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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 June 2016 to 29 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Study was conducted in accordance with OECD, EU and ISO test guidlines in an accredited GLP laboratory.
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Test item: 207368/A
Identification: Hepteen Base®
Appearance: Clear amber liquid (determined by Charles River Den Bosch)
Batch: LT5C30Y170
Purity/Composition: 99.7%
Test item storage: At room temperature
Stable under storage conditions until: 30 November 2016 (retest date)
Purity/composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Specific gravity/density: 0.9130
Solubility in water: Insoluble

Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Reference item – preparation of stock and test concentrations
The batch of activated sludge was checked for sensitivity by testing the reference item 3,5-dichlorophenol.
A 3,5-dichlorophenol solution with a final concentration of 1 g/L in Milli-RO water was prepared. The pH as used for the test was 7.9 in the combined limit/range-finding test and 8.0 in the final test. The 3,5-dichlorophenol stock solution was stored in a freezer until use. The reference item solution was defrosted at room temperature and diluted to reach the test concentrations. Four concentrations were tested: 2.0, 4.0, 8.0 and 16 mg/L.

Test item – preparation of test solutions
The batch of Hepteen Base® tested was a clear amber liquid with a purity of 99.7%. No correction was made for the purity/composition of the test item. The test item was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water. Therefore, 1 litre test bottles were filled with 200 mL of test item mixtures in Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for approximately 24 hours. Subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added resulting in the required loading rates. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Source: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
Preparation of the sludge: The sludge was coarsely sieved (1 mm) and allowed to settle.
The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3 g/L of sludge, as used for the test). The pH was 7.4 on the day of testing. The batch of sludge was used one day after collection; therefore, 50 mL of synthetic medium was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
Medium: Adjusted ISO-medium, formulated using RO water (tap water purified by reverse osmosis; GEON Waterbehandeling, Berkel- Enschot, The Netherlands) with the following composition:
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L
Rationale: Recognized by international guidelines as the recommended test system.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Remarks on exposure duration:
After the 3-hour contact time, the oxygen consumption was recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.
Post exposure observation period:
Not specified
Hardness:
Not specified
Test temperature:
20 - 22°C
pH:
7.4 - 7.7
Dissolved oxygen:
During the experimental period, the dissolved oxygen concentrations in all test vessels were > 60% of the saturated dissolved oxygen concentration.
Salinity:
Not specified
Conductivity:
Not specified
Nominal and measured concentrations:
Combined limit/range-finding test
In a combined limit/range-finding test loading rates of 10, 100 and 1000 mg/L were tested. The highest loading rate was tested in triplicate, lower loading rates consisted of one replicate. In addition, a blank control (6 replicates) and a nitrification control (2 replicates) were included. Furthermore, an abiotic control (1 replicate) and the highest loading rate with a nitrification inhibitor (3 replicates) were tested.

Final test Loading rates
Hepteen Base®: Five loading rates: 46, 100, 220, 460 and 1000 mg/L.
Replicates: 5 replicates per test group and 6 replicates for the control.
Blank controls: Test medium without test item, treated in the same way as the test item solutions.
Details on test conditions:
Test procedure and conditions
Contact time: 3 hours, during which aeration and stirring took place.
Vessels: All glass open bottles/vessels.
Milli-RO / Milli-RO water: Tap water purified by reverse osmosis (Millipore Corp., Bedford, Mass., USA).
Synthetic medium (=sewage feed): 16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
Dissolved in Milli-RO water, made up to 1 litre and filtered.
The pH was within 7.5 ± 0.5.
Air supply: Clean, oil-free air.
Aeration: The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.

The final test was a standard test that did not include a nitrification inhibitor or abiotic conditions.

Oxygen recording
Determination of oxygen was performed with multiple oxygen probes connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system.


Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
other: NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
other: ELR10
Effect conc.:
143 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
other: ELR20
Effect conc.:
257 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
other: ERL50
Effect conc.:
787 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
Combined limit/range-finding test
The combined limit/range-finding test showed no inhibition of the respiration rate at a loading rate of 10 mg/L and 5 and 66% inhibition of the respiration rate at a loading rate of 100 and 1000 mg/L, respectively. Therefore, the expected ELR50 was between a loading rate of 100 and 1000 mg/L.
There was no oxygen uptake from abiotic processes and the result at 1000 mg/L with a nitrification inhibitor showed that there was heterotrophic inhibition of the respiration rate. Based on the results of the total and heterotrophic respiration, nitrification inhibition was calculated to be 96%. There were no spurious results (i.e. bi-phasic concentration related respiration curves, distorted dose-responses or unexpectedly low EC50 values) observed in the combined limit/range-finding. Therefore, N-allylthiourea was not used in the final test.
All test conditions and acceptability criteria prescribed by the study plan were met.

