the dry matter of the inoculum was determined as 2.18 g suspended solids/L, giving a concentration of 1.09 g suspended solids/L in the test.
Test type:
not specified
Water media type:
other: tap water
Total exposure duration:
3 h
Post exposure observation period:
After 3 hours, the content of the first vessel was poured in a 250 mL narrow-neck bottle and the respiration rate was determined by measurement of the O2-concentration over a period of max. 5 minutes. The respiration rate was measured over a period of only 5 minutes. In the guideline, a period of 5 to 10 minutes or until the oxygen concentration falls below 2 mg/L is recommended. As the linearity of all regression linear curves (r2 of 0.99 or more) is given, the measurement period can be considered as sufficiently long.
Hardness:
0.88 mmol/L
Test temperature:
18.5 – 20.7 °C (first experiment (pre-test))
19.1 – 21.4 °C (second experiment (main test))
pH:
The pH of the nutrient solutions was 7.0.
The pH of the water used to prepare the solutions was 8.01.
Conductivity:
210 µS/cm
Nominal and measured concentrations:
In the first experiment (pre-test) the test item was tested using 4 concentrations ranging from 1000 to 1 mg/L (1000, 100, 10 and 1 mg/L) nominal concentration.
In the second experiment (main test) the test item was tested using 5 concentrations ranging from 1000 to 10 mg/L (1000, 320, 100, 32 and 10 mg/L) nominal concentration.
Details on test conditions:
In the blank control vessels, 16 mL nutrient solution (synthetic sewage) was mixed with 234 mL water. The positive control vessels and the treatments were prepared by putting the appropriate amount of positive control solution or test item solution into the respective test vessel, adding 16 mL nutrient solution and water to reach a total volume of 250 mL. Then, 250 mL inoculum was added in 5 minutes intervals and the mixtures were aerated.
After 3 hours, the content of the first vessel was poured in a 250 mL narrow-neck bottle and the respiration rate was determined by measurement of the O2-concentration over a period of max. 5 minutes . The following vessels were measured likewise in 5 minutes intervals.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol (CAS-No. 591-35-5) was used as positive control. A stock solution in deionised water containing 501 mg/L of 3,5-Dichlorophenol was freshly prepared for each experiment.
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks on result:
other: confidence interval 350 - 860 mg/L
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
190 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks on result:
other: confidence interval 100 - 270 mg/L
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
320 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Details on results:
For evaluation of the results the nominal concentrations were used because the difference between real concentration and nominal concentration can be stated as not significant.
For the calculation of the EC10 and EC50, the percentage inhibition was plotted versus concentration in a Gauß-logarithmic diagram. EC10 and EC50 were determined from the x values of the regression line at y = 10% and y = 50%.
The coefficient of variation of oxygen uptake rate in blank control replicates was not more than 30% at the end of the test.
The blank controls oxygen uptake rate was more than 20 mg oxygen per gram of activated sludge (dry weight of suspended solids) in 1 hour.
Results with reference substance (positive control):
Concentrations of the positive control were calculated using the concentration of the stock solution and the dilution factor.
Reported statistics and error estimates:
For the treatments with the test item concentrations 1000 and 320 mg/L, it was tested whether the differences between treatment and blank control were significant. For this determination, the values of the O2 consumption were used. The difference between treatment 320 mg/L and the blank control was considered as not significant as the calculated t-value laid below the tabulated t-value and O2 consumption in the treatment was higher than in the blank control. Therefore, the concentration 320 mg/L is stated as NOEC.

Content of Test Vessels in the second experiment (main test):

Vessel No.

Content

Concentration (mg/L)

nominal

Nutrient Solution (mL)

Tap Water (mL)

Stock Solution added (mL)

Concen­tration of Stock Solution (mg/L)

Inoculum (mL)

1

Blank Control

0

16

234

0

--

250

2

Blank Control

0

16

234

0

--

250

3

Positive Control

5

16

229

5

501

250

4

Positive Control

10

16

224

10

501

250

5

Positive Control

20

16

214

20

501

250

6

Positive Control

40

16

194

40

501

250

7

Blank Control

0

16

234

0

--

250

8

Blank Control

0

16

234

0

--

250

Vessel No.

Content

Concentration (mg/L) nominal

Nutrient Solution (mL)

Tap Water (mL)

Amount added (mg)

Calculated amount (mg)

Inoculum (mL)

9

Test Item

1000

16

234

500.1

1000.2

250

10

Test Item

1000

16

234

499.8

999.6

250

11

Test Item

1000

16

234

500.3

1000.6

250

12

Test Item

1000

16

234

500.6

1001.2

250

13

Test Item

1000

16

234

499.8

999.6

250

14

Test Item

320

16

234

159.6

319.2

250

15

Test Item

320

16

234

160.4

320.8

250

16

Test Item

320

16

234

160.9

321.8

250

17

Test Item

320

16

234

159.8

319.6

250

18

Test Item

320

16

234

159.6

319.2

250

19

Test Item

100

16

234

50.2

100.4

250

20

Test Item

100

16

234

50.1

100.2

250

21

Test Item

100

16

234

50.2

100.4

250

22

Test Item

100

16

234

50.3

100.6

250

23

Test Item

100

16

234

49.7

99.4

250

Vessel No.

