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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-01-27 to 2016-01-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The test item (10 and 32 mg/L) was weighed out onto object slides and transferred with these into the Erlenmeyer flasks. The other concentrations were weighed out directly in the Erlenmeyer flasks. 200 mL demineralised water were added and stirred overnight.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Activated sludge of Municipal sewage treatment plant of 31137 Hildesheim, Germany
Pretreatment : The sludge was washed twice with chlorine free tap water and adjusted to a dry sludge concentration of 3.0 g/L ± 10 %. The sludge was used within 24 h after sampling.
Date of receipt: 2016-01-27
Dry sludge concentration 2.74 g/L, corresponding to 1.37 g/L in the test vessel
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20.5 °C
pH:
Activated sludge: 7.26
Synthetic waste water: 7.32
Nominal and measured concentrations:
Nomial: 10 - 32 - 100 - 320 - 1000 mg/L
Details on test conditions:
TEST SYSTEM
Replicates: 5
Vehicle: None
Pretreatment: Stirring overnight
Control: Test medium without test or reference item
Replicates: (conrol): Triplicates at the beginning of the test and triplicates at the end of the test
Test vessels : 500 mL Erlenmeyer flasks, DIN 12380
Nutrient solution: Synthetic waste water according to OECD Guideline 209
Volume of the test medium : 500 mL
Spacing factor: 3.2
Aeration: Permanent, to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flakes in suspension.

TEST PARAMETER
After 3 h incubation, the oxygen concentration of each control, reference item and test item replicate was determined by means of oximeter immediately after transferring the solution to the measuring cell and the oxygen depletion was recorded for at least 3 minutes on a flatbed recorder.
Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate p.a.
Key result
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 heterotrophic respiration
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
676 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Details on results:
From the oxygen depletion the oxygen uptake rates were calculated in mg O2/L-h, additionally the specific respiration rates based on the dry sludge concentration were calculated in mg O2/g-h. The inhibition of the oxygen uptake rates of the test and reference item concentrations in % was calculated in comparison to the oxygen uptake rates of the control (see below table). The mean inhibion of respiration for the test item replicates ranged from -10 % to 19 %.
Results with reference substance (positive control):
In the reference test the EC50-value for copper (II) sulphate pentahydrate was determined by probit analysis with:
EC50 = 115 mg/L
95% Confidence Interval = 111 – 119 mg/L This is in the recommended range of validity of 53 - 155 mg/L.

                  Oxygen Uptake Rates and Inhibition of the Respiration of the
Control and Test ItemConcentrations

 

Test
Concentration
[mg/L]

Repl.

Oxygen Uptake Rate R
[mg O2/L
×h]

Inhibition

[%]

Mean Inhibition
[%]

 

Control

1

25.6

2

25.6

3

27.6

4

27.6

5

28.8

6

29.6

Test Item

10

1

26.0

5

2

2

26.8

3

3

26.8

3

4

28.0

-2

5

27.2

1

32

1

28.0

-2

-3

2

28.0

-2

3

28.4

-3

4

28.4

-3

5

28.8

-5

100

1

28.4

-3

-6

2

28.4

-3

3

28.4

-3

4

30.8

-12

5

30.0

-9

320

1

30.0

-9

-10

2

30.4

-11

3

29.6

-8

4

30.4

-11

5

30.8

-12

1000

1

21.2

23

19

2

24.0

13

3

22.4

19

4

22.0

20

5

22.4

19

Repl. = Replicate

            Coefficient of variation of oxygen uptake rates of the control: 5.9%

Suspended solids in the test vessel: 1.37 g/L

 

Remark: Up to a test concentration of 320 mg/L test item, there was an increasing stimulation of the respiration of the activated sludge. Therefore, 320 mg/L is regarded as NOEC.

EC-Values with Confidence Interval

 

EC-values

[mg/L]

Confidence interval
P = 95 [%]
[mg/L]

NOEC*

320

̶

EC10

676

n. d.

EC20

> 1000

̶̶̶̶̶̶

EC50

> 1000

̶

EC80

> 1000

̶

*) No statistically significant inhibition, ANOVA, Dunnett`s Method

               n.d.: The confidence interval could not be determined.

Validity criteria fulfilled:
yes
Conclusions:
The test item is not toxic to activated sludge of a municipal sewage treatment plant up to the concentration of 320 mg/L. The EC10 is 676 mg/L. The EC20, EC50 and EC80 of the test item are >1000 mg/L
Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item from2016-01-27to 2016-01-28, with the definitive exposure phase on 2016-01-28 at the test facility. The test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the test item concentrations10 - 32 - 100 - 320 - 1000 mg/L. Ther espiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The NOECis320mg/L.The test item is not toxic to activated sludge of a municipal sewage treatment plant up to the concentration of 320 mg/L. The EC10is 676 mg/L. The EC20, EC50and EC80of the test item are >1000 mg/L..

Description of key information

A Respiration Inhibition Test with activated sludgeaccording to OECD Guideline No. 209 was carried out with the test itemfrom2016-01-27to 2016-01-28, with the definitive exposure phase on 2016-01-28 at the test facility.The test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the test item concentrations10 - 32 - 100 - 320 - 1000 mg/L. Ther espiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and theinhibitory effects of the test and reference item were determined in comparison to the control respiration rates.The NOECis320mg/L.The test item is not toxic to activated sludge of a municipal sewage treatment plant up to the concentration of 320 mg/L. The EC10is 676 mg/L. The EC20, EC50and EC80of the test item are >1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
676 mg/L

Additional information