Final test
Inhibition of the respiration rate
No statistically significant inhibition of the respiration rate of the sludge was recorded at or below a loading rate of 100 mg Hepteen Base® per litre. At higher loading rates the inhibitory effect of Hepteen Base® on aerobic waste water (activated sludge) bacteria increased with increasing loading rate, ranging from 22% inhibition at 220 mg/L to 57% at 1000 mg/L.

Experimental conditions
The pH in all test vessels, before addition of sludge was between 7.4 and 7.7. After the 3 hour exposure period the pH was between 7.3 and 8.1.
The temperature continuously measured in the temperature control vessels ranged between 20 and 22°C during the test, and complied with the requirements as laid down in the study plan (20 ± 2°C).
Results with reference substance (positive control):
The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L for total respiration (4.0 mg/L in the final test)
Reported statistics and error estimates:
Not specified

Final test – Overview of the results

 

 

Treatment

 

Loadingrate (mg/L)

pH

Mean respiration rate

% Inhibition of the respiration rate (mean value)

 

Start

 

End

 

(mg O2/L h)

 

(mg O2/g h)¹

Blank control

 

7.6

7.5

37.77

25.18

 

T1

46

7.6

7.5

41.70

27.80

-10.39

T2

100

7.7

7.5

38.40

25.60

-1.65

T3

220

7.7

7.5

29.60

19.73 *

21.64

T4

460

7.6

7.6

25.63

17.09 *

32.14

T5

1000

7.5

7.6

16.09

10.72 *

57.41

¹) The amount of suspended solids in the final test mixture was 1.5 g/L.

* Statistically significantly different compared to control

 

Effect parameters

Parameter

Hepteen Base® Loading rate (mg/L)

NOELR

100

ELR10

143 (96 – 187 mg/L)

ELR20

257 (198 – 310 mg/L)

ELR50

787 (672 – 961 mg/L)

(  ) Between brackets the 95% confidence intervals are given.

Combined limit/range-finding test – Respiration rate/inhibition, pH values

 

Replicate

 

Loading rate (mg/L)

pH

Respiration rate

% Inhibition respiration rate

(mean value)

 

Start

 

End

 

(mg O2/L.h)

 

(mg O2/g.h)¹

C 1

0

7.6

8.0

42.94

28.63

 

C 2

0

7.6

7.6

46.32

30.88

 

C 3

0

7.6

7.6

46.85

31.23

 

C 4

0

7.6

7.6

44.47

29.65

 

C 5

0

7.6

7.6

39.41

26.27

 

C 6

0

7.6

7.7

44.41

29.61

 

C Mean

 

 

 

44.07 (RTB)

29.38

 

SD

 

 

 

2.69

1.79

 

CV (%)

 

 

 

6

6

 

 

 

 

 

 

 

 

CN 1

0

7.6

8.4

22.20

14.80

 

CN 2

0

7.6

8.2

30.06

20.04

 

CN Mean

 

 

 

26.13 (RHB)

17.42

 

 

 

 

 

 

 

 

R 1

2.0

7.6

8.3

27.63

18.42

37.30

R 2

4.0

7.6

8.3

26.03

17.35

40.93

R 3

8.0

7.6

8.2

18.94

12.63

57.02

R 4

16

7.6

8.2

11.28

7.52

74.40

 

 

 

 

 

 

 

T 1

10

7.6

7.5

60.07

40.05

-36.32

T 2

100

7.6

7.5

41.78

27.85

5.19

T 3a

1000

7.6

7.7

12.68

8.45

71.23

T 3b

1000

7.6

7.7

17.44

11.63

60.42

T 3c

1000

7.6

7.6

15.19

10.13

65.53

T3 Mean

 

 

 

15.10 (RT)

10.07 *

65.73 (IT)

 

 

 

 

 

 

 

TN a

1000

7.6

7.7

15.43

10.29

40.95

TN b

1000

7.6

7.8

16.03

10.69

38.65

TN c

1000

7.6

7.8

11.52

7.68

55.91

TN Mean

 

 

 

14.33 (RH)

9.55

45.17 (IH)

 

 

 

 

 

 

 

TA

1000

7.6

7.4

0#

0#

100

 

C:

Blank control

¹

The amount of suspended solids in the final test mixture was

 

 

 

1.5 g/L.