Content

Concentration (mg/L)

nominal

Nutrient Solution (mL)

Tap Water (mL)

Amount added (mg)

Calculated amount (mg)

Inoculum (mL)

24

Test Item

32

16

234

15.9

31.8

250

25

Test Item

32

16

234

15.9

31.8

250

26

Test Item

32

16

234

16.1

32.2

250

27

Test Item

32

16

234

16.1

32.2

250

28

Test Item

32

16

234

16.1

32.2

250

29

Test Item

10

16

234

4.9

9.8

250

30

Test Item

10

16

234

5.1

10.2

250

31

Test Item

10

16

234

4.9

9.8

250

32

Test Item

10

16

234

5.1

10.2

250

33

Test Item

10

16

234

4.9

9.8

250

34

Blank Control

0

16

234

--

--

250

35

Blank Control

0

16

234

--

--

250

Results of the second experiment (main test):

Vessel No.

Content

Concentration in mg/L

nominal

O2consumption in mg/(L*min)

O2consumption in mg/(L*h)

Inhibition in %

pH meeasured at the end of the test

1

Blank Control

0

0.7024

42.142

0

8.1

2

Blank Control

0

0.7713

46.279

0

8.1

3

Positive Control

5

0.4971

29.826

31.7

8.2

4

Positive Control

10

0.3127

18.764

57.0

8.2

5

Positive Control

20

0.1557

9.345

78.6

8.3

6

Positive Control

40

0.0800

4.799

89.0

8.3

7

Blank Control

0

0.6504

39.022

0

8.2

8

Blank Control

0

0.7296

43.773

0

8.1

9

Test Item

1000

0.0278

1.666

96.2

4.6

10

Test Item

1000

0.0583

3.497

92.0

4.5

11

Test Item

1000

0.0550

3.301

92.4

4.5

12

Test Item

1000

0.0232

1.395

96.8

4.5

13

Test Item

1000

0.0294

1.767

96.0

4.5

14

Test Item

320

0.7550

45.299

-3.7

7.7

15

Test Item

320

0.7025

42.148

3.5

7.8

16

Test Item

320

0.7595

45.569

-4.3

7.8

17

Test Item

320

0.7541

45.245

-3.6

7.8

18

Test Item

320

0.7431

44.587

-2.1

7.8

19

Test Item

100

0.7737

46.420

-6.3

8.0

20

Test Item

100

0.7575

45.452

-4.1

8.0

21

Test Item

100

0.6835

41.011

6.1

8.0

22

Test Item

100

0.7034

42.204

3.4

8.1

23

Test Item

100

0.6415

38.488

11.9

8.1

24

Test Item

32

0.7666

45.997

-5.3

8.1

25

Test Item

32

0.7391

44.346

-1.5

8.1

26

Test Item

32

0.7621

45.727

-4.7

8.1

27

Test Item

32

0.7468

44.810

-2.6

8.1

28

Test Item

32

0.7860

47.163

-8.0

8.0

29

Test Item

10

0.7508

45.048

-3.1

8.1

30

Test Item

10

0.7357

44.141

-1.1

8.1

31

Test Item

10

0.7544

45.265

-3.6

8.1

32

Test Item

10

0.7528

45.168

-3.4

8.1

33

Test Item

10

0.7735

46.411

-6.3

8.1

34

Blank Control

0

0.7763

46.578

0

8.1

35

Blank Control

0

0.7378

44.269

0

8.1

Validity criteria fulfilled:
yes
Remarks:
No observations were made which might cause doubts concerning the validity of the study outcome.
Conclusions:
The following results for the test item CDTA were determined:
3h NOEC = 320 mg/L
3h EC10 = 190 mg/L (confidence interval 100 - 270 mg/L)
3h EC50 = 500 mg/L (confidence interval 350 - 860 mg/L)
As justified above, it is reasonable to assume the the values for tripotassium hydrogen ethylenediaminetetraacetate are the same or even higher.

Note: According to the guideline, NOEC is determined by comparing the respective treatment with the blank control. Statistically insignificant variation is considered as “no observed effect”, although the EC10 which is read from the graph toxicity vs. concentration may lie lower.
Executive summary:

Two experiments were performed.

