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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-10-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Name of test material (as cited in study report): Aldehyde C10 decyclic food grade, decanal
- Substance type: Colourless liquid
- Physical state: Liquid
- Impurities (identity and concentrations): No data
- Composition of test material, percentage of components: No data
- Isomers composition: No data
- Purity test date: No data
- Lot No: VE00097618
- Expiration date of the lot/batch: 2011-06-11
- Stability under test conditions: No data
- Storage condition of test material: No data
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- The test substance was not soluble enough in water to allow the preparation of an aqueous solution with tap water.
- Different solutions were prepared by dissolving the test material in ethanol in order to always add the same volume of ethanol (5 mL) to each cylinder.
- The following solutions were prepared:
-- A1: Solution of 1000 mg of test material in 100 mL ethanol
-- A2: Solution of 315 mg of test material in 100 mL ethanol
-- A3: 2 mL of solution A1 diluted to 20 mL with ethanol
-- A4: 2 mL of solution A2 diluted to 20 mL with ethanol
-- A5: 2 mL of solution A3 diluted to 20 mL with ethanol
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source: Waste water treatment plant, Bois-de-Bay, 1242, Satigny, Switzerland.
- Laboratory culture: The sludge was kept under aerobic conditions until being used the day after it was obtained.
- pH: Between 7 and 8
- The suspended solid content was determined by drying and weighing 50 mL of the sludge:
- Dry weight of suspended solids: 3.22 g/L.
- By adding 200 mL of sludge to 500 mL test vessels, the final concentration of suspended solids was 1.29 g/L, i.e. in the range of 0.8 to 1.6 g/L as recommended by ISO-8192-1986

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 ± 2 °C
pH:
7.8 to 8.2
Details on test conditions:
TEST SYSTEM
- Test vessels: 500 mL scaled vessels
- Type: Closed
- Aeration: Clean compressed air supply
- No. of vessels per concentration (replicates): 1

SYNTHETIC SEWAGE FEED
- A synthetic sewage feed was made by dissolving the following masses of substances in 1 L tap water:
-- 16.0 g peptone
-- 11.0 g yeast extract powder
-- 3.0 g urea
-- 0.7 NaCl
-- 0.4 g CaCl₂.2H₂O
-- 0.3 g MgSO₄.7H₂O
-- 2.8 g K2HPO₄

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Test concentrations: See table under "Any other information on materials and methods incl. tables" below.

REFERENCE SUBSTANCE
A solution of 3,5 -dichlorophenol was prepared by dissolving 250 mg of 3,5 -dichlorophenol in 5 mL of 1.0 mol/L NaOH, and 0.5 mol/L H₂SO₄ was added while stirring to the point of incipient precipitation. Finally, the mixture was diluted to 500 mL with distilled water. The pH, 7.8 was in the recommended range of 7 to 8.

TEST PROCEDURE
- Each vessel test consisted of 16 mL of synthetic sewage feed, plus relevant stock solution(s) (see below), made up to 300 mL with water. 200 mL of sludge was then added and the mixture was aerated continuously for 3 hrs by bubbling air into it.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
ca. 70 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:
NOEC
Effect conc.:
31.6 mg/L
Nominal / measured:
nominal
Details on results:
- Only at test concentrations of 100 mL was a significant inhibition of respiration observed.
- The results are given in table 1 (attached) and graphed in appendix 1 (also attached)
Results with reference substance (positive control):
- Results with reference substance valid
- Relevant effect levels: 5 mg/L produced 9.5 % inhibition; 30 mg/L produced 80.4 % inhibition.
Reported statistics and error estimates:
For estimating the EC₅₀ (3 hr) a linear relationship between % inhibition and the common logarithm of the test substance concentration was assumed for concentrations ranging from 31.6 mg/L (vessel S2) to 100 mg/L (vessel S1). A linear regression was performed (least squares method) and the parameters obtained:

% inhibition = 131.48 × log conc. - 191.46

From this the EC₅₀ was calculated.
Validity criteria fulfilled:
yes
Remarks:
1. Reference substance Ec₅₀ is between 5 mg/L and 80.4 mg/L. 2. The difference between the 2 controls C1 and C2 was 9.5 %, within the validity range (15 % max).
Conclusions:
The test substance was assessed for activated sludge respiration inhibition according to OECD guideline 209. The results showed that the 3 h EC50 of the test substance was 70 mg/L, based on respiration rate. The NOEC was 31.6 mg/L.

Description of key information

The test substance was assessed for activated sludge respiration inhibition according to OECD guideline 209.  The results showed that the 3 h EC50 of the test substance was 70 mg/L, based on respiration rate. The NOEC was 31.6 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
70 mg/L
EC10 or NOEC for microorganisms:
31.6 mg/L

Additional information

The test substance was assessed for activated sludge respiration inhibition according to OECD 209. Five concentrations were tested: 1, 3.2, 10, 31.6 and 100mg/L. Only at a test concentration of 100 mg/L was significant inhibition of the respiration observed. Therefore the NOEC is considered to be 31.6 mg/L. For estimating the EC50 (3 hours) a linear relationship between % inhibition and the common logarithm of the test substance concentration was assumed for concentrations ranging from 31.6 to 100mg/l. Using this equation the concentration corresponding to 50% inhibition was calculated to be ~ 70 mg/l.