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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 September 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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)
Qualifier:
according to
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Volatile: No
Stability in water: Unknown
Solubility in water: Unknown
Specific Gravity: 1.0 at 25°C
Analytical monitoring:
not specified
Details on sampling:
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 (2 replicates) was included. Furthermore, an abiotic control and the highest loading rate with a nitrification inhibitor were tested.
Vehicle:
yes
Details on test solutions:
The batch of Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene tested was a clear red-brown viscous liquid with a purity of >99%. No correction was made for the purity/composition of the test substance.
The test substance was not completely soluble in test medium at the loading rates initially prepared. Therefore, 1-Litre test bottles were filled with 200 ml of test substance 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, 250 ml sludge and Milli-RO water up to 500 ml were added resulting in the required loading rates. Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Test system: Micro-organisms in activated sludge.
Source: Municipal sewage treatment plant: 'Waterschap Aa en Maas', Heeswijk-Dinther, The Netherlands, receiving predominantly domestic sewage.
Number of micro-organisms: Number of micro-organisms was determined as the amount of Mixed Liquor Suspended Solids (MLSS) per litre test medium.
Preparation of the sludge: The sludge was coarsely sieved (1 mm), washed and diluted with ISO-medium. 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 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 type:
flow-through
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
No post exposure period specified within the report.
Hardness:
Not specified within the report.
Test temperature:
The temperatures measured in the various vessels ranged between 18 and 21°C.
pH:
The pH in all test series, before addition of sludge was between 7.3 and 7.5. After the 3 hour exposure period the pH was between 7.0 and 8.2.
Dissolved oxygen:
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.
Salinity:
Not applicable.
Nominal and measured concentrations:
Nominal concentrations.
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-Q water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges (Milli-Q) (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-Q water, made up to 1 litre and filtered. The pH was within 7.5 ± 0.5.
Inhibitor of nitrification: A 2.32 g/l solution of N-allylthiourea (ATU, Merck Schuchardt OHG, Hohenbrunn, Germany) was prepared. 2.5 ml of this solution was added to 500 ml final test medium (final ATU concentration: 11.6 mg/l).
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.
Test set up: 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 (2 replicates) was included. Furthermore, an abiotic control and the highest loading rate with a nitrification inhibitor were tested.
Performance of the test: The synthetic medium (16 ml) and an appropriate amount of the test substance loading were mixed and made up to 250 ml with Milli-RO water in a 1 litre bottle. The pH was determined. Thereafter 250 ml activated sludge was added. This was the start of the test. The mixture was then aerated during the contact time, using a pipette as an aeration device.
After the 3-hour contact time, the oxygen consumption was recorded for 10 - 11 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The pH and temperature were determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference substance concentrations/loading rates and controls.
Oxygen recording: Determination of oxygen was performed with 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
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
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 Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene per liter. Thus, the EC50 was above the highest loading rate tested (1000 mg/l).
There was no oxygen uptake from abiotic processes and the results at 1000 mg/l with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was comparable to the total inhibition.
The temperatures measured in the various vessels ranged between 18 and 21°C and were within the ranges prescribed by the protocol (rounded to integer). The pH in all test series, before addition of sludge was between 7.3 and 7.5. After the 3 hour exposure period the pH was between 7.0 and 8.2.
Results with reference substance (positive control):
Results reported in table form - detailed under Any other information.
Reported statistics and error estimates:
Results reported in table form - detailed under Overall remarks

Study results

Flask

Conc./ loading rate (mg/l)

Temp (°C)

pH before addition of sludge

pH after 3 h contact time

Respiration rate

(mg O2/l h)

Respiration rate

(mg O2/g h)1

% Inhibition respiration relative to the control

(mean value)

C 1

0

20.7

7.5

7.9

37

25

 

C 2

0

19.7

7.5

8.0

28

19

 

Mean

 

 

 

 

33

22

 

SD

 

 

 

 

6

4

 

CV (%)

 

 

 

 

20

20

 

CN

0

18.6

7.5

8.2

19

13

42

R 1

5.0

19.9

7.5

8.1

21

14

35

R 2

12

20.3

7.5

8.1

19

13

42

R 3

30

19.5

7.5

8.2

5

3

85

T1

10

18.6

7.3

7.7

28

19

14

T2

100

19.2

7.3

7.7

31

21

5

T3a

1000

18.9

7.3

7.7

23

15

29

T3b

1000

19.1

7.3

7.7

26

17

20

T3c

1000

17.9

7.3

7.8

24

16

26

T3 mean

 

 

 

 

24

16

25

TA

1000

19.8

7.3

7.0

0

0

100

TN

1000

18.0

7.3

8.1

16

11

162

C:           Blank Control

CN:        Nitrification control

R:           Reference substance, 3,5-dichlorophenol

T:           Test substance, Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene

SD:        Standard deviation

CV:        Coefficient of variation

TA:        Abiotic control of test substance

TN:        Test substance with N-allythiourea

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

2             Relative to CN

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this present test Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 1000 mg/l (NOEC).

