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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Nov 2017 - 10 Nov 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
Version 2010
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The batch of 3-Methyl Sulfolane tested was a clear colourless to pale yellow liquid. No correction was made for the purity or composition of the test item. A stock solution of 10 g/L was prepared by adding 2.4998 g test item to 250 mL of Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). Magnetic stirring for 15 minutes was applied to accelerate dissolution and to ensure homogeneity. Volumes of the clear and colourless stock solution (pH 5.8) corresponding to the test concentration were then added to the test media (16 mL synthetic medium and made up to 250 mL with Milli-RO). Thereafter, 250 mL of activated sludge was added resulting in the required concentrations. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring. Any residual volumes were discarded.
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. The pH was 7.6 on the day of testing.
- Pretreatment: 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.
- Initial biomass concentration: A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids, i.e. 3.0 g/L of sludge. The amount of suspended solids in the final test mixture was 1.5 g/L.
Test type:
static
Water media type:
other: Tap water purified by reverse osmosis (Millipore Corp., Bedford, Mass., USA).
Limit test:
yes
Total exposure duration:
3 h
Remarks on exposure duration:
Optimal contact between the test item and test medium was ensured by applying continuous aeration and stirring during the 3-hour exposure period.
Test temperature:
20 - 22°C
pH:
T 0hr control: 7.3
T 3hr control: 7.0 - 7.6

T 0hr test item: 7.4
T 3hr test item: 7.2 - 7.5

T 0hr 3,5 DCP: 7.3 - 7.4
T 3hr 3,5 DCP: 7.9 - 8.0
Dissolved oxygen:
>60-70% of air saturation at test start (60% of air saturation is > 5 mg/L at 20°C)
Nominal and measured concentrations:
Nominal: 10, 100 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: all glass open bottles/vessels
- Total fill volume: 500 mL (250 mL test solution (incl. synthetic medium) + 250 mL activated sludge in test medium)
- Aeration: yes (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): 1 replicate for the lower concentrations and 3 for the highest concentration
- No. of vessels per control (replicates): 6
- No. of vessels per positive control (replicates): 4
- No. of vessels per abiotic control (replicates): 1
- Sludge concentration (weight of dry solids per volume): 1.5 g/L sludge
- Nutrients provided for bacteria: synthetic medium acc. to OECD 209
- Nitrification inhibitor used : no

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 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

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Details on termination of incubation: 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.
- 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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : oxygen uptake

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: loading rates of 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: yes; The combined limit/range-finding test showed no inhibition at 10 and 100 mg/L, and an average of 3% inhibition of the respiration rate at 1000 mg/L. Therefore, the EC50 was above the highest concentration tested (1000 mg/L).
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
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:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
Inhibition of the Respiration Rate:
The combined limit/range-finding test showed no inhibition at 10 and 100 mg/L, and an average of 3% inhibition of the respiration rate at 1000 mg/L. Therefore, the EC50 was above the highest concentration tested (1000 mg/L). No statistically significant inhibition of the respiration rate of the sludge was recorded at a concentration of 1000 mg 3-Methyl Sulfolane per litre. Therefore the NOEC was considered to be the highest concentration tested. There was no significant oxygen uptake from abiotic processes.

Acceptability of the Test:
1. The mean blank control oxygen uptake rate exceeded 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (23 mg oxygen per one gram of activated sludge). The coefficient of variation of oxygen uptake in blank control replicates did not exceed
30% at the end of the definitive test (6%).
2. The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L for total respiration (4.9 mg/L).
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 (4.9 mg/L)
Reported statistics and error estimates:
EC50:
For the reference item, calculation of the EC50 value was based on a 3-parameter logistic cumulative distribution function (CDF) using non-linear regression analysis, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the reference item. For 3-Methyl Sulfolane, no EC50-value could be calculated because the test item proved to be non-toxic (EC50 > 1000 mg/L)

NOEC determination:
An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the blank control revealed significant inhibition of the respiration rate (Two-sample t-test Procedure, α=0.05, α=0.05, one-sided, smaller).

Calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Results - Respiration Rate/Inhibition

Replicate

Concentration

(mg/L)

Respiration rate

% Inhibition

respiration rate

(mean value)

(mg O2/L.h)

(mg O2/g.h)¹

C 1

0

37.94

25.29

 

C 2

0

33.44

22.29

 

C 3

0

34.99

23.33

 

C 4

0

35.11

23.41

 

C 5

0

32.28

21.52

 

C 6

0

35.61

23.74

 

C Mean

 

34.90

23.26 (RC)

 

SD

 

1.94

1.29

 

CV (%)

 

6

6

 

 

 

 

 

R 1

1.0

31.04

20.69

11.05

R 2

3.2

20.18

13.45

42.17

R 3

10

11.75

7.83

66.33

R 4

32

3.99

2.66

88.57

 

 

 

 

 

T 1

10

39.13

26.09

-12.14

T 2

100

45.01

30.01

-28.99

T 3a

1000

31.31

20.87

10.27

T 3b

1000

32.76

21.84

6.12

T 3c

1000

37.88

25.25

-8.55

T3 Mean

33.98

22.66 (RT)

2.61 (IT)

 

 

 

 

 

TA

1000

0.94

0.63

97.31

C:      Control
R:      Reference item, 3,5-dichlorophenol

T:      Test item, 3-Methyl Sulfolane

TA:    Abiotic control of 3-Methyl Sulfolane

SD:    Standard deviation

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

RC:     Total respiration in the control

RT:      Total respiration with 3-Methyl Sulfolane

IT:      % inhibition of total respiration relative to RC

CV:    Coefficient of variation

Validity criteria fulfilled:
yes
Remarks:
see details on results
Conclusions:
Under the conditions of the test 3-Methyl Sulfolane was not toxic to waste water bacteria (activated sludge) at 1000 mg/L (NOEC). The EC50 was thus above 1000 mg/L.
Executive summary:

A study was performed to assess the effect of 3-Methyl Sulfolane on the respiration of activated sewage sludge according to OECD 209 and GLP guidelines. In a combined limit/range finding test, micro-organisms in activated sludge (in test medium) were exposed to test concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for 3 hours. Optimal contact between the test item and test medium was ensured by applying continuous stirring and aeration during that period. In parallel, an untreated control (6 replicates), reference substance (3,5 -dichlorophenol, 4 replicates) and abiotic control (1 replicate) were tested. After the 3-hour contact time, oxygen consumption was recorded for a period of approximately 10 minutes (no aeration but continuous stirring).The study met the acceptability criteria prescribed by the study plan and was considered valid. There was no oxygen uptake from abiotic processes and the reference item 3,5- dichlorophenol showed normal sensitivity. Under the conditions of this present test 3-Methyl Sulfolane was not toxic to waste water (activated sludge) bacteria at a concentration of 1000 mg/L (NOEC).The EC50 was determined to be above 1000 mg/L.

Description of key information

A study was performed to assess the effect of 3-Methyl Sulfolane on the respiration of activated sewage sludge according to OECD 209 and GLP guidelines. In a combined limit/range finding test, micro-organisms in activated sludge (in test medium) were exposed to test concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for 3 hours. Optimal contact between the test item and test medium was ensured by applying continuous stirring and aeration during that period. In parallel, an untreated control (6 replicates), reference substance (3,5 -dichlorophenol, 4 replicates) and abiotic control (1 replicate) were tested. After the 3-hour contact time, oxygen consumption was recorded for a period of approximately 10 minutes (no aeration but continuous stirring).The study met the acceptability criteria prescribed by the study plan and was considered valid. There was no oxygen uptake from abiotic processes and the reference item 3,5- dichlorophenol showed normal sensitivity. Under the conditions of this present test 3-Methyl Sulfolane was not toxic to waste water (activated sludge) bacteria at a concentration of 1000 mg/L (NOEC).The EC50 was determined to be above 1000 mg/L.

Key value for chemical safety assessment

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

Additional information

Since no effects were observed at the highest dose tested (1000 mg/L), the EC50 is thus > 1000 mg/L.