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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Dec 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
Adopted 22 July 2010
Deviations:
no
GLP compliance:
yes
Test material information:
Composition 1
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Test vessels were prepared individually by weighing out the appropriate amount of the test item (0.0050, 0.0500, 0.5000, 0.5000, 0.5000, and 0.5000 g as active ingredient) and directly adding it to the respective vessels.
- Eluate:
no
- Controls:
yes, negative and abiotic control
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected:
New Bedford Waste Water Treatment Plant, New Bedford, Massachusetts, USA
- Preparation of inoculum for exposure:
The inoculum received 1.75 mL of an undiluted synthetic sewage feed, was aerated overnight, and used for testing the next day. The inoculum was prepared one day before test initiation and had a final pH value of 7.23.
- Pretreatment:
The sludge solution was passed through a 2-mm stainless steel sieve, concentrated by centrifuging for 10 minutes at 1000 rpm, and the resulting supernatant poured off. The sludge was washed with 600 mL of laboratory well water and centrifuged at 1000 rpm for 10 minutes, for a total of four times, with the supernatant being discarded each time. A small amount of the washed sludge was weighed and dried to calculate the quantity of wet sludge required to be suspended in 3.5 L of laboratory well water to obtain an activated sludge containing a solid level of three grams per liter (± 10%).
- Initial biomass concentration: 3 g/L
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
19.9 °C
pH:
7.46 (abiotic control)
7.65 - 7.88 (remaining test vessels)
Nominal and measured concentrations:
nominal: 10, 100, 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
1 L glass beakers
- Aeration:
yes
- No. of vessels per concentration (replicates):
1 (10 mg/L and 100 mg/L), 3 (1000 mg/L)
- No. of vessels per control (replicates):
2
- No. of vessels per abiotic control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
according to the guideline

EFFECT PARAMETERS MEASURED:
Following approximately three hours of aeration, an aliquot of each sludge mixture was transferred to a 50 mL cylinder which was placed inside a 20 °C water bath placed over a stir plate. The pH of the sludge mixture was measured before the respiration measurement. The cylinders received a stir bar and were fitted with a dissolved oxygen probe after receiving the sample. The probes were attached to a Strathkelvin Instruments (SI) Model 928 oxygen system and a computer which ran the Strathkelvin software. Immediately after the dissolved oxygen probes were attached to the Strathkelvin system, the computer program was initiated so that oxygen depletion rates were measured starting at time zero. The oxygen depletion rate was terminated after a 10 min period. The sludge mixtures were stirred continuously while the oxygen measurements were taken. The temperature of the environmental chamber in which the test was conducted was monitored using a minimum/maximum thermometer. Solution pH was measured with a Yellow Springs Instrument (YSI) Model pH100 pH meter. The SI Multisample Activated Sludge Respirometer was used for determination of O2 consumption. The oxygen depletion rate was measured in a single sample from each beaker such that there was one replicate per beaker with zero headspace.

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
10
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
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
Details on results:
- Blank controls oxygen uptake rate:
At test termination, the respiration rate for the two control vessels was determined to be 51.8 and 46.9 mg O2/L/h, which met the acceptability criterion (i.e., within 30% of each other during the test).
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 = 8.3 mg/L
- Other: Respiration rate in the 3,5-dichlorophenol test vessels (1.0, 3.0, 10 and 30 mg/L) was 41.8, 38.5, 20.8, and 12.9 mg O2/L/hr, respectively. Compared to the controls, respiratory inhibition for the 3,5-dichlorophenol test vessels was 15.3, 22.0, 57.9, and 73.9%, respectively.
Reported statistics and error estimates:
No statistical analysis was necessary since no effects were observed. The EC10 value (equivalent to a No-Observed-Effect-Concentration, NOEC) could not be mathematically determined due to an inverted dose curve.

Table 1: Validity criteria

Criterion from the guideline

Outcome

Validity criterion fulfilled

The blank controls (without the test substance or reference substance) oxygen uptake rate should not be less than 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour.

32.9 mg O2/g sludge dw/h

yes

The coefficient of variation of oxygen uptake rate in control replicates should not be more than 30% at the end of definitive test.

7 %

yes

Table 2: The pH, respiration rate, percent inhibition and EC50 values determined in all test vessels during the activated sludge respiration inhibition test.

Sample type

pH

Respiration rate

(mg O2/L/h)

% inhibition

Abiotic

7.46

- 0.2

NA

Control 1

7.65

51.8

NA

Control 2

7.72

46.9

NA

3,5 dichlorophenol (mg/L)

1.0

7.73

41.8

15.3

3.0

7.80

38.5

22.0

10

7.85

20.8

57.9

30

7.88

12.9

73.9

EC50: 8.3 mg/L

 

 

 

10

7.83

36.7

25.6

100

7.83

41.0

16.9

1000

7.84

42.4

14.1

1000

7.84

40.3

18.3

1000

7.84

38.9

21.2

EC50: > 1000 mg/L

 

 

 

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.

Description of key information

EC50 (3 h) > 1000 mg/L (nominal, activated sludge microorganisms, OECD 209)

Key value for chemical safety assessment

Additional information

One experimental study is available investigating the toxicity of 2-(1,1-dimethylethyl)-6-[[3-(1,1-dimethylethyl)-2-hydroxy-5-methylphenyl]methyl]-4-methylphenyl acrylate (CAS 61167-58-6) to activated sludge microorganisms. Activated sludge from a sewage treatment plant treating predominantly domestic sewage was used for the study. Three nominal concentrations of 10, 100 and 1000 mg/L were tested. The respiration rate was continuously measured for 10 min after 3 h exposure. Due to the inverted dose response curve no EC10 value could be statistically determined. However, the EC50 (3 h) was > 1000 mg/L (nominal).