The EC50 exceeded a loading rate of 1000 mg/l.
Executive summary:

The influence of Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene 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 on the OECD guideline No. 209, 2010. In addition, the procedures were designed to meet the test methods of theCouncilRegulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C11 and ISO Standard 8192 (2007).

 

The batch of Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene tested was a clear red-brown viscous liquid with a purity of >99%.No correction was made for the purity/composition of the test substance.The test substance was not completely soluble in test medium at the loading rates initially prepared. Therefore, 1-Litre test bottles were filled with 200 ml of test substance mixtures in Milli-RO water 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, synthetic medium, sludge and Milli-RO water were added resulting in the required loading rates. Optimal contact between the test substance and test medium was ensured applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was recorded for 10 - 11 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 concentrations consisted of one replicate. In addition, blank-controls and a nitrification control of the sludge were included. Furthermore, an abiotic control and the highest concentration with a nitrification inhibitor were tested.

 

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 Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene per liter (Two Sample t-Test : α=0.05). Thus, the EC50was above the highest loading rate tested (1000 mg/l).

 

There was no oxygen uptake from abiotic processes and the results at 1000 mg/l with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was comparable to the total inhibition.

The batch of activated sludge was checked for sensitivity by testing the reference substance 3,5-dichlorophenol, which showed normal sensitivity.

 

The study met the acceptability criteria prescribed by the protocol and was considered valid.

 

In conclusion, Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 1000 mg/l (NOEC).

 

The EC50exceeded a loading rate of 1000 mg/l.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
Not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted to ISO test standard but not in compliance with GLP, however data included for OECD SIDS Dossier. Although this study was probably not conducted to GLP, the test parameters used were based on a known and well established procedure. Read across to supporting substance, CAS No. 68442-68-2, by structural analogue.
Justification for type of information:
Please refer to IUCLID Section 13 for the read-across justification.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Deviations:
not specified
GLP compliance:
no
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Melting Point: Value: ca. 6 degree C; Remark: Solidifying point
Boiling Point: Value: > 300 degree C at 1013 hPa
Density: Type: density; Value: ca. 1.1 g/cm3 at 20 degree C; Remark: Specific Gravity is 1.08-1.10
Vapour Pressure: Value: < 100 hPa at 50 degree C
Partition Coefficient: log Pow: 4.64 at 22 degree C
Water Solubility: Value: .41 mg/l at 20 degree C; Qualitative: of very low solubility
Flash Point: Value: > 100 degree C
Analytical monitoring:
no
Details on sampling:
Not specified
Vehicle:
not specified
Details on test solutions:
Not specified
Test organisms (species):
activated sludge
Details on inoculum:
Not specified
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
Not specified
Hardness:
Not specified
Test temperature:
Not specified
pH:
Not specified
Dissolved oxygen:
Not specified
Salinity:
Not applicable.
Nominal and measured concentrations:
Not specified
Details on test conditions:
Not specified
Reference substance (positive control):
not specified
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 10 000 mg/L
Nominal / measured:
not specified
Conc. based on:
not specified
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
Not specified
Results with reference substance (positive control):
Not specified
Reported statistics and error estimates:
Not specified
Validity criteria fulfilled:
yes
Conclusions:
3h EC50 > 10000 mg/l
Executive summary:

Study conducted to ISO testing standard but not in compliance with GLP, however data is included for OECD SIDS dossier. 3h E50 > 10000 mg/l. Read across to supporting substance, CAS No. 68442 -68 -2, by structural analogue.

Although this study was not conducted to GLP, the test parameters used were based on a known and well established procedure.

Description of key information

Key value determined using OECD no. 209, EU Method C11 and ISO standard no. 8192. 

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

The test substance was not completely soluble in test medium at the loading rates initially prepared. Therefore, 1-Litre test bottles were filled with 200 ml of test substance mixtures in Milli-RO water 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, synthetic medium, sludge and Milli-RO water were added resulting in the required loading rates. Optimal contact between the test substance and test medium was ensured applying continuous aeration and stirring during the 3-hour exposure period.

 

Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 1000 mg/l (NOEC).

 

The EC50exceeded a loading rate of 1000 mg/l.