CN:

Nitrification control

RTB:

Total respiration blank

R:

Reference item, 3,5-dichlorophenol

RHB:

Heterotrophic respiration in the nitrification control

T:

Test item, Hepteen Base®

RT:

Total respiration with Hepteen Base®

TA:

Abiotic control of Hepteen Base®

RH:

Heterotrophic respiration with Hepteen Base®

TN:

Hepteen Base® with N-allylthiourea

IT:

% inhibition of total respiration relative to RTB

SD:

Standard deviation

IH:

% inhibition of heterotrophic respiration relative to RHB

CV:

Coefficient of variation

*

Statistically significantly different compared to control

#

No respiration, therefore expressed as 0 mgO2/L.h

 

 

 

Final test – Respiration rate/inhibition, pH values

 

Replicate

 

Loading rate (mg/L)

pH

Respiration rate

% Inhibition respiration rate

(mean value)

 

Start

 

End

 

(mg O2/L.h)

 

(mg O2/g.h)¹

C 1

0

7.7

7.7

35.65

23.77

 

C 2

0

7.6

7.5

39.41

26.27

 

C 3

0

7.6

7.5

32.11

21.41

 

C 4

0

7.6

7.4

40.06

26.71

 

C 5

0

7.6

7.5

35.49

23.66

 

C 6

0

7.6

7.3

43.92

29.28

 

C Mean

 

 

 

37.77 (RTB)

25.18

 

SD

 

 

 

4.18

2.79

 

CV (%)

 

 

 

11

11

 

 

 

 

 

 

 

 

R 1

2.0

7.7

8.1

22.59

15.06

40.20

R 2

4.0

7.7

8.0

17.67

11.78

53.22

R 3

8.0

7.7

8.0

17.75

11.83

53.01

R 4

16

7.7

8.0

10.28

6.85

72.79

 

 

 

 

 

 

 

T 1a

46

7.7

7.5

41.82

27.88

-10.71

T 1b

46

7.6

7.5

37.79

25.19

-0.04

T 1c

46

7.6

7.4

45.36

30.24

-20.08

T 1d

46

7.6

7.5

42.10

28.07

-11.45

T 1e

46

7.6

7.6

41.42

27.61

-9.65

T 1 Mean

 

 

 

41.70 (RT)

27.80

-10.39 (IT)

 

 

 

 

 

 

 

T 2a

100

7.7

7.6

35.15

23.43

6.94

T 2b

100

7.7

7.4

37.80

25.20

-0.07

T 2c

100

7.7

7.3

45.44

30.29

-20.30

T 2d

100

7.7

7.5

35.64

23.76

5.65

T 2e

100

7.7

7.5

37.95

25.30

-0.47

T 2 Mean

 

 

 

38.40 (RT)

25.60

-1.65 (IT)

¹:            The amount of suspended solids in the final test mixture was 1.5g/L.

C:  Blank control

SD:        Standard deviation CV: Coefficient of variation

R:           Reference item,3,5-dichlorophenol T: Test item, HepteenBase®

¹:            The amount of suspended solids in the final test mixture was1.5g/L

RTB:  Total respiration blank

RT:          Total respiration with HepteenBase®

IT:           % inhibition of total respiration relative to RTB

 

Final test – Respiration rate/inhibition, pH values

 

Replicate

 

Loading rate (mg/L)

pH

Respiration rate

% Inhibition respiration rate

(mean value)

 

Start

 

End

 

(mg O2/L.h)

 

(mg O2/g.h)¹

T 3a

220

7.7

7.5

32.43

21.62

14.15

T 3b

220

7.7

7.6

23.68

15.79

37.31

T 3c

220

7.7

7.6

29.23

19.49

22.62

T 3d

220

7.7

7.5

28.91

19.27

23.46

T 3e

220

7.6

7.5

33.75

22.50

10.65

T 3 Mean

 

 

 

29.60 (RT) *

19.73

21.64 (IT)

 

 

 

 

 

 

 

T 4a

460

7.7

7.6

25.26

16.84

33.13

T 4b

460

7.6

7.5

23.67

15.78

37.34

T 4c

460

7.7

7.6

26.70

17.80

29.32

T 4d

460

7.6

7.6

20.45

13.63

45.86

T 4e

460

7.6

7.5

32.08

21.39

15.07

T 4 Mean

 

