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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
08 Mar 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
adopted in 1984
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was directly added to the test medium.
Test organisms (species):
activated sludge
Details on inoculum:
- Source of inoculum: Activated sludge was collected from the sewage treatment plant of Therwil, Switzerland on 06 Mar 1989.
- Preparation of inoculum for exposure: The sludge was separated from the aqueous layer by settling.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
18 - 20 °C
Nominal and measured concentrations:
Nominal: 0, 1.1, 3.3, 10.7, 32.0, 100.2 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: BOD flasks with gas inlet
- Size, fill volume: 250 mL, 200 mL
- Aeration: Yes
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Concentration of sludge: 1.6 g/L

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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable)
- Microbial respiration was measured after 3 h of incubation.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approx. 3.2
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Duration:
3 h
Dose descriptor:
other: IC20
Effect conc.:
75 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
IC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
- Validity criteria are fulfilled.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: IC50 = 13 mg/L

Table 1: Inhibition of microbial respiration

Concentration (mg/L)

Consumption Rate (mg/L*h)

Inhibition (%)

Blank 1

40.62

2

Blank 2

42.21

-2

1.1

41.62

0

3.3

43.97

-6

10.7

41.79

-1

32.0

35.70

14

100.2

30.00

28

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Sep 2009
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:
adopted in 1984
Deviations:
yes
Remarks:
Higher temperature during the test (Guideline: 18 - 20 °C, Test: 23.3 - 24.4 °C)
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
adopted in 2008
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution containing 10 g/L was prepared using the following procedure: 10 g test item was mixed with 1.1 g NaOH (45%). 40 mL deionised water (80 – 100 °C) was added. After homogenisation with a glass stirrer, 85 mL deionised water (room temperature) was added. Then, the vessel was stirred on a magnetic stirrer. 0.5 M NaOH was added until the resulting solution was clear. The pH of the solution was adjusted to 7.8 ± 0.2 with 1 M HCl. the solution was transferred into a volumetric flask and filled up to 1000 mL with deionised water.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source of the inoculum: The sludge was taken from the activation basin of the sewage plant in Edenkoben, Germany
- Laboratory culture: Upon arrival, the sludge was washed with tap water three times, centrifuged and resuspended in tap water.
- Suspended solids: 4.2 g/L
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
pH:
7.7 - 8.1
Nominal and measured concentrations:
Nominal: 0, 1.0, 3.2, 10, 32, 100, 320, 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Beakers
- Material, fill volume: Glass, 500 mL
- Aeration: Purified air at 0.75 L/min
- No. of vessels per concentration (replicates): 2 for treatment 1000 mg/L, 1 for all other treatments and positive control
- Concentration of suspended solids: 1.7 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water
- Total organic carbon: < 1.0 mg/L
- Metals (mg/L): Na 4.7, K 2.6, Ca 33.1, Mg 2.1, other metals < 0.0005 to < 0.01
- Pesticides: < LOD
- Chloride: 9.0 mg/L
- Conductivity: 189 µS/cm
- Intervals of water quality measurement: pH was measured at test end.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable)
- Respiration rate was measured after 3 h of incubation

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
130 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL = 62 - 310 mg/L
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
1 300 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL = 480 - 7200 mg/L; extrapolated
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
- The inhibition caused by the test item did not rise above 45%. Therefore the EC50(3h) was determined by extrapolation.
- All validity criteria are fulfilled.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: 9.1 mg/L, 95% CL = 7.2 - 11.0 mg/L

Table 1: Inhibition (%) of microbial respiration after 3 h of incubation

Concentration

% Inhibition

Control

-

1.0

-3.2

3.2

1.2

10

1.7

32

16.9

100

25.5

320

24.0

1000 (1)

43.8

1000 (2)

45.3

Description of key information

EC50 (3h) > 1000 mg/L (OECD 209)

NOEC (3h) = 10 mg/L (OECD NOEC (3h) = 10 mg/L (OECD 209)

NOEC (3h)

NOEC (3h) = 10 mg/L (OECD 209)

Key value for chemical safety assessment

EC50 for microorganisms:
1 300 mg/L
EC10 or NOEC for microorganisms:
10 mg/L

Additional information

Two studies investigating the toxicity to microorganism of N-methyl-N-(C18-(unsaturated)alkanoyl)glycine (EC 701-177-3) are available.

The key study was carried out according to OECD 209 under GLP-conditions with domestic activated sludge as inoculum. In this static test seven test concentrations were used (1.0, 3.2, 10, 32, 100, 320 and 1000 mg/L). The dry matter was determined as 4.2 g suspended solids/L, giving a concentration of 1.7 g suspended solids/L in the test. The highest inhibition of respiration rate was determined for the highest tested concentration (1000 mg/L nominal) with 45.3% and 43.8% inhibition. Thus, the inhibition of respiration rate is < 50%. In the study report the EC50 was extrapolated with an EC50 (3h) of 1300 mg/L. But as an extrapolation is imprecise, the EC50 (3h) > 1000 mg/L (nominal) could be stated. The NOEC (3h) was reported as 10 mg/L.

The supporting study was conducted according to OECD 209 under non-GLP conditions. Five test concentrations (1.1, 3.3, 10.7, 32.0, 100.2 mg/L) were inoculated to activated sludge. In this study, the IC50 (3h) for respiration inhibition was above the highest test concentration of 100 mg/L. A NOEC of 10 mg/L is reported.