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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10-18 January 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No 209.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
minor deviation: slightly lower average oxygen uptake rate for the control replicates (19 instead of 20 mg O2/g dry weight/hour)
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
no data
Analytical monitoring:
no
Details on sampling:
not applicable
Vehicle:
no
Details on test solutions:
APPLICATION OF TEST SOLUTION: test substance was weighed directly in the incubation vessels.

PREPARATION AND APPLICATION OF POSITIVE CONTROL SOLUTION
- Positive control was added to the incubation vessels using a stock solution of approximately 1.0 g/L. The stock solution was prepared by dissolving 0.1046 g of 3,5-dichlorophenol in approximately 40 mL of deionised water with the addition of a few drops of 3 M NaOH. Finally the pH was corrected with H2SO4 to approximately pH 7 and the solution was diluted further to 100 mL with deionized water.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Preparation of inoculum for exposure: activated sludge was obtained from an activated sludge plant treating predominantly domestic wastewater (WWTP Nieuwgraaf, Duiven, The Netherlands). Prior to use the activated sludge was homogenised with a syringe. The dry weight of the homogenised activated sludge was determined and subsequently concentrated by settlement to the required dry weight concentration.
- Dry weight of the activated sludge in the incubation vessels: 1.5 g/L
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
not applicable
Hardness:
no data
Test temperature:
20.0-20.6 °C
pH:
6.7-7.0
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
Nominal concentrations: 19.6, 39.3, 78.6, 157.1, 314.2, 628.5 and 1257.0 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks closed with Teflon coated lids, with a working volume of 50 mL
- Incubation conditions: homogenized activated sludge was incubated in a shaking water bath (100 rpm, 20 °C) for 3 h with various concentrations of the test compound and synthetic sewage.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized water containing no more than 0.01 mg/L Cu and 1 mg/L non-purgeable organic carbon was prepared in a water purification system.

SYNTEHTIC SEWAGE FEED:
- Composition of synthetic sewage feed:
It was made by dissolving the following amounts of substances in 1 L of deionized water: 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.3 g CaCl2, 0.2 g MgSO4.7H2O, 2.8 g K2HPO4
- In the incubation vessels 1.6 mL of the synthetic sewage feed was added in a total volume of 50 mL.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): respiration rate of activated sludge

MEASURING EQUIPMENTS:
- Respiration rates of the activated sludge were measured in a Biological Oxygen Monitor YSI 5300. The BOM consisted of a thermostated vessel (volume: 10 mL, temperature: 20 °C) with a magnetic stirrer and an oxygen electrode.
- The pH was measured using an EUTECH pH meter.
- The temperature was measured with a Keithley 871A digital thermometer.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Test concentrations: 0 (control), 19.6, 39.3, 78.6, 157.1, 314.2, 628.5 and 1257.0 mg/L
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (0, 2.5, 5.1, 10.2, 20.3 and 40.6 mg/L)
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
190 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: calculated using all concentrations
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
126 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: without using the two highest concentrations
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
24 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:
other: EC20
Effect conc.:
49 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:
other: EC80
Effect conc.:
740 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
see tables 6.1.7/1 and 6.1.7/2
Results with reference substance (positive control):
- Results with reference substance valid: yes
- Relevant effect levels: EC50 (3 h): 11 mg/L
Reported statistics and error estimates:
- EC values were computed from the best-fitted line (least square method) through the points given by the probit of the percentage inhibition and the logarithm of the concentration of the substance.
- All computations were performed with the program TOXCALC (Tidepool Scientific Software 1994-1999).

Table 6.1.7/1: Respiration rates of the activated sludge, inhibition percentages, and pH at various concentrations of 3,5-dichlorophenol after 3 h contact time

 

Concentration

(mg/L)

Activity

(mg O2/g/min)

Inhibition (%)

pH at start

pH at end

Temperature at end

(°C)

Control

0.2926

-

6.6

7.0

20.4

Control

0.3067

-

6.7

7.0

20.5

2.5

0.2750

8

6.6

7.0

20.2

5.1

0.2222

26

6.7

7.0

20.0

10.2

0.1833

39

6.7

7.0

20.3

20.3

0.0593

80

6.7

6.9

20.6

40.6

0.0370

88

6.7

6.9

20.4

Concentration stock solution 3,5-dichlorophenol = 1015 mg/L

pH stock solution 3,5-dichlorophenol = 7.1

 

Table 6.1.7/2: Respiration rates of the activated sludge, inhibition percentages and pH at various concentrations of test material after 3 h contact time

 

Concentration

(mg/L)

Activity

(mg O2/g/min)

Inhibition (%)

pH at start

pH at end

Temperature at end

(°C)

Control

0.3259

-

6.7

7.0

20.4

Control

0.3444

-

6.7

7.0

20.5

Control*

0.3367

-

6.7

7.0

20.2

Control*

0.3133

-

6.7

7.0

20.4

19.6*

0.3267

-1

n.d.

