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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: New study performed to GLP and to internationally accepted guidelines
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Test organisms (species):
activated sludge of a predominantly domestic sewage
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
15 min
pH:
Measurements of the pH of the aqueous stock solutions of the synthetic sewage, the reference substance and of the sample of activated sludge (4 g/L) before the start of the test are given below.
Synthetic sewage: pH7.41
3,5-DCP stock solution: pH7.40
Activated sludge: pH6.87
Reference substance (positive control):
yes
Remarks:
3,5-DCP
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
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
Remarks:
respiration rate
Details on results:
The mean specific respiration rate of the control cultures was 29.5 mgO2/h with a coefficient of variation of 14.5%.

Kalama® K-FLEX® PG did not significantly inhibit the respiration rates of samples of activated sludge at 10 and 100 mg/L, but inhibition (19%) was statistically significant at nominal 1000 mg/L. The EC20, EC50, and EC80 (with 95% confidence intervals) could not be estimated for Kalama® K-FLEX® PG because there was insufficient inhibition to identify a concentration-response relationship up to 1000 mg/L and therefore no further testing was undertaken. Consequently, the EC50 for inhibition must be greater than 1000 mg/L, the highest concentration employed in the study. As the level of inhibition (19%) at 1000 mg/L was statistically significant from the control, the no observed effect concentration (NOEC) for Kalama® K-FLEX® PG was considered to be 100 mg/L.
Results with reference substance (positive control):
Sludge respiration rates were progressively reduced in the presence of increasing concentrations of 3,5-DCP. The three-hour 50% effect concentration (EC50) for 3,5-DCP was calculated to be 8.7 mg/L (95% confidence limits of 3.32 and 22.6 mg/L).These results show that the sample of activated sludge employed was sensitive to inhibition and that the test was valid.
Validity criteria fulfilled:
yes
Conclusions:
Kalama® K-FLEX® PG did not significantly inhibit the respiration rates of samples of activated sludge at 10 and 100 mg/L, but a statistically significant level of inhibition (19%) was observed at nominal 1000 mg/L. Consequently, the EC50 for inhibition must be greater than 1000 mg/L, the highest concentration employed in the study. As the level of inhibition (19%) at 1000 mg/L was statistically significant from the control, the no observed effect concentration (NOEC) for Kalama® K-FLEX® PG was considered to be 100 mg/L.
Executive summary:

Kalama® K-FLEX® PG did not significantly inhibit the respiration rates of samples of activated sludge at 10 and 100 mg/L, but a statistically significant level of inhibition (19%) was observed at nominal 1000 mg/L. Consequently, the EC50 for inhibition must be greater than 1000 mg/L, the highest concentration employed in the study. As the level of inhibition (19%) at 1000 mg/L was statistically significant from the control, the no observed effect concentration (NOEC) for Kalama® K-FLEX® PG was considered to be 100 mg/L.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 April 1997 - 10 April 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted in accordance with EC, OECD, and US EPA test guidelines, and in compliance with GLP.
Justification for type of information:
A discussion and report on the read across strategy is presented as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test item was added directly to test vessels containing carbon filtered tap water. The mixtures were treated with ultra-sound for 10 minutes, and shaken for 10 minutes before the addition of synthetic sewage and the microbial inoculum.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: Maintained under aerobic conditions until required.
- Preparation of inoculum for exposure: Aliquots (25 mL) of the activated sludge were filtered through dried and preweighed filter papers which were dried again at 110°C for at least one hour, allowed to cool in a desiccator and reweighed. The mixed liquor suspended solids (MLSS) content of the activated sludge was then calculated. Synthetic sewage (50 mL/L) was added and the mixture aerated overnight. On the day of the test, the MLSS content was determined and adjusted to 4 g/L by the addition of dechlorinated tap water. The pH of the activated sludge was measured after its MLSS content had been adjusted to 4 g/L.
- Initial biomass concentration: 4 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
200-250 mg/L CaCO3
Test temperature:
18.4 to 20°C preliminary test
19.0 to 20.9°C main test
pH:
7.7 to 8.3 preliminary test
7.8 to 8.3 main test
Nominal and measured concentrations:
Preliminary test: 1, 10, and 100 mg/L nominal
Main test: 100 mg/L (in triplicate) nominal.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: One-litre glass beaker, 500 mL fill volume
- Aeration: Aerated throughout the exposure period using a pasteur pipette connected to a laboratory supply of oil-free compressed air.

