Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-01-07
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Principles of method if other than guideline:
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Phosphorpentoxid
- Molecular formula: P4O10
- Molecular weight: 283.9 g/mol
- Physical state: Solid, white powder
- Analytical purity: 99.7 % (m/m)
- Purity test date: 2009-09-23
- Lot/batch No.: DEH2 018965
- Expiration date of the lot/batch: 2011-09-08
- Storage condition of test material: Stored in a desiccator at room temperature, protected from light.

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Agitation
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loading:100 - 180 - 320 - 580 - 1000 mg/L without adjustment of pH
+ 320 - 580 - 1000 mg/L adjusted to pH 7
- Controls: 2 control samples without test item were included, one at the beginning and one at the end of the test.

Test organisms

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

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h

Test conditions

Hardness:
Not determined
Test temperature:
19.9 °C
pH:
6.74
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 out in each test vessel)



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 Tests
Nominal Test Item
Concentration [mg/L] Inhibition
[%]
10 (pH: 7.27) -5
100 (pH: 5.35  7.00) -2
1000 (pH: 2.16  7.08) 42
10 -2
100 5
1000 98






Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate p.a.

Results and discussion

Effect concentrations
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 other: mg/L (with adjustment of pH); 215 (209-220) mg/L without adjustment
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: 101 mg/L
Reported statistics and error estimates:
The EC-values of the test item (without adjustment of pH) were calculated by sigmoidal dose-response regression using software GraphPad Prism4, GRAPHPAD SOFTWARE, INC. Calculation of the confidence intervals for the EC50-value was carried out using standard procedures.

The EC-values of the test item (with adjustment to pH 7) and the EC50-value of the reference item were calculated by probit analysis using software SIGMAPLOT (Windows), SPSS CORPORATION. Probit values were taken from WEBER (1986).

Any other information on results incl. tables

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

0.0

100

580

0.0

100

320

2.8

91

180

21.2

31

100

26.0

15

1000 #

17.2

44

580 #

24.4

20

320 #

27.6

10

Control 1

29.2

Control 2

32.0

Reference item

180

8.4

73

100

15.2

50

58

22.4

27

The EC20-, EC50- and EC80-values were calculated by sigmoidal dose-response regression for the test item concentrations without adjustment of pH and by probit analysis for the test item concentrations adjusted to pH 7.

Table 6:    EC-Values with Confidence Interval of Phosphorpentoxid

Values without adjustment of pH

Values with adjustment of pH

EC-values

Confidence interval

EC-values

Confidence interval

[mg/L]

P = 95 [%]

[mg/L]

[mg/L]

P = 95 [%]

[mg/L]

EC20

147

-

524

-

EC50

215

209 - 220

>1000

>1000

EC80

275

-

>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

8.68

0

0.0

580

8.73

0

0.0

320

8.47

7

2.8

180

7.29

53

21.2

100

6.57

65

26.0

1000 #

7.12

43

17.2

580 #

7.54

61

24.4

320 #

6.32

69

27.6

Control 1

7.03

73

29.2

Control 2

7.52

80

32.0

Reference item

180

8.13

21

8.4

100

7.85

38

15.2

58

7.58

56

22.4

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Executive summary:

A Respiration Inhibition Test according to OECD Guideline No. 209 was performed with the test item Phosphorpentoxid (batch-no. DEH2 018965) on 2010-01-07 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 without adjustment of pH and 320 - 580 - 1000 mg/L adjusted to pH 7 to differentiate between pH effects and test item effects. 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 of the test item ranged from 15 % to 100 %. The neutralised test item concentrations showed inhibitions from 10 % to 44 %. The EC20-, EC50- and EC80-values were calculated by sigmoidal dose-response regression for the test item concentrations without adjustment of pH and by probit analysis for the test item concentrations adjusted to pH 7.

Table 1:           EC-Values with Confidence Interval of Phosphorpentoxid

Values without adjustment of pH

Values with adjustment of pH

EC-values

Confidence interval

EC-values

Confidence interval

[mg/L]

P = 95 [%]

[mg/L]

[mg/L]

P = 95 [%]

[mg/L]

EC20

147

-

524

-

EC50

215

209 - 220

>1000

>1000

EC80

275

-

>1000

-

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 101 mg/L.