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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-01-31 to 2017-11-20
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Reference:
Composition 0
Qualifier:
according to
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Version / remarks:
01 Jun 2007
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
Calculation of inhibition of oxygen consumption
GLP compliance:
no
Remarks:
Routine measurement conducted in company lab with sewage sludge from the company sewage treatment plant; Because of the specific conditions of the facility's water treatment plant, the company lab is preferred over external laboratories.
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
201601013
- Expiration date of the lot/batch:
06.01.2019
- Purity:
99.9 %

Analytical monitoring:
not required
Details on sampling:
Not applicable.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
At the start of the test, the 1st vessel was filled with 16 mL of synthetic sewage, deoinizied water and activated sludge. This procedure was repeated every 10 minutes with increasing concentrations of the test substance. Finally the test ended with the second control. Thirty minutes after the test start, the oxygen consumption content of the first vessel was determined. This procedure was repeated on the contents of the other vessels at 10 minutes intervals. After a contact time of 3 hours an identical procedure as outlined before was followed.
Test organisms (species):
activated sludge, industrial
Details on inoculum:
Activated sludge from the industrial waste water treatment plant Lenzing AG Stufe 1 was collected one day before the test started.
- Preparation of inoculum for exposure: First, the sludge was washed three times with 0.9 % NaCl solution. Afterwards the sludge was aerated, synthetic medium and deionizied water were added and used within 24 hours. The pH was measured. No adjustment is necessary.
- Sludge concentration (suspended solids): 1500 mg/L ± 10 %



Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
None
Hardness:
Not reported.
Test temperature:
22 ± 2 °C
pH:
Not reported in detail. It is mentioned, that no deviation from the standard procedure occured. The pH of the solution has to be 7.5 ± 0.5.
Dissolved oxygen:
Not reported.
Salinity:
Not reported.
Conductivity:
Not reported.
Nominal and measured concentrations:
Nominal test concentrations: 10.0, 100.0, 500.0, 1000.0, 1500.0 and 2000.0 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
Glass beakers
- Material, size, headspace, fill volume:
1000 mL
- Aeration:
Yes
- No. of vessels per concentration (replicates):
1
- No. of vessels per control (replicates):
2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
refer to guideline ÖNORM EN ISO 8192.

OTHER TEST CONDITIONS
- Adjustment of pH:
No

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : total
oxygen consumption

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
irregular spacing
- Range finding study
: Yes. Similar to range-finding test, a screening test was performed with nominal concentrations of 10, 100 and 1000 mg/L.
- Test concentrations (nominal):

1. test: 5.0, 500.0 and 1500 mg/L
2. test: 10.0, 1000.0 and 2000.0 mg/L
Definitive test: 10.0, 100.0, 500.0, 1000.0 and 2000.0 mg/L
- Results used to determine the conditions for the definitive study: Yes. The concentrations for the definitive test were determined to be 10.0, 100.0, 500.0, 1000.0 and 2000.0 mg/L
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
636 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
The results of the second test and definitive test show, that the percentage inhibition decreased at higher concentration levels (refer to tables in "Any other information on results incl. tables" below). At a concentration of 1000 mg/L the inhibition was 57 % / 68 % (2. test / definitive test). While the percentage inhibition at a concentration of 2000 mg/L was deteremined to be 39 % / 29 % (2. test / definitive test). The reason for this decrease of the percentage inhibition is unclear. Incorrectness during the test can be excluded. The test was repeated twice and the same observation was made. For the determination of the EC50 of the test substance, the 2000 mg/L-value was excluded.

- Any observations (e.g. precipitation): During the screening or other tests, no deviations from the standard procedure was observed. But the activated sludge was colored into dark brown with increasing concentrations.

Results with reference substance (positive control):
- Results with reference substance valid? Yes

Respiration rates and inhibition of respiration of activated sludge in presence of propyl gallate after a contact time of 30 minutes and 3 hours:

1. Test     Concentration mg/L
BW 1 Stand
(25 mg/L)
5 500 1500 BW 2
after 30 min in mg/L 10min (in mg/L h) 2 (12) 0.6 (1.2) 2 (12) 1.7 (10.2) 2.6 (13.2) 2 (12)
% inhibition n.i. 70 n.i. n.i. n.i. n.i.
after 3 hours in mg/L 10min  (in mg/L h) 2.6 (15.6) 0.6 (3.6) 2.5 (15) 1.8 (10.8) 1.7 (10.2) 2.6 (15.6)
% inhibition n.i. 77 n.i. 31 35 n.i.
N.i.= no inhibition (i. e., inhibition < 10 % was defined as no inhibition)  
BW = Control; Stand = Reference substance

2. Test     Concentration mg/L
BW 1 Stand
(25 mg/L)
10 1000 2000 BW 2
after 30 min in mg/L 10min (in mg/L h) 2 (12) 0.8 (4.8) 1.9 (11.4) 1.7 (10.2) 2.3 (13.8) 2 (12)
% inhibition n.i. 60 n.i. 15 n.i. n.i.
after 3 hours in mg/L 10min (in mg/L h) 2.7 (16.2) 1.2 (7.2) 2.4 (14.4) 1.2 (7.2) 1.7 (10.2) 2.9 (17.4)
% inhibition n.i. 57 14 57 39 n.i.
 N.i.= no inhibition (i. e., inhibition < 10 % was defined as no inhibition)  
BW = Control; Stand = Reference substance

Definitive test     Concentration mg/L
BW 1 Stand
(25 mg/L)
10 100 500 1000 2000 BW 2
after 30 min mg/L 10min  (in mg/L h) 3.2 (19.2) 1.4 (8.4) 3.2 (19.2) 3.1 (18.6) 2.9 (17.4) 2.7 (16.2) 3.8 (22.8) 3.5 (21)
% inhibition n.i. 58 n.i. n.i. 13 19 n.i. n.i.
after 3 hours in mg/L 10min  (in mg/L h) 6.1 (36.6) 1 (6) 5.3 (31.8) 4.8 (28.8) 3.9 (23.4) 2 (12) 4.4 (26.4) 6.3 (37.8)
% inhibition n.i. 84 15 23 37 68 29 n.i.
 N.i.= no inhibition (i. e., inhibition < 10 % was defined as no inhibition)  
BW = Control; Stand = Reference substance
Validity criteria fulfilled:
yes
Remarks:
The validity criteria were met in the screening as well as in the three main tests.
Conclusions:
The inhibition of the respiration rate of activated sludge in the presence of propyl gallate was investigated according to ÖNORM EN ISO 8192:2007 and OECD guideline 209. The concentrations tested were 10.0, 100.0, 500.0, 1000.0 (and 2000 mg/L). The respiration rate was measured after a contact time of 30 minutes and 3 hours. The EC50 value after 3 hours exposure was determined to be 636 mg/L.
Executive summary:

The inhibition of the respiration rate of activated sludge in the presence of propyl gallate was investigated according to ÖNORM EN ISO 8192:2007 and OECD guideline 209. The concentrations tested were 10.0, 100.0, 500.0, 1000.0 (and 2000 mg/L). The respiration rate was measured after a contact time of 30 minutes and 3 hours. The EC50 value after 3 hours exposure was determined to be 636 mg/L.

Description of key information

The EC50for respiration inhibition of sewage sludge by propyl gallate was determined to be 636 mg/L (guideline study, ÖNORM EN ISO 8192).

Key value for chemical safety assessment

EC50 or LC50 for microorganisms:
636 mg/L

Additional information