Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test item name: Fatty acid, tall-oil, reaction products with diethylenetriamine, maleic anhydride, tetraethylenepentamine and triethylenetetramine
Batch number : TEE3316/22
Molecular weight: > 700
Declared purity: 97 %
Storage conditions: Room temperature/dark conditions
Stability under storage conditions: Stable

Sampling and analysis

Analytical monitoring:
not specified

Test solutions

Vehicle:
not specified

Test organisms

Test organisms (species):
activated sludge
Details on inoculum:
The study was performed using activated sludge collected from the recirculation tank of ALSI (Alto Lambro Servizi Idrici) wastewater treatment plant treating mainly domestic wastewater. Four litres of activated sludge were kept on June 18, 2010 and transferred to ChemService laboratory where the sample was immediately put under aeration and shaking. The reception temperature of the inoculum was 22.8 °C
The sludge was washed by centrifugation, the supernatant liquid phase decanted and the solid material re-suspended in tap water. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated as 6.57%.

Based on this ratio, calculated amounts of wet sludge were suspended in the test water to obtain a concentration equivalent to 4.0 g dry material per litre. The obtained suspension was held under constant aeration, room temperature and dark conditions for one day prior to the test initiation. Moreover 50 mL per litre of a synthetic feeding prepared immediately before use (for composition and preparation see Appendix B was added to the sludge inoculum.

The pH of sludge inoculum used for the test was 7.19 and its temperature was 21.8 °C.
The temperature during the holding period was measured twice a day using a digital thermometer. It was in the range 21.3 – 21.9 °C .

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h

Test conditions

Test temperature:
range of 21.3 - 21.5 °C
pH:
At the end of the test period, the pH values were in the range of 7.68 – 7.98 (Tables 7 and 8) and the temperature, recorded in each test vessel, was in the range 21.3 – 21.5 °C
Dissolved oxygen:
The concentration of dissolved oxygen did not drop below 2.5 mg/L during the incubation period. Prior to the measurement of the respiratory rate, the oxygen concentration always exceeded 6.5 mg/L.
Nominal and measured concentrations:
The test item was tested at five concentrations in the following geometric series: 10.0, 17.8, 31.6, 56.2 and 100.0 mg/L.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol pestanal analytical standard

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:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate

Any other information on results incl. tables

The test concentrations were not measured during the test, therefore all reported results are related to the nominal concentrations of the test item.

 

The respiratory rate expressed as oxygen consumption was measured after three hours of exposure. The oxygen decrease measurement was carried out one per minute for a period of 10 minutes. The measured oxygen concentrations in the test solutions and in the controls are reported in Table 1.

The reference substance 3,5-dichlorophenol was tested in parallel at three concentrations under the same test conditions to check the response of the sludge activate batch.

The measured oxygen concentrations in the test solutions are reported in Table 2.

When the oxygen concentration dropped below 2.5 mg O2/L, the measurement was stopped according to the mentioned guideline.

Tab. 1 Oxygen consumption by activated sludge in presence of different concentrations of

Fatty acid, tall-oil, reaction products with diethylenetriamine, maleic anhydride, tetraethylenepentamine

Test item concentration

(mg/L)

Oxygen concentration (mg/L)

T0

T1

T2

T3

T4

T5

T6

T7

T8

T9

T10

0 (control)

6.41

5.61

4.70

3.95

3.06

2.58

n.d.

n.d.

n.d.

n.d.

n.d.

10.0

6.48

5.53

4.72

4.02

3.41

2.75

n.d.

n.d.

n.d.

n.d.

n.d.

17.8

6.44

5.60

4.78

4.51

3.26

2.74

n.d.

n.d.

n.d.

n.d.

n.d.

31.6

6.38

5.54

4.85

4.05

3.52

2.65

n.d.

n.d.

n.d.

n.d.

n.d.

56.2

6.52

5.77

4.93

4.30

3.72

2.94

2.56

n.d.

n.d.

n.d.

n.d.

100.0

6.63

5.84

5.09

4.38

3.65

3.01

2.57

n.d.

n.d.

n.d.

n.d.

0 (control)

6.62

5.82

5.11

4.58

3.74

2.96

2.51

n.d.

n.d.

n.d.

n.d.

n. d.: not determined

Tab. 2 Oxygen consumption by activated sludge in presence of different concentrations of 3,5-dichlorophenol

Reference substance concentration

(mg/L)

Oxygen concentration (mg/L)

T0

T1

T2

T3

T4

T5

T6

T7

T8

T9

T10

5.0

6.43

5.77

5.24

4.54

3.82

3.04

2.75

n.d.

n.d.

n.d.

n.d.

15.0

6.52

4.82

4.63

4.41

4.22

4.02

3.82

3.64

3.29

3.13

2.51

50.0

6.55

5.68

5.59

5.48

5.35

5.24

5.11

5.01

4.89

4.77

4.23

n. d.: not determined

The respiratory rates for the test concentrations and for the controls are reported in Table 3 together with the percentage of respiration inhibition in the test concentrations compared to the control.

