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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 JUN 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
performed acc. to outdated guideline version with fewer replicates and fewer validity criteria
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
1984
Deviations:
yes
Remarks:
reference item used: copper (II) sulphate pentahydrate instead of 3,5-dichlorophenol
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
pH in water 13-14
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Stock solutions: none, direct weighing
Preparation of test solution: 200 mL inoculum,16 mL synthetic waste water, 284 mL autoclaved tap water, test item concentrations by direct weighing
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Inoculume: activated sludge
- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant of Sarstedt, Germany
- Date of receipt: 2002-06-04
- Nutrient solution: Synthetic waste water according to OECD 209
- Preparation of inoculum for exposure: The sludge was washed with autoclaved tap water and diluted corresponding to a dry weight of 4.0 g/L ± 10%.
- Initial biomass concentration: 3.81 g/L
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
21°C
pH:
7.09
Nominal and measured concentrations:
Nominal test concentrations: 420 - 756 - 1344 - 2436 - 4200 mg a.i./L (1000 - 1800 - 3200 - 5800 - 10000 mg/L test item)
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flasks, DIN 12440
- Fill volume: 500 mL
- Aeration: permanent
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): na
- No. of vessels per abiotic control (replicates): na
- Sludge concentration (weight of dry solids per volume): 3.81 g/L
- Nutrients provided for bacteria: Synthetic waste water according to OECD 209

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: not reported
- Water quality parameters:

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: not reported
- Details on termination of incubation: no

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Respiration rates / oxygen depletion: oxygen concentration [mg O2/L]), measured at 0 and 3 hours

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study: yes
- Test concentrations: 10 - 100 - 1000 mg/L test item
- Results used to determine the conditions for the definitive study: yes

TEST PERFORMANCE
A range-finding test (NON-GLP-state) was carried out with three concentrations: 10 - 100 - 1000 mg/L. A sample of activated sludge washed twice with autoclaved tap water and diluted corresponding to a dry weight of 4.0 ± 10 %, was used for the test procedure. Five concentrations within the range 1000 - 10000 mg/L in a series with a dilution factor of 1.8 were tested. 2 controls without test item were included. The respiration rates of all samples were determined subsequent to an incubation period of 3 h.
Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate
Duration:
3 h
Dose descriptor:
IC50
Effect conc.:
844 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: CI 95%: 815 - 876
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
420 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Details on results:
A summary table of results is provided in 'any other information on results'.

The test substance inhibited the respiration activities of bacteria in test concentrations >= 756 mg a.i./L. The lowest test concentration at 420 mg a.i./L did not show any adverse effect. Inhibition of respiration followed a dose-response pattern. The derived 3h-EC50 was 844 mg a.i./L and the 3h-NOEC was 420 mg a.i./L.

Validity criteria
Validity criteria were fulfilled (OECD 209 adopted 1984):
• The two control respiration rates differ not more than 15 % in the study: in this study they differed by 11%.

In addition, validity criteria could not be fulfilled for OECD 209 adopted 2010:
• The blank controls (without the test substance or reference substance) oxygen uptake rate should not be less than 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour: in this study 4.2 - 4.7 mgO2/g*h.
• The EC50 (3 hours) of copper(II)sulphate pentahydrate is in the accepted range of 53-155 mg/L: in this study EC50 = 131 mg/L.
Results with reference substance (positive control):
Once every 7 days a reference test is carried out. The reference item was tested in three concentrations within the concentration range 58 - 180 mg/L with a dilution factor of 1.8. The EC50-value was determined by probit analysis. The EC50-value of the reference item was EC50 = 131 mg/L. The EC50-value of the reference item copper(II)-sulphate-pentahydrate was in the confidence range of 54 - 146 mg/L.
Reported statistics and error estimates:
The EC-values of the test and reference item were calculated with probit analysis using software SIGMAPLOT (Windows) rel. 6.00, 2000, SPSS CORPORATION. Probit values were taken from WEBER (1986). Confidence intervals were calculated according to a standard procedure (BREITIG & TÜMPLING 1982).

