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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 June 2010 to 17 June 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline 209
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes
Analytical monitoring:
no
Details on sampling:
not relevant
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:The test substance was pipetted (eppendorf, Multipette plus) directly in the incubation vessels
- Controls: positive control: 3,5-dichlorophenol
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Unadapted secondary activated sludge from the waste water treatment plant of Nieuwgraaf in Duiven, NL (predominantly domestic wastewater)
Homogenized with a syringe prior to use
Dry weight of activated sludge = 1.7 mg/L
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
no post exposure duration period
Hardness:
not relevant
Test temperature:
20.1 to 21.1°C
pH:
6.9 to 7.6
Salinity:
not relevant
Conductivity:
not relevant
Nominal and measured concentrations:
Nominal concentrations: 0 (control), 9.2, 18.3, 36.6, 73.3, 146.6, 293.1, 586.2
Details on test conditions:
TEST SYSTEM
- Test vessel: 250-ml and 1L Erlenmeyer flasks
- Type (delete if not applicable): closed with Teflon coated lids
- Material, size, headspace, fill volume: working volumes of 5 and 200 mL; incubation in a shaking water bath (100 rpm)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized water containing no more than 0.01 mg/L Cu

SYNTEHTIC SEWAGE FEED AND STOCKS:
- Amounts of substances in 1 liter of deionized water: 16g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.3g CaCl2, 0.2 g MgSO4.7H2O, 2.8 g K2HPO4
- Quantity added: 1.6 mL of synthetic sewage feed is added in a total volume of 50 mL and 6.4 mL in a total volume of 200 mL
- The test substance was added directly into the incubation vessels

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

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Test concentrations: 0 (control), 9.2, 18.3, 36.6, 73.3, 146.6, 293.1, 586.2 mg/L
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (quality: 98,9%)
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
ca. 225 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: respiration rate
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
ca. 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: respiration rate
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
ca. 28 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: respiration rate
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
ca. 1 785 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: respiration rate
Details on results:
The calculated respiration rates and inhibition percentages of the test item are presented in Table II and the results of the graphical probit analysis are shown in Figure 2.
Results with reference substance (positive control):
The calculated respiration rates and inhibition percentages of dichlorophenol are presented in Table I and the results of the graphical probit analysis are shown in Figure 1.
Reported statistics and error estimates:
no additionnal data

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

 

Concentration

mg/L

 

Activity

mg O2/g.min

 

Inhibition%

pH

start

test

 

pH

end

test

 

Temperature

end test

(°C)

 

Control

0.282

--

6.8

7.0

20.6

Control

0.253

--

6.8

7.0

20.3

Control*

0.276

--

6.8

7.1

20.9

Control*

0.259

--

6.8

7.1

21.1

1.5*

0.235

12

6.8

6.9

21.0

3.0

0.165

38

6.9

7.2

20.4

5.9

0.135

49

6.9

7.2

20.7

11.8

0.106

60

6.9

7.2

20.4

23.6

0.053

80

6.9

7.2

20.8

47.2

0.035

87

6.9

7.1

20.1

 * = Inhibition percentages at concentrations indicated by the asterisk are calculated with the controls indicated by the asterisk.Table 2: Respiration rates of the activated sludge, inhibition percentages and pH at various concentrations of2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-2-buten-1-ol after 3 hours contact time

Concentration

mg/L

 

Activity

mg O2/g.min

 

Inhibition%

pH

end

test

 

Temperature

end test

(°C)

 

Control (1)

0.394

-- 

7.4

20.5

Control (2)

0.400

--

7.5

20.4

Control (2)

0.406

-- 

7.4

20.7

9.2 (2+3)

0.388

4

7.6

20.8

18.3 (2+3)

0.341

15

7.6

20.7

36.6 (1+2)

0.294

26

7.5

20.5

73.3 (1+2)

0.206

48

7.5

20.3

146.6 (1+2)

0.188

53

7.6

20.9

293.1 (1+2)

0.194

51

7.6

20.6

586.2 (1+2)

0.188

53

7.6

20.6

(1 2 3)= Inhibition percentages at concentrations indicated by the numbers are calculated with the controls indicated by the same numbers

Validity criteria fulfilled:
yes
Remarks:
the control respiration rates were within 15% of each other; the EC50 of the reference compound was 7 mg/L
Conclusions:
The EC50 of the test item for activated sludge after 3 hours contact time is 225 mg/L.
The EC10, EC20 and EC80 are 10, 28 and 1785 mg/L, respectively.
Executive summary:

In a 3 hour acute toxicity study, activated sludgewere exposed to the test item at nominal concentrations of 0 (control), 9.2, 18.3, 36.6, 73.3, 146.6, 293.1, 586.2 mg/L under static conditions in accordance with the OECD 209 guideline.

The EC50  and EC10values based on respiration rate inhibition were 225 and 10 mg/L, respectively.

 

This toxicity study satisfies with the guidelines requirements for activated sludge toxicity study and the test substance can be classified as non harmful for activated sludge.

Results Synopsis

Test Organism: Activated sludge (origin: wastewater treatment plant, predominantely domestic wastewater)

Test Type (Flowthrough, Static, Static Renewal): static

 

3-hr EC50: 225 mg a.i./L               

3-hr EC10: 10 mg a.i./L                  

Endpoint(s) Effected: respiration rate inhibition

Description of key information

The substance exhibits an EC50 of 225 mg/L and an EC10 of 10 mg/L for STP microorganisms.

Key value for chemical safety assessment

EC50 for microorganisms:
225 mg/L
EC10 or NOEC for microorganisms:
10 mg/L

Additional information

This toxicity study is considered as acceptable and satisfies with the guidelines requirements for activated sludge toxicity study. 2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-2 buten-1-ol is not harmful to activated sludge.