- No. of vessels per concentration (replicates): One during preliminary test, three in main test
- No. of vessels per control (replicates): Two
- Biomass loading rate: 4 g/L


OTHER TEST CONDITIONS
- Adjustment of pH: pH was measured, but not reference to adjustment was made.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Biochemical oxygen demand.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not applicable - main test used a single (limit) concentration only.
- Range finding study
- Test concentrations: 1, 10, and 100 mg/L nominal
- Results used to determine the conditions for the definitive study:Yes
Reference substance (positive control):
yes
Remarks:
3,5 Dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: Tested at 3.0, 10, and 32.0 mg/L; 3-hour 50% effect concentrations were found to be 14.5 mg/L in the preliminary test and 19.0 mg/L in the main test.
Validity criteria fulfilled:
yes
Conclusions:
DPGDB had no inhibitory effect on the respiration rate of activated sludge at any ofthe concentrations employed in the test.

The EC20, EC50 and EC80 of the test material could not therefore be calculated but these must be greater than a nominal concentration of 100 mg/L, the highest tested level.
Executive summary:

Key data is available for propylene glycol dibenzoate (PGDB). This data is supported by relevant information available from a structural analogue Dipropylene glycol dibenzoate (DPGDB). The justification for read across is presented as an attachment included in Section 13 of the IUCLID dossier.

An activated sludge respiration inhibition test was conducted to determine the effect on sewage micro-organisms of the test material DPGDB. The study was conducted in accordance with EC, OECD, and US EPA test guidelines, and in compliance with GLP.

DPGDB had no inhibitory effect on the respiration rate of activated sludge at any ofthe concentrations employed in the test.

The EC20 and EC50 of the test material could not therefore be calculated but these must be greater than 100 mg/L, the highest tested level.

Description of key information

Key data is available for propylene glycol dibenzoate (PGDB). This data is supported by relevant information available from a structural analogue Dipropylene glycol dibenzoate (DPGDB). The justification for read across is presented as an attachment included in Section 13 of the IUCLID dossier.

A GLP, OECD Guideline 209 study activated sludge respiratory inhibition test was performed with propylene glycol dibenzoate. The EC50 was found to be >1000 mg/L and NOEC was determined to be 100 mg/L.

Two studies conducted according to international guidelines and in compliance with GLP, are available for DPGDB. The activated sludge respiration inhibition test demonstrated a NOEC ≥ 100 mg/L, the highest concentration tested. The assay on growth of the bacteria Pseudomonas putida demonstrated a NOEC ≥ 10 mg/L.

Key value for chemical safety assessment

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

Additional information

Key data is available for propylene glycol dibenzoate (PGDB). This data is supported by relevant information available from a structural analogue Dipropylene glycol dibenzoate (DPGDB). The justification for read across is presented as an attachment included in Section 13 of the IUCLID dossier.

PGDB did not significantly inhibit the respiration rates of samples of activated sludge at 10 and 100 mg/L, but a statistically significant level of inhibition (19%) was observed at nominal 1000 mg/L. Consequently, the EC50 for inhibition must be greater than 1000 mg/L, the highest concentration employed in the study. As the level of inhibition (19%) at 1000 mg/L was statistically significant from the control, the no observed effect concentration (NOEC) for PGDB was considered to be 100 mg/L (Huntingdon Life Sciences, 2014d; Klimisch score = 1).

An activated sludge respiration inhibition test was conducted to determine the effect on sewage micro-organisms of the test material DPGDB (Huntingdon Life Sciences, 1998d; Klimisch score = 1). The study was conducted in accordance with EC, OECD, and US EPA test guidelines, and in compliance with GLP. DPGDB had no significant inhibitory effect on the respiration rate of activated sludge at any of the concentrations employed in the tests. The NOEC was determined to be ≥ 100 mg/L, the highest concentration tested.

 

A second study that supports this result was performed to assess the effect of DPGDB on the growth of the bacteria Pseudomonas putida (Safepharm Laboratories Ltd.,1998a, Klimisch score = 1). The method followed that described in the German Water Hazard Classification Scheme Bewertung Wassergefahrdender Stoffe LTWS Nr 10 and ISO 10712, and was conducted to GLP. Exposure of Pseudomonas putida to DPGDB gave EC10 and EC50 values of greater than 10 mg/L. The "no-observed effect level" (NOEC) was ≥ 10 mg/L.