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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
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 (Carbon and Ammonium Oxidation))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
not required
Vehicle:
no
Details on test solutions:
Test system: Micro-organisms in activated sludge.

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 (3.0 g/L of sludge, as used for the test). The pH was 7.6 on the day of testing. The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium (= sewage feed) 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
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's- Hertogenbosch, The Netherlands, receiving predominantly domestic sewage. 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 (3.0 g/L of sludge, as used for the test). The pH was 7.6 on the day of testing. The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium (= sewage feed) was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
20°C - 21°C (min-max)
pH:
7.0 - 8.2 (min - max)
Nominal and measured concentrations:
0 (6 replicates) ; 10 (1 replicate) ; 100 (1 replicate) ; 1000 mg/L (3 replicates) nominal
Details on test conditions:
Contact time: 3 hours, during which aeration and stirring took place.

Vessels: All glass open bottles/vessels.

Milli-RO water: Tap water purified by reverse osmosis (Millipore Corp., Bedford, Mass., USA).

Synthetic medium: 16 g peptone
(=sewage feed): 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.

Performance of the test
The synthetic medium (16 mL) made up to 50 mL with Milli- RO and 200 mL test item solution were mixed (total volume 250 mL). The pH was determined. Thereafter, 250 mL activated sludge was added. This was the start of the test. 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. The pH was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference item concentrations and controls. The medium temperature was recorded continuously in two temperature control vessels. The temperature control vessels were identically prepared compared to the control vessels. A temperature control vessel with a REES sensor was placed in each fume cupboard of the climate room. Determination of oxygen was performed with multiple oxygen sensors 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:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
93% purity
Basis for effect:
inhibition of total respiration
Details on results:
The combined limit/range-finding test showed no inhibition at the two lowest loading rates and an average of 10% inhibition of the respiration rate at 1000 mg/L. Therefore, the ELR50 was above the highest loading rate tested (1000 mg/L). In the combined limit/range-finding test no statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg N- NITROSODIPHENYLAMINE per litre. Therefore, the highest loading rate tested was considered to be the NOELR (1000 mg/L). There was no oxygen uptake from abiotic processes.
Results with reference substance (positive control):
The EC50 of 3,5-dichlorophenol was 10 mg/L, which is in the accepted range of 2 to 25 mg/L for total respiration.
Reported statistics and error estimates:
Results were analysed with Toxrat v3.2.1

  
Results: Respiration Rate/Inhibition, pH Values

Replicate

Loading rate (T)

Concentration (R)

(mg/L)

pH

Respiration rate

% Inhibition

respiration rate

(mean value)

Start

End

(mg O2/L.h)

(mg O2/g.h)¹

C 1

0

7.2

8.1

52.31

34.87

 

C 2

0

7.2

8.0

56.55

37.70

 

C 3

0

7.2

8.0

46.07

30.71

 

C 4

0

7.2

8.0

54.09

36.06

 

C 5

0

7.1

7.9

40.98

27.32

 

C 6

0

7.1

7.9

51.49

34.33

 

C Mean

 

 

 

50.25

33.50 (RC)

 

SD

 

 

 

5.72

3.81

 

CV (%)

 

 

 

11

11

 

 

 

 

 

 

 

R 1

1.0

7.2

8.2

48.40

32.27

3.68

R 2

3.2

7.2

8.2

38.22

25.48

23.94

R 3

10

7.2

8.1

25.82

17.21

48.62

R 4

32

7.2

8.2

11.40

7.60

77.31

 

 

 

 

 

 

 

T 1

10

7.1

8.0

55.09

36.73

-9.64

T 2

100

7.2

8.0

52.63

35.09

-4.74

T 3a

1000

7.1

7.9

40.27

26.85

19.86

T 3b

1000

7.1

8.0

42.20

28.13

16.02

T 3c

1000

7.2

7.9

53.64

35.76

-6.75

T3 Mean

 

 

45.37

30.25 (RT)