 

 

25.63 (RT) *

17.09

32.14 (IT)

 

 

 

 

 

 

 

T 5a

1000

7.7

7.6

15.20

10.13

59.76

T 5b

1000

7.6

7.5

16.31

10.87

56.82

T 5c

1000

7.5

7.6

15.47

10.31

59.05

T 5d

1000

7.4

7.6

16.77

11.18

55.60

T 5e

1000

7.4

7.5

16.68

11.12

55.84

T 5 Mean

 

 

 

16.09 (RT) *

10.72

57.41 (IT)

T:    Test item, HepteenBase®

¹:      The amount of suspended solids in the final test mixture was1.5g/LRT:  Total respiration with HepteenBase®

IT:    % inhibition of total respiration relative toRTB

*:     Statistically significantly different compared tocontrol

 

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this present test Hepteen Base® was not toxic to waste water bacteria (activated sludge) at or below a loading rate of 100 mg/L (NOELR).
The ELR10 was at a loading rate of 143 mg/L (95% confidence interval: 96 – 187 mg/L).
The ELR20 was at a loading rate of 257 mg/L (95% confidence interval: 198 - 310 mg/L).
The ELR50 was at a loading rate of 787 mg/L (95% confidence interval: 672 – 961 mg/L).
Executive summary:

The influence of Hepteen Base® on the respiration rate of activated sludge was investigated after a contact time of 3 hours.

The study procedures were based on the OECD guideline No. 209, 2010. In addition, the procedures were designed to meet the test methods of the Council Regulation (EC) No. 440/2008 of 30 May 2008, Amended 7 December 2015, Publication No. L142, Part C11 and ISO Standard 8192 (2007).

The batch of Hepteen Base® tested was a clear amber liquid with a purity of 99.7%. The test item was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water. Therefore, the test item – Milli-RO water mixtures were magnetically stirred for a period of approximately 24 hours. Subsequently, synthetic medium, sludge and Milli-RO water were added resulting in the required loading rates. Optimal contact between the test item and test medium was ensured applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.

The final test was performed based on the result of a preceding combined limit/range-finding test. Loading rates, ranging from 46 to 1000 mg/L, increasing with a factor 3.2 were tested. Five replicates per loading rate and six replicates for an untreated control group were tested.

No statistically significant inhibition of the respiration rate of the sludge was recorded at or below a loading rate of 100 mg Hepteen Base® per litre. At higher loading rates the inhibitory effect of Hepteen Base® on aerobic waste water (activated sludge) bacteria increased with increasing loading rate, ranging from 22% inhibition at 220 mg/L to 57% at 1000 mg/L.

 

Parameter

Hepteen Base® Loading rate (mg/L)

NOELR

100

ELR10

143 (96 – 187 mg/L)

ELR20

257 (198 – 310 mg/L)

ELR50

787 (672 – 961 mg/L)

(  ) Between brackets the 95% confidence intervals are provided.

Description of key information

Activated Sludge Respiration Inhibition Test (Carbon and Ammonium Oxidation)

Under the conditions of this present test the test material was not toxic to waste water bacteria (activated sludge) at or below a loading rate of 100 mg/L (NOELR).

The ELR10 was at a loading rate of 143 mg/L (95% confidence interval: 96 – 187 mg/L).

The ELR20 was at a loading rate of 257 mg/L (95% confidence interval: 198 - 310 mg/L).

The ELR50 was at a loading rate of 787 mg/L (95% confidence interval: 672 – 961 mg/L).

 

Key value for chemical safety assessment

EC50 for microorganisms:
787 mg/L
EC10 or NOEC for microorganisms:
100 mg/L

Additional information

Activated Sludge Respiration Inhibition Test (Carbon and Ammonium Oxidation)

Study was conducted in accordance with the following guidelines in an accredited GLP laboratory:

OECD guidelines for Testing of Chemicals, Section 2: Effects on biotic systems, Guideline no. 209, "Activated Sludge, Respiration Inhibition Test " (Carbon and Ammonium Oxidation), adopted 22 July 2010.

Council regulation (EC) No 440/2008 of 30 May 2008, Part C: Methods for the determination of ecotoxicity, Publication No. L142, C 11:"Biodegradation: Activated sludge respiration inhibition test", Amended by EC No. 2016/266 of 7 December 2015, Publication No.L54.

ISO Standard 8192, Water Quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation(2007).