In the first experiment (pre-test) the test item was tested using 4 concentrations ranging from 1000 to 1 mg/L nominal concentration. For the treatment 1000 mg/L, 5 replicates were used; for the other treatments, 1 replicate each.

Duration of the test was 3 hours. Activated sludge was used as inoculum. It was taken from a domestic sewage treatment plant and washed before usage. The dry matter was determined as 2.92 g suspended solids/L, giving a concentration of 1.46 g suspended solids/L in the test.3,5-Dichloro-phenol was used as positive control.

Because significant inhibition was observed, an additional experiment had to be performed.

In the second experiment (main test) the test item was tested using 5 concentrations ranging from 1000 to 10 mg/L nominal concentration. For all treatments 5 replicates were used. The dry matter of the inoculum was determined as 2.18 g suspended solids/L, giving a concentration of 1.09 g suspended solids/L in the test.

All validity criteria were met. For the estimation of the EC50 of the positive control, the fits showed good statistical correspondence of the data with the dose-response-equation. The positive control gave an EC50 of 8.8 mg/L (first experiment pre-test) and 8.4 mg/L (second experiment main test) which lie within the recommended range of 2 – 25 mg/L stated in the OECD guideline. The coefficient of variation of oxygen uptake rate in control replicates was below 30% at the end of the test. The oxygen uptake rate of the blank controls was above 20 mg O2per gram activated sludge in 1 hour.

For evaluation of the results the nominal concentrations were used because the difference between real concentration and nominal concentration can be stated as not significant.

Because no inhibition was observed in the 3 lowest concentrated treatments, the 2 lowest concentrated treatments were not used for evaluation to receive a linear regression.

No observations were made which might cause doubts concerning the validity of the study outcome.

The result of the test can be considered valid.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Tripotassium hydrogen ethylenediaminetetraacetate dissociates into potassium cations and anions of edetic acid.
The first are known to be non-toxic as potassium is an essential element for all species.
The EU risk assessment reports collected data of toxicity studies on microorganisms for edetic acid and several salts (Na2H2EDTA, Na4EDTA).
Those substance dissociate into non-toxic cations and the same edetic acid anions as tripotassium hydrogen ethylenediaminetetraacetate.
As the relevant species are exactly the same, also the toxicity is the same and thus a read-across approach is justified.
Qualifier:
no guideline available
Principles of method if other than guideline:
These tests are not well-documented. It is unclear whether the test organisms are sufficiently supplied with trace nutrients. It cannot be excluded that, similar to algae, the effects were caused by nutrient deficiency.
Nevertheless the toxiciy might be overestimated but not underestimated.
The reliable respiratory test (according to OECD 209) confirms the low toxicity of edetic acid and it derivatives towards microorganisms.
GLP compliance:
no
Specific details on test material used for the study:
Additionally Na2H2EDTA and Na4EDTA) were evaluated.
Analytical monitoring:
no
Test organisms (species):
other: several species were used. OECD 209 was performed with activated sludge.
Key result
Duration:
16 h
Dose descriptor:
NOEC
Remarks:
species: Pseudomonas putida
Effect conc.:
105 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Remarks:
referred to edetic acid
Duration:
30 min
Dose descriptor:
EC10
Effect conc.:
48 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Remarks:
referred to edetic acid
Remarks on result:
not determinable because of methodological limitations
Remarks:
Due to the short test duration and limit test details this value was not considered as relevant.
Key result
Duration:
20 h
Dose descriptor:
NOEC
Remarks:
species: Uronema parduczi
Effect conc.:
13 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Remarks:
referred to edetic acid
Key result
Duration:
48 h
Dose descriptor:
NOEC
Remarks:
species: Chilomonas paramaecium
Effect conc.:
510 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Remarks:
referred to edetic acid
Key result
Duration:
72 h
Dose descriptor:
NOEC
Remarks:
species: Entosiphon sulcatum
Effect conc.:
28 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Remarks:
referred to edetic acid
Key result
Duration:
30 min
Dose descriptor:
EC10
Remarks:
activated sludge according to OECD 209
Effect conc.:
>= 500 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Remarks:
referred to edetic acid
Details on results:
The TGK value (toxic limit concentration) is regarded as the NOEC value.
Conclusions:
In conclusion, the NOEC values reported by the risk assessment report are 13 mg/L or higher.

Description of key information

Based on several studies on edetic acid or their derivatives the lowest EC50 was 500 mg/L and the NOEC was 13 mg/L, respectively.

The EC50 value was confirmed by a study according to OECD 409 on CDTA containing the same ammonium groups. The NOEC of the latter is significantly higher. Nevertheless, in a worst case approach the lowest available NOEC is considered as relevant for the assessment.

Key value for chemical safety assessment

EC50 for microorganisms:
500 mg/L
EC10 or NOEC for microorganisms:
13 mg/L

Additional information