9.71 (IT)

 

 

 

 

 

 

 

TA

1000

7.1

7.0

0.00#

0.00#

100.00

C:          Control

R:          Reference item, 3,5-dichlorophenol      

T:          Test item, N-NITROSODIPHENYLAMINE (1: 10 mg/L ; 2: 100 mg/L ; 3: 1000 mg/L)     

TA:       Abiotic control of N-NITROSODIPHENYLAMINE        

SD:       Standard deviation 

CV:       Coefficient of variation                         

RT:         Total respiration with N-NITROSODIPHENYLAMINE

RC:         Total respiration in the control

IT:           % inhibition of total respiration relative to RC

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

#             No respiration, therefore expressed as 0 mg O2/L.h (see paragraph 6.1)

Validity criteria fulfilled:
yes
Conclusions:
There were no inhibitory effect of n-nitrosodiphenylamine (CAS 86-30-6) on total respiration of activated sludge.
Executive summary:

The objective of the study was to evaluate n-nitrosodiphenylamine for its ability to adversely affect aerobic microbial treatment plants and, if possible, to determine the ELR50and/or the no-observed effect loading rate (NOELR). The influence of n-nitrosodiphenylamine on the respiration rate of activated sludge was investigated after a contact time of 3 hours. The study procedures described in this report were based onthe OECD guideline No. 209, 2010. The batch of n-nitrosodiphenylamine tested consisted of brown granules with a purity of 93.0% (HPLC). No correction was made for the purity/composition of the test item.

The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, weighed amounts were added to the test bottles containing Milli-RO water. The test item – Milli-RO water mixtures were magnetically stirred for a short period and 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 by applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.

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. Furthermore, at 1000 mg/L an abiotic control (1 replicate) was tested. Responses were compared to the controls (6 replicates).

Nostatistically significant inhibition of the respiration rate of the sludge was recordedat a loading rate of 1000 mg n-nitrosodiphenylamine per litre.Therefore, the ELR50was above the highest concentration tested (1000 mg/L).

There was no oxygen uptake from abiotic processes. The batch of activated sludge was tested for sensitivity with the reference item 3,5- dichlorophenol, and showed normal sensitivity. The study met the acceptability criteria prescribed by the study plan and was considered valid. Under the conditions of this present test, n-nitrosodiphenylamine was not toxic to waste water (activated sludge) bacteria at a loading rate of 1000 mg/L (NOELR).

Description of key information

The objective of the study was to evaluate n-nitrosodiphenylamine for its ability to adversely affect aerobic microbial treatment plants and, if possible, to determine the ELR50and/or the no-observed effect loading rate (NOELR). The influence of n-nitrosodiphenylamine on the respiration rate of activated sludge was investigated after a contact time of 3 hours. The study procedures described in this report were based onthe OECD guideline No. 209, 2010. The batch of n-nitrosodiphenylamine tested consisted of brown granules with a purity of 93.0% (HPLC).No correction was made for the purity/composition of the test item.

The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, weighed amounts were added to the test bottles containing Milli-RO water. The test item – Milli-RO water mixtures were magnetically stirred for a short period and 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 by applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.

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. Furthermore, at 1000 mg/L an abiotic control (1 replicate) was tested.Responses were compared to the controls (6 replicates).

Nostatistically significant inhibition of the respiration rate of the sludge was recordedat a loading rate of 1000 mg n-nitrosodiphenylamine per litre.Therefore, the ELR50was above the highest concentration tested (1000 mg/L).

There was no oxygen uptake from abiotic processes. The batch of activated sludge was tested for sensitivity with the reference item 3,5- dichlorophenol, and showed normal sensitivity. The study met the acceptability criteria prescribed by the study plan and was considered valid. Under the conditions of this present test, n-nitrosodiphenylamine was not toxic to waste water (activated sludge) bacteria at a loading rate of 1000 mg/L (NOELR).

Key value for chemical safety assessment

Additional information