WEBER, E. (1986): Grundriß der biologischen Statistik, 9. Auflage.
BREITIG, G., TÜMPLING, W. (editors, 1982): Methoden der Wasseruntersuchungen Bd.II. VEB GUSTAV FISCHER VERLAG, Jena.

Respiration rates and inhibition of respiration of test and reference item concentrations

  concentration
[mg/L]
concentration
[mg a.i./L]
respiration rate
[mgO2/L*h]
inhibition
[%]
test item 10000 4200 0 100
5800 2436 1.2 93
3200 1344 0.8 95
1800 756 5.2 69
1000 420 18.4 -8
control 1 -   16.0 -

control 2

-   18.0 -

reference item

180 5.2 69
100 11.6 32
58 14.8 13

Difference of the control respiration rates: 11 %

Validity criteria fulfilled:
yes
Remarks:
adopted 1984 (partly for updated guideline from 2010)
Conclusions:
3h-EC50 (STP microorganisms, respiration inhibition) = 844 mg a.i./L; 3h-NOEC (STP microorganisms, respiration inhibition) = 420 mg a.i./L.
Executive summary:

A Respiration Inhibition Test with activated sludge was performed with the test substance according OECD 209 (1984) and GLP principles. Test system was activated sludge of a municipal treatment plant. The test method was static. Nominal concentrations of the test item were as follows: 420 - 756 - 1344 - 2436 - 4200 mg a.i./L. The respiration rates of control, reference and test item were measured after a contact time of three hours, and the inhibitory effects of the test and reference concentrations were determined in comparison to the control respiration rates.

After 3 hours of exposure, respiration inhibition ranged from 0 to 100%: The test substance inhibited the respiration activities of bacteria in test concentrations >= 756 mg a.i./L. The lowest test concentration at 420 mg a.i./L did not show any adverse effect. Inhibition of respiration followed a dose-response pattern. The derived 3h-EC50 was 844 mg a.i./L and the 3h-NOEC was 420 mg a.i./L.  

In order to check the activity of the test system and the test conditions a reference test was carried out with copper-(II)-sulphate-pentahydrate. The EC50-value for the reference item was 131 mg/L and therefore laid in the valid range.

Validity criteria of OECD 209 adopted 1984 were fulfilled. The additional validity criterion for the updated guideline from 2010 regarding the minimum respiration rate of the controls could not be met. However, this violation was not taken as reason to assume singnificant distortion of results. Furthermore, the assessment was considererd conservative because observed effects were likely linked to pH-effect rather than inherent toxicity of the test substance. The results of the study were considered acceptable for the hazard assessment for STP microorganisms.  

Description of key information

3h-EC50 (STP microorganisms, respiration inhibition) = 844 mg a.i./L; 3h-NOEC (STP microorganisms, respiration inhibition) = 420 mg a.i./L (nominal, OECD 209, 2002)

Key value for chemical safety assessment

EC50 for microorganisms:
844 mg/L
EC10 or NOEC for microorganisms:
420 mg/L

Additional information

A reliable key study is available on the activated sludge respiration inhibition of the submission substance (RL2). Respiration inhibition with activated sludge was performed with the test substance according OECD 209 (1984) and GLP principles. Nominal concentrations of the test item were as follows: 420 - 756 - 1344 - 2436 - 4200 mg a.i./L. The respiration rates of control, reference and test item were measured after a contact time of three hours, and the inhibitory effects of the test and reference concentrations were determined in comparison to the control respiration rates. After 3 hours of exposure, respiration inhibition ranged from 0 to 100%. The derived 3h-EC50 was 844 mg a.i./L and the 3h-NOEC was 420 mg a.i./L. 

Validity criteria of OECD 209 adopted 1984 were fulfilled. The additional validity criterion for the updated guideline from 2010 regarding the minimum respiration rate of the controls could not be met. However, this violation was not taken as reason to assume significant distortion of result. Furthermore, the assessment was considered conservative because observed effects are likely linked to pH-effect rather than inherent toxicity of the test substance. The results of the study were considered acceptable for the hazard assessment for STP microorganisms.