 

The test meets the validity criteria indicated by the mentioned guidelines, being always³6.5 mg/L just before the measurement of the respiration rates, and the respiration rate of the two controls differs of 10.6%, less than the maximum value of 15% accepted by guideline.

 

The respiratory rate for the reference substance concentrations together with the percentage of respiration inhibition in the test concentrations compared to the control are reported in Table 4.

Tab. 3 Respiratory rates for the test concentrations and for the controls and active sludge respiration inhibition caused by test item after 3-h contact.

Test item concentration

(mg/L)

Rate of

respiration

(mg O2/L/h)

Respiration inhibition

(%)

0 (control)

46.0

----

10.0

44.8

-2.8

17.8

44.4

-2.0

31.6

44.8

-2.8

56.2

43.0

1.3

100.0

43.4

0.2

0 (control)

41.1

----

Tab. 4 Respiratory rates for the reference substance concentrations and active sludge respiration inhibition in comparison to controls after 3-h contact.

Reference substance concentration

(mg/L)

Rate of

respiration

(mg O2/L/h)

Respiration inhibition

(%)

5.0

40.7

6.5

15.0

12.7

70.9

50.0

6.8

84.3

The EC50calculation based on percentage of respiration inhibition was performed using a linear interpolation analysis for both the reference substance and test item. The statistical analysis was carried out by the mean of CETIS elaboration software v1.026D.

The EC50for 3,5-dichlorophenol was found to be 11.76mg/L, thus complying with criteria indicated by the mentioned guidelines.

The NOEC, and the EC50values for Fatty acid, tall-oil, reaction products with diethylenetriamine, maleic anhydride, tetraethylenepentamine were found to be greater than the maximum concentration tested, namely 100 mg/L.

At the end of the test period, the pH values were in the range of 7.68 – 7.98 (Tables 5 and 6) and the temperature, recorded in each test vessel, was in the range 21.3 –as reported in Table 7.

Tab. 5 pH values in test item suspensions and in controls at the end of the test

Test Item Concentrations

(mg/L)

0 (control)

10.0

17.8

31.6

56.2

100.0

0 (control)

pH test end

7.93

7.97

7.98

7.94

7.95

7.97

7.95

Tab. 6 pH values in reference substance solutions at the end of the test

Reference substance concentrations(mg/L)

5.0

15.0

50.0

pH test end

7.89

7.77

7.68

Tab. 7 Temperature values recorded in each test vessel before the reading of oxygen consumption

Test Concentration (mg/L)

Temperature (°C)

0 (control)

21.3

5.0 (3.5-dichlorophenol)

21.5

15.0 (3.5-dichlorophenol)

21.4

50.0 (3.5-dichlorophenol)

21.4

10.0 (test item)

21.5

17.8 (test item)

21.4

31.6 (test item)

21.3

56.26 (test item)

21.4

100.0 (test item)

21.3

0 (control)

21.4

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The EC50 and NOEC were determined to be greater than the nominal tested concentration of product > 100.0 mg/L
Executive summary:

The toxicity of the test item “Fatty acid, tall-oil, reaction products with diethylenetriamine, maleic anhydride, tetraethylenepentamine and triethylenetetramine” to activated sludge was assessed in a respiration inhibition study according to:

- EU Commission Directive 92/69/EEC, C.11 (1992),

- OECD Guideline for Testing of Chemicals, No. 209 (1984).

 

For this purpose, aerobic activated sludge collected from the recirculation tank of a municipal wastewater treatment plant was incubated for 3-hours with different concentrations of the test item under defined conditions.

 

The test item concentrations were 10.0, 17.8, 31.6, 56.2 and 100.0 mg/L. In addition to the five test item concentrations, two untreated controls were incubated under the same conditions.

 

In order to check the normal sensitivity of the batch of activate sludge, 3,5-dichlorophenol was used as a reference item. For this purpose three nominal concentrations of 3,5-dichlorophenol were tested, 5.0, 15.0 and 50.0 mg/L respectively.

 

The EC50of 3,5-dichlorophenol was found to be 11.76 mg/L, thus complying with the validity range indicated by the referenced guidelines (EC50: 5-30 mg/L).

 

The respiratory rate expressed as oxygen consumption was measured after three hours of exposure using a portable oximeter.

 

The inhibitory effect of the test item is expressed as a percent of the mean respiratory rate of two controls. The results are related to the nominal concentrations of the test item.

 

The respiration rate of the two controls was found to differ by 10.6%, thus complying with the validity criteria indicated by the mentioned guidelines (maximum difference between two replicates of control = 15%).

Based on the results, the NOEC (£15% inhibition), the EC50and the LOEC for the test item were assessed to be greater than the highest tested concentration, i.e. greater than 100 mg/L.

At the start of the test the pH, of test water was 7.41.

The pH values and temperature of test solutions were measured in each test flask during the oxygen determination : the pH was in the range 7.68 – 7.98while the temperature was found to be in the range 21.3 – 21.5°C, i.e. within the specified range (20.0±).