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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:
2010-04-26 to 2010-04-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
In contrast to the guideline, it was decided to use copper (II) suphate pentahydrate instead of the prescribed 3,5-dichlorophenol. The usage of dichlorophenol is unsuitable for routine lab use.
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Directly weighing, stirring for 24 hours
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loading: 100 - 180 - 320 - 580 - 1000 mg/L
- Controls: 2 control samples without test item were included, one at the beginning and one at the end of the test.
Test organisms (species):
activated sludge
Details on inoculum:
- Preparation of inoculum for exposure: Non adapted activated sludge from the sewage plant at Sarstedt (Municipal sewage treatment plant of 31157 Sarstedt, Germany)
- Pretreatment: Washed twice with autoclaved tap water and adjusted to a dry weight concentration of 4.0 g/L +/- 10 %
- Dry sludge concentration: 4.12 g/L
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
Not determined
Test temperature:
20.8 °C
pH:
6.72
Salinity:
Not determined
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flask, DIN 12380
- Aeration: Permanent
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Composition of test medium: 200 mL inoculum + 16 mL synthetic waste water (acc. to the guideline) + 284 mL autoclaved tap water + test substance concentration (directly weighing of 500 mg)



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Determination of respiration rates subsequently to an incubation period of 3 h

TEST CONCENTRATIONS
- Range finding study
Inhibitions in the Preliminary Test
Nominal Test Item
Concentration [mg/L] Inhibition*[%]
10 -1
100 6
1000 43





Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate p.a.
Key result
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
292 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: EC50: 115 mg/L
Reported statistics and error estimates:
The EC20-value of the test item was calculated by sigmoidal dose-response regression using software GraphPad Prism4, GRAPHPAD SOFTWARE, INC. The EC50 and EC80-values could not be determined since these values were outside the tested concentration range.
The EC50-value of the reference item was calculated by probit analysis using software SIGMAPLOT (Windows), SPSS CORPORATION. Probit values were taken from WEBER (1986).

From the oxygen depletion the respiration rates were calculated in mgO2/(L×h).

The inhibition of the respiration rates from the test and reference item concentrations in % was calculated in comparison with the respiration rates from the control.

Respiration Rates and Inhibition of Respiration of Test and Reference Item Concentrations

Concentration

Respiration rate

Inhibition

[mg/L]

[mgO2/(L×h)]

[%]

Test item

1000

17.6

46

580

21.2

35

320

25.6

21

180

27.6

15

100

30.4

6

Control 1

32.0

Control 2

32.8

Reference item

180

10.8

67

100

18.4

43

58

23.6

27

EC-Values with Confidence Interval of the test item

EC-values

Confidence interval

[mg/L]

P = 95 [%]

[mg/L]

EC20

292

-

EC50

> 1000

n.d.

EC80

> 1000

-

Raw Data and Respiration Rates of Test Item, Control and Reference Item

Concentration

Oxygen-concentration*

Measured distance**

Respiration rate

[mg/L]

[mgO2/L]

[mm/3 min]

[mgO2/(L×h)]

Test item

1000

9.04

44

17.6

580

7.92

53

21.2

320

6.89

64

25.6

180

6.67

69

27.6

100

6.53

76

30.4

Control 1

5.62

80

32.0

Control 2

6.95

82

32.8

Reference item

180

8.66

27

10.8

100

7.16

46

18.4

58

6.60

59

23.6

Validity criteria fulfilled:
yes
Conclusions:
The test item is not toxic at concentrations
Executive summary:

A Respiration Inhibition Test according to OECD Guideline No. 209 was performed with the test item. The test was performed under static conditions with the nominal concentrations 100 - 180 - 320 - 580 - 1000 mg/L. The respiration rates of control, reference and test item concentrations 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 inhibition ranged from 6 % to 46 %. The EC20 value is given in Table 1, due to the low inhibitions, the EC50- and EC80-values of the test item could not be calculated (Table 1).

Table 1:           EC-Values with Confidence Interval of the test item

EC-values

Confidence interval

[mg/L]

P = 95 [%]

[mg/L]

EC20

292

-

EC50

> 1000

n.d.

EC80

> 1000

-

                                                         n.d. = not determinable

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC50-value for the reference item was 115 mg/L.

Validity criteria of the test guideline were fulfilled.

Description of key information

Toxicity to microorganisms - OECD 209 – EC20: 292 mg/l, EC50: >1000 mg/l, 3h - PARAD consortium, NOACK, 2010)

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

Toxicity to microorganisms - OECD 209 – EC20: 292 mg/l,EC50: >1000 mg/l, 3h - PARAD consortium, NOACK, 2010, S. Fiebig)

A Respiration Inhibition Test according to OECD Guideline No. 209 was performed with the test item Photomer 4149 F (batch-no. S700040031) from 2010-04-26 to 2010-04-27 at Dr.U.Noack-Laboratorien, D-31157 Sarstedt.

Test system was activated sludge of the municipal treatment plant of D-31157 Sarstedt. The test was performed under static conditions with the nominal concentrations 100 - 180 - 320 - 580 - 1000 mg/L. The respiration rates of control, reference and test item concentrations 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 inhibition ranged from 6 % to 46 %. Due to the low inhibitions, the EC50- and EC80-values of the test item could not be calculated. The EC20 was determined to be EC20: 292 mg/l, whereas the EC50 was determined to be EC50: >1000 mg/l.

 

Evaluation: valid without restrictions, Klimisch-I , Key study