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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes
Specific details on test material used for the study:
Identifier: EXP1200078
Appearance: Very dark brown (almost black) viscous liquid
Batch: E00275-350
Sample Expiration Date: end-2013
Purity:100%
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Reference Stock Solution Preparation
A stock solution of the reference substance, 3,5-dichlorophenol, was prepared at 500 mg/L on 20 November 2012 by
dissolving 500.2 mg of the reference substance in 10 mL of 1N NaOH and then adding 20 mL of NANOpure¿ water.
The NANOpure¿ water is not known to contain any contaminants that are reasonably expected to be present at levels capable of interfering with the study.

While stirring, 9.1 mL of 1N H2SO4 was added to reach the point of incipient precipitation. The solution of
3,5 dichlorophenol was then diluted to a final volume of 1 L with NANOpure¿ water.
The pH of the reference stock solution was 7.8. The reference stock solution was stored at an ambient temperature and given an expiration date of 16 May 2013. The reference substance solution was administered to the reference group by volumetric addition. Volumetric addition is the most appropriate route of administration of aqueous solutions.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Activated sludge collected from the Cambridge Wastewater Treatment Facility, Cambridge, Maryland on 20 February 2013
was utilized as the inoculum for the test. The Cambridge facility receives wastes from predominately domestic sources. The sludge was sieved using a 2 mm screen and allowed to settle for 30 minutes. The supernatant above the settled solids was
removed and the total suspended solids (TSS) concentration of the settled sludge was determined. Total suspended solids in
the settled sludge were adjusted to a nominal concentration of approximately 3000 mg/L by dilution with municipal water.
Fifty mL of synthetic sewage (Appendix I of Protocol) was added to each liter of adjusted sludge each day of storage.
The sludge was maintained at a temperature of 20 ¿ 2¿C and continuously aerated until utilized in the test. The pH and
total suspended solids concentration of the activated sludge were determined on the day of use in the study.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
3 hours
Hardness:
no data
Test temperature:
Temperatures measured throughout the period of sludge maintenance and during the test ranged from 20.0 – 21.2C.
pH:
7.9
Dissolved oxygen:
8.4 - 8.7 mg/L in abiotic control
Salinity:
no data
Nominal and measured concentrations:
The reference group was dosed with 3,5 dichlorophenol, a known inhibitor of respiration, at concentrations of 3, 15 and 50
mg/L. The treatment group was dosed with EXP1200078 at concentrations of 10, 100, and 1000 mg/L.
Details on test conditions:
Test Conditions and Apparatus
Control, reference, and treatment test mixtures were incubated at 20 ± 2ºC and aerated for three hours at a rate sufficient to provide aerobic conditions and maintain solids in suspension. The mixtures were prepared and aerated in 1000 mL Erlenmeyer flasks and then transferred to fill 300 mL biochemical oxygen demand (BOD) bottles to conduct
dissolved oxygen (DO) measurements. Test mixtures were identified by project number, test substance identification, test concentration, and bottle number.

Test Inoculum
Activated sludge collected from the Cambridge Wastewater Treatment Facility, Cambridge, Maryland on
20 February 2013 was utilized as the inoculum for the test. The Cambridge facility receives wastes from predominately
domestic sources. The sludge was sieved using a 2 mm screen and allowed to settle for 30 minutes. The supernatant above the settled solids was removed and the total suspended solids (TSS) concentration of the settled sludge was determined. Total suspended solids in the settled sludge were adjusted to a nominal concentration of approximately 3000 mg/L by dilution with municipal water. Fifty mL of synthetic sewage (Appendix I of Protocol) was added to each liter of adjusted sludge
each day of storage. The sludge was maintained at a temperature of 20 ¿ 2¿C and continuously aerated until utilized in the test. The pH and total suspended solids concentration of the activated sludge were determined on the day of use in the
study.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (CAS 591-35-5)
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
not determinable
Remarks:
No effects noted in respiration under given exposure conditions.
Details on results:
An inhibitory dose response effect was not observed for the treatment group; therefore, the EC50 value for EXP1200078 is greater than 1000 mg/L, the highest concentration tested. The abiotic treatment mixture dosed with 1000 mg/L of
EXP1200078 had a respiration rate of –2.4 mg O2/L/hr showing there was no significant chemical uptake or release of
oxygen resulting from abiotic reactions of the test substance.
Results with reference substance (positive control):
The respiration rates in the two controls were 111.0 and 168.0 mg O2/L/hr. Both controls had respiration rates above 25.2
mg O2/L/hr, which is the low limit based on the validity criteria for mixtures with 1.26 g dry weight of suspended solids
per liter. The coefficient of variation of the two control respiration rates was 28.9%, and was within the 30% limit
established for the test. The validity of the test was further supported by the results from the 3,5-dichlorophenol reference
group, which resulted in an EC50 value of 16.3 mg/L, with 95 percent confidence limits of 3 mg/L and 50 mg/L. The EC50
for the reference substance was within the 2 to 25 mg/L range considered acceptable for the test.

Respiration Rates and Percent Inhibitions

 

 

 

 

Nominal

 

Respiration

 

Percent

 

 

 

 

Concentration

 

Rate

 

Respiration

Treatment

 

 

 

mg/L

 

mg O2/L/hr1

 

Inhibition1

Control 1

 

 

 

0

 

111.0

 

NA

Control 2

 

 

 

0

 

168.0

 

NA

3,5-dichlorophenol

 

3

 

96.0

 

31.2%

3,5-dichlorophenol

 

15

 

74.1

 

46.9%

3,5-dichlorophenol

 

50

 

20.1

 

85.6%

EXP1200078

 

 

10

 

88.6

 

36.5%

EXP1200078

 

 

100

 

684.0

 

-390.3%

EXP1200078

 

 

1000

 

1080.0

 

-674.2%

EXP1200078

 

 

1000

 

900.0

 

-545.2%

EXP1200078

 

 

1000

 

936.0

 

-571.0%

EXP1200078 Abiotic Treatment

 

1000

 

-2.4

 

NA

1Calculations performed using MS Excel 2010 in full precision mode. Manual calculations may differ slightly.

NA - Not Applicable

 

 

 

 

 

 

 
Validity criteria fulfilled:
yes
Conclusions:
An inhibitory dose response effect was not observed for the treatment group; therefore, the EC50 value for EXP1200078 is greater than 1000 mg/L, the highest concentration tested. The abiotic treatment mixture dosed with 1000 mg/L of
EXP1200078 had a respiration rate of –2.4 mg O2/L/hr.
Executive summary:

The potential effect of EXP1200078 on activated sludge microorganisms maintained in an aerobic environment was assessed by the Activated Sludge Respiration Inhibition Test Method (OECD Guideline 209). Testing was done with three different concentrations and a control (0, 10, 100 and 1000 mg/L), and dichrlorophenol was used as a positive control. The positive control performed within previous limits, and there was no effect from the test material at any of the concentrations inluding the highest (1000 mg/L). Therefore, EXP1200078 does not impact microorganisms within the limits of this study.

Description of key information

A GLP compliant study in accordance with the OECD (209) reported that there was no appreciable inhibition of respiration measured at the concentration tested, thus EC 50 value can be presumed to be greater than 1000 mg/l.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

In a GLP compliant study conducted in accordance with the OECD 209 guideline, the test substance was evaluated to determine its potential to inhibit the respiration of activated sewage sludge. No appreciable inhibition of respiration was measured at the concentrations tested. Therefore, an EC50value can be presumed to be greater than 1000 mg/l.