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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
Cited as Directive 87/302/EEC, part C, p. 118 (Biodegradation: Activated sludge respiration inhibition test)
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test substance were added directly into the test vessels.
Test organisms (species):
activated sludge
Details on inoculum:
- Laboratory culture: obtained from municipal wastewater treatment plant of Marl-Ost (Germany), which predominantely treats domestic sewage.
- Preparation of inoculum for exposure: The activated sludge were centrifuged four times for 5 min at 2000 rpm and cleaned afterwards with 600 mL tap water.
- Initial biomass concentration: 3.7 g suspended solids/L
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
18 - 20.6 °C
pH:
7.8 - 8.4
Nominal and measured concentrations:
Nominal: 100.8, 127.4, 190.8, 269.6 and 352.4 mg/L
Details on test conditions:
TEST SYSTEM
- Aeration: continuously
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water
- Intervals of water quality measurement: at the end of the incubation phase (after 3h)

EFFECT PARAMETERS MEASURED: O2 consumption was measured for 8 minutes after an incubation time of 3h.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
> 350 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:
EC50
Effect conc.:
> 350 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: EC50 (3h) = 5.5 mg/L

Table 1: Respiration inhibition of control, test and reference substance

 Treatment

Nominal concentration [mg/L]

Measuring time

Inhibition [%]

Test item

352.4

5

-6.6

Test item

269.6

6

-8.0

Test item

190.8

6

-6.6

Test item

127.4

6.5

-7.7

Test item

100.8

5

-9.7

Reference

5

6.5

45.9

Reference

15

7.5

77.9

Reference

30

8

86.1

Control

 

6.5

 

Control

 

7

 

Validity criteria fulfilled:
yes
Conclusions:
A 3h respiration inhibition test with domestic activated sludge revealed that DEHA is not inhibitory to aquatic microorganisms up to the saturation limit in water.
Executive summary:

A 3h activated sludge respiration inhibition test was performed according to EU method C.11 under GLP conditions. Different test concentrations up to ~ 350 mg/L were tested. No respiration inhibition but stimulation was observed in all treatments compared to the control. Thus, the 3h-EC50 value is determined to be above the highest concentration tested : >350 mg/L.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
05 Sep 1988
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Only short documentation available
Qualifier:
according to guideline
Guideline:
other: DIN 38412, part 27
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
- stock solution: 10000 mg/L mixed with 100 mg/l Tween 80 and stired for 2 hours.
- After a short standing time the test dispersion separates back in its components.
Test organisms (species):
Pseudomonas putida
Details on inoculum:
- age of bacteria culture: 4 hours
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
30 min
Test temperature:
25 °C
Nominal and measured concentrations:
Nominal: 2500, 5000 and 10000 mg/L
Duration:
30 min
Dose descriptor:
other: EC20
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
30 min
Dose descriptor:
EC10
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
Until the highest test concentration (1000 mg/l) no effect on the bacteria measured.

Description of key information

The inhibition of the degradation activity of activated sludge is not anticipated when bis(2-ethylhexyl) adipate is introduced in appropriate low concentrations.

Key value for chemical safety assessment

Additional information

Two studies investigating the toxicity of DEHA to aquatic microorganisms are available. The key study was performed according to EU method C.11 under GLP conditions (Hüls 1996). Different test concentrations up to 352.4 mg/L were tested. No respiration inhibition but stimulation was observed in all treatments compared to the control. Thus, the 3h-EC50 value is determined to be above the highest concentration tested of 350 mg/L.

Similar results measured in a ROBRA test according to DIN 38412 part 27, in which the highest test concentration of 10000 mg/L caused no effect on the respiration rate of the microorganisms (BASF 1988).