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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07.02.12-15.02.12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: sludge was taken from ESN (Stadtentsorgung Neustadt) STP D-67435 NW-Lachen-Speyerdorf
- Method of cultivation:
- Preparation of inoculum for exposure:
- Pretreatment: sludge was filtrated, washed with tap water and afterwards resuspended in tap water. Sludge was aerated during the whole period from sampling until usage in test. Sludge was fed daily with 50 mL/L synthetic sewage feed.
- Initial biomass concentration: 1.25 g suspended solids/L, respectively 1.51 g suspended solids/L, dry matter in the test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
1.08 mmol
Test temperature:
16.9 - 18.8 °C (pre-test)
18.9 - 19.7 °C (main study)
pH:
8.18
Nominal and measured concentrations:
pre-test: 1000, 100, 10, 1 mg/L (nominal)
main study: 3200, 1000, 320, 100, 32, 10 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: beakers
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume: glass, 500 mL
- Aeration: purified air, using Pasteur pipettes, flow approx. 0.75 L/min
- Type of flow-through (e.g. peristaltic or proportional diluter): -
- Renewal rate of test solution (frequency/flow rate): -
- No. of organisms per vessel: 250 mL inoculum
- No. of vessels per concentration (replicates): positive control: one replicate(/concentration; pre-test: one replicate (lower concentrations), five replicates (highest concentrations); main study: two replicates/treatment
- No. of vessels per control (replicates): two replicates before and two after measuring positive control and test item, respectively
- No. of vessels per vehicle control (replicates): -
- Biomass loading rate:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water
- Total organic carbon: 0.5 mg/l
- Particulate matter:
- Metals:< 0.007 mg/L
- Pesticides: < LOD
- Chlorine: 12 mg/L
- Alkalinity: not stated
- Ca/mg ratio: 7
- Conductivity: 223 µS/cm
- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Adjustment of pH: -
- Photoperiod: -
- Light intensity: -

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : inhibition of respiration rate

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations:1000, 100, 10, 1 mg/L (nominal)
- Results used to determine the conditions for the definitive study:
Reference substance (positive control):
yes
Remarks:
3,5 DCP (CAS# 591-35-5)
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
7 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% CI 3-13 mg/L
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
28 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% CI 14-44 mg/L
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
360 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% CI 280-510 mg/L
Results with reference substance (positive control):
pre-test: positve control (3,5-DCP) 7.8 mg/L
main test: positive control (3,5-DCP) 11.3 mg/L
Reported statistics and error estimates:
linear regression

Vessel No

content

concentration in mg/L

O2consumption in mg//L*min)

O2consumption in mg//L*h)

Inhibition in %

pH

1

control

0

0.5680

34.078

0

8.3

2

control

0

0.5909

35.452

0

8.2

3

positive control

5.0

0.4870

29.221

24.9

8.2

4

positive control

10.1

0.3492

20.950

46.2

8.2

5

positive control

20.2

0.1937

11.624

70.1

8.4

6

positive control

40.4

0.1176

7.059

81.9

8.4

7

control

0

0.6507

39.044

0

8.3

8

control

0

0.6829

40.971

0

8.2

9

test item

3201.2

0.1721

10.325

73.5

8.3

10

test item

3200.2

0.1927

11.560

70.3

8.3

11

test item

1000.2

0.2320

13.918

64.2

8.4

12

test item

1000.4

0.2350

14.102

63.8

8.3

13

test item

320.4

0.3145

18.872

51.5

8.3

14

test item

321.0

0.3072

18.433

52.6

8.3

15

test item

100.4

0.3919

23.517

39.6

8.3

16

test item

98.8

0.4042

24.254

37.7

8.3

17

test item

31.4

0.5397

32.380

16.8

8.2

18

test item

33.0

0.5313

31.879

18.1

8.2

19

test item

10.2

0.6529

39.174

-0.7

8.2

20

test item

11.2

0.6462

38.774

0.4

8.2

21

control

0

0.6979

41.875

0

8.2

22

control

0

0.7010

42.061

0

8.2

Validity criteria fulfilled:
yes
Remarks:
The results of the positive controls were within the stated range of tests with 3,5-DCP. Furthermore the CV of the oxygen control were less than 30 % and the O2 uptake of the blanks was more than 20 mg O2/ g activated sludge.
Conclusions:
Based on experimental observations the effect concentrations EC10(3h) = 7 mg/L and EC50(3h)= 360 mg/L were calculated. All validity criteria were fulfilled and hence the study is considered adequate for regulatory purposes.
Executive summary:

Two valid experiments were performed. In the pre-test, the test item was tested using four concentrations (ranging from 1000 to 1 mg/L nominal concentration). Duration of the test was three hours. Activated sludge was used as inoculum. It was taken from a domestic sewage treatment plant and washed before usage. The dry matter was determined as 2.5 g suspended solids/, giving a concentration of 1.25 g suspended solids/L in the test.

3,5 -dichlorophenol was used as positive control. Four concentrations were tested; an EC50 of 7.8 mg/L (95% confidence interval: < 12 mg/L) was determined, which lies within the range of 2 -25 mg/L.

As inhibition > 20% was observed, a study plan amendment was issued and a main study was performed. The main study was performed likewise using six concentrations, ranging from 3200 to 10 mg/L nominal concentration. the dry matter of the activated sludge was determined as 3.02 g suspended solids/L, giving a concentration of 1.51 g suspended solids/L in the test. The EC50 of the positive control was determined as 11.3 mg/L (95% confidence interval: 8.6 - 14.0 mg/L), which lies within the range of 2 -25 mg/L.

The following results for 2,2'-azobis(2 -amidinopropane)-dihydrochloride (= 2,2'-azobis[2 -methylpropionamidine] dihydrochloride) were determined:

3h EC10 = 7 mg/L (95% confidence interval: 3 -13 mg/L)

3h EC20 = 28 mg/L (95% confidence interval: 14 -44 mg/L)

3h EC50 = 360 mg/L (95% confidence interval: 280 -510 mg/L)

Description of key information

Key value is based on experimental results observed in an OECD 209 guideline study.

Key value for chemical safety assessment

EC50 for microorganisms:
360 mg/L
EC10 or NOEC for microorganisms:
7 mg/L

Additional information