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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 May 2015
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))
Version / remarks:
2010
Deviations:
yes
Remarks:
The temperature was outside the range as prescribed by the protocol. The validity criteria were met and this deviation is considered not to have influenced the study integrity.
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test substance 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 substance mixtures in Milli-RO water (tap-water purified by reverse osmosis) 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 substance and test organisms was ensured applying continuous aeration and stirring.
- Controls: Control without test item or reference item; Reference item control; Nitrification control, Abiotic control
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's -Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: 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 7.4 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:
no
Total exposure duration:
3 h
Test temperature:
21.9 - 23.5°C
pH:
At start (before addition of sludge): 7.4 - 7.5
After 3 h: 7.3 - 8.2
Nominal and measured concentrations:
Nominal loading rates: 10, 100 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: all-glass open bottles, 500 mL fill volume
- Aeration: continuous aeration with clean, oil-free air. 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.
- No. of vessels per concentration (replicates): 3 for highest test loading rate, 1 for lower loading rates
- No. of vessels per control (replicates): 6
- No. of vessels per reference control (replicates): 1 replicate per concentration (3 concentrations used)
- No. of vessels per abiotic control (replicates): 1 (at highest loading rate)
- No. of vessels for nitrification control (replicates): 3 (at highest loading rate)
- Sludge concentration (weight of dry solids per volume): 1.5 g/L suspended solids in the final test mixture.
- Nitrification inhibitor used: ATU (N-allylthiourea), final concentration in vessels: 11.6 mg/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (tap water purified by reverse osmosis)
- Synthetic medium: according to OECD 209.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: not specified.

EFFECT PARAMETERS MEASURED
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 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.
Oxygen recording: determination of oxygen was performed with multiple oxygen sensors connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system.


TEST CONCENTRATIONS
- The test was performed as a Combined Limit/Range Finding test.
- Spacing factor for test concentrations: 10
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
other: NOEL
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
other: EL50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
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/L. Therefore, was the EL50 above the highest loading rate tested (1000 mg/L).
- There was no oxygen uptake from abiotic processes and the result at 1000 mg/L with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was not inhibited. Based on the results of the total and heterotrophic inhibition, nitrification was calculated to be 63% inhibited. Since the heterotrophic respiration was stimulated and nitrification is calculated using the total and heterotrophic respiration rate the inhibition of nitrification could be overestimated. Since no effect for total respiration was observed the effect on nitrification was not required according to the guidelines and was therefore not further investigated.

Results with reference substance (positive control):
EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L for total respiration (i.e. 3.3 mg/L)
Reported statistics and error estimates:
- ECx values Reference item: probit analysis
- NOEL determination: Student-t test for Homogeneous Variances, α=0.05, one-sided, smaller
Calculations were performed with ToxRat Professional v. 2.10.05 (ToxRat Solutions® GmbH, Germany).

Table 1: Results

Replicate

Loading rate (mg/l)

Start

pH

 

End

Respiration rate

(mg O2/l.h)

 

(mg O2/g.h)¹

% Inhibition respiration rate

(mean value)

C 1

0

7.5

 

 

7.7

40

27

 

C 2

0

7.5

 

 

7.4

47

31

 

C 3

0

7.5

 

 

7.4

52

35

 

C 4

0

7.5

 

 

7.4

50

33

 

C 5

0

7.5

 

 

7.3

60

40

 

C 6

0

7.5

 

 

7.4

45

30

 

C Mean

 

 

 

 

 

49 (=RTB)

33

 

SD

 

 

 

 

 

7

5

 

CV (%)

 

 

 

 

 

14

14

 

 

 

 

 

 

 

 

 

 

CN 1

0

7.5

 

 

8.2

21

14

57

CN 2

0

7.5

 

 

8.0

26

17

47

CN Mean

 

 

 

 

 

24 (=RHB)

16

 

 

 

 

 

 

 

 

 

 

R 1

2.0

7.5

 

 

8.0

35

23

29

R 2

5.0

7.5

 

 

8.0

13

9

73

R 3

12

7.5

 

 

8.0

11

7

78

 

 

 

 

 

 

 

 

 

T 1

10

7.4

 

 

7.4

62

41

-27

T 2

100

7.4

 

 

7.4

62

41

-27

T 3a

1000

7.4

 

 

7.8

51

34

-4

T 3b

1000

7.4

 

 

7.8

41

27

16

T 3c

1000

7.4

 

 

7.9

33

22

33

T3 Mean

 

 

 

 

 

42 (=RT)

28

15 (=IT)

 

 

 

 

 

 

 

 

 

TN a

1000

7.5

 

 

8.1

31

21

-32

TN b

1000

7.5

 

 

8.1

31

21

-32

TN c

1000

7.5

 

 

8.1

35

23

-49

TN Mean 

 

 

 

 

 

32 (=RH)

22

-38 (=IH)

 

 

 

 

 

 

 

 

 

TA

1000

7.4

 

 

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 substance, 3,5-dichlorophenol      RHB: Heterotrophic respiration in the nitrification control

T:     Test substance, URALAC P 1920C               RT:   Total respiration with URALAC P 1920C

TA: Abiotic control of URALAC P 1920C               RH:   Heterotrophic respiration with URALAC P 1920C

TN: URALAC P 1920C 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                                             

#      No respiration, therefore expressed as 0 mg            

O2/l.h

                                          

 

Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks' section for details on validity criteria.
Conclusions:
The test substance was not toxic to waste water (activated sludge) bacteria at a loading rate of 1000 mg/L (NOEL). The EL50 was above 1000 mg/L.
Executive summary:

In a 3 -h activated sludge respiration inhibition study conducted according to OECD Guideline 209 and according to GLP Principles, microorganisms in activated sludge were exposed to the test item at nominal loading rates of 10, 100 and 1000 mg/L. There was no statistically significant inhibition of the respiration rate of the sludge, and no oxygen uptake from abiotic processes. The test item was not toxic at a loading rate of 1000 mg/L (NOEL), and the EL50 was >1000 mg/L. The study met all validity criteria and is considered reliable without restriction.

Description of key information

The test item was not toxic to microorganisms from activated sludge at a loading rate of 1000 mg/L (NOEL).

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

In a 3 -h activated sludge respiration inhibition study conducted according to OECD Guideline 209 and according to GLP Principles, microorganisms in activated sludge were exposed to the test item at nominal loading rates of 10, 100 and 1000 mg/L. There was no statistically significant inhibition of the respiration rate of the sludge, and no oxygen uptake from abiotic processes. The test item was not toxic at a loading rate of 1000 mg/L (NOEL), and the EL50 was >1000 mg/L. The study met all validity criteria and is considered reliable without restriction.

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