7.0

20.4

39.3

0.2667

20

n.d

6.9

20.4

78.6

0.2300

31

n.d

6.9

20.2

157.1

0.1167

65

n.d

6.8

20.0

314.2

0.0741

78

n.d

6.7

20.3

628.5

0.0733

78

n.d

6.7

20.3

1257.0*

0.0788

76

n.d

6.7

20.3

* = corresponding control and reference concentration

n.d. = not determined, the pH was not measured at the start to prevent evaporation of the test substance.

Validity criteria fulfilled:
yes
Remarks:
two control respiration rates were within 15 % of each other; EC 50 (3 h) of 3,5-dichlorophenol was in the accepted range 5-30 mg/L
Conclusions:
The 3h-EC50 of nopyl acetate for inhibition of respiration of activated sludge was calculated to be 190 mg/L (with all concentrations) and 126 mg/L (without using the two highest concentrations). Therefore nopyl acetate was considered not harmful to activated sludge.
Executive summary:

A respiration inhibition study was performed to assess the effect of nopyl acetate on STP microorganisms according to OECD Guideline No 209 and in compliance with GLP. The test material was tested at concentrations of 19.6, 39.3, 78.6, 157.1, 314.2, 628.5 and 1257.0 mg/L (nominal) and the inoculum (microbial source) was activated sludge from a predominantly domestic sewage. Toxicity to microorganisms was assessed by measuring the respiration rate. Two controls without test substance were included in the test design, one at the start and the other at the end of the test series. 3,5-dichlorophenol was used as a reference substance at concentrations of 2.5, 5.1, 10.2, 20.3 and 40.6 mg/L.

The EC50 of the reference compound, 3,5-dichlorophenol was 11 mg/L and the coefficient of variation of the replicates of the control oxygen uptake rates was 7%. The average oxygen uptake rate for the control replicates was slightly lower (19 mg O2/g dry weight/h) than the prescribed rate. The accuracy of the oxygen uptake rate measurements was not affected by the slightly lower oxygen uptake rates, as shown by the low coefficient of variation for the replicates of the control oxygen uptake rates, and herewith did not invalidate the test. 

 

The 3h-EC50 of nopyl acetate for inhibition of respiration of activated sludge was 190 mg/L. The EC10, EC20 and EC80 were 24, 49 and 740 mg/L, respectively. The dose response curve of nopyl acetate did not follow the shape of a classical dose response curve. The maximum inhibition obtained with nopyl acetate was approximately 78% at highest concentrations tested. The EC values were therefore also computed without using the two highest concentrations. The 3h-EC50 calculated with the second computation was 126 mg/L. The EC10, EC20 and EC80 were 40, 59 and 268 mg/L, respectively, with both computations.

 

The 3h-EC50 of nopyl acetate for inhibition of respiration of activated sludge was 190 mg/L (with all concentrations) and 126 mg/L (without using the two highest concentrations). Therefore nopyl acetate was considered not harmful to activated sludge.

Description of key information

A respiration inhibition study was performed to assess the effect of nopyl acetate on STP microorganisms according to OECD Guideline No 209. 
The 3h-EC50 of nopyl acetate for inhibition of respiration of activated sludge is 190 mg/L.
The 3h-EC10 of nopyl acetate for inhibition of respiration of activated sludge is 40 mg/L.
Therefore nopyl acetate is considered not harmful to activated sludge.

Key value for chemical safety assessment

EC50 for microorganisms:
190 mg/L
EC10 or NOEC for microorganisms:
40 mg/L

Additional information

Key study conducted according to OECD 209 Guideline under GLP without deviation.

Nopyl acetate was tested at nominal concentrations of 19.8, 39.3, 78.6, 157.1, 314.2, 628.5, and 1257.0 mg/L and the inoculum (microbial source) was activated sludge of a predominantly domestic sewage. The validity criteria were fulfilled and the observed 3h-EC50 and 3h-EC10 values can be used for chemical safety assessment.

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