Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 hours
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant study conducted according to OECD guideline 209 (1984)

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:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Batch number - LPBS2A2005Purity - 100.2%
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):Not applicable

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
Not applicable

Test solutions

Details on test solutions:
A stock solution of the test substance (nominally 1.5 g/L) was prepared by dissolving 301.5 mg (dry weight) in 200 mL deionised water. Appropriate amounts of this stock solution were added to the different samples at the beginning of the incubation.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
A sample of activated sludge was collected on the day before the test. The container for transportation was only maximally 2/3 filled in order to maintain the contact with air. At arrival in the laboratory (less than 1 hour after sample collection), the supernatant over the sludge was decanted, the sludge was diluted with tap water and aerated by means of compressed air. 50 mL of synthetic sewage concentrate were added per litre each day. On the day of the test the sludge was allowed to sediment and the supernatant was decanted. The concentration of suspended solids was determined by filtering 3 mL of the sample through a pre-dried and pre-weighed glass microfibre filter. After drying at 110 °C and reweighing the amount of solids was calculated.On the basis of this calculation the concentration of suspended solids was adjusted to 4 g/L with tap water. This suspension was used as inoculum for the samples. The inoculum was aerated until use.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
Not applicable

Test conditions

Hardness:
Not applicable
Test temperature:
The temperature in the samples was 18 °C
pH:
The pH of the standardised activated sludge was 7.81. The pH of the incubation mixtures after the 3 hour incubation was between 8.32 and 8.46, the pH of the negative control samples was 8.38 and 8.41. The test substance did not influence the pH.
Dissolved oxygen:
Respiration rates (in mg O2.L-1.h-1 ) were determined in aliquots of the incubation mixtures. Bottles with narrow neck and a volume of about 100 mL were filled with the sample and positioned on a magnetic stirrer. Then the oxygen electrode (equipped with a magnetic stirring wheel) was inserted in a way that no air remained in the bottle and that the electrode sealed the bottle neck to avoid contact of the sample with the atmosphere. The decline of oxygen concentration was measured by recording the concentrations of oxygen at various times.The respiration rates of the two negative control samples were within 15 % of each other, the actual individual values were 93.9 and 106.1 % of their calculated mean value. The respiration rates of all samples with the test substance were in the range of 98.2 to 119.7 % of the control value.
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal exposure concentrations were 0 (control), 7.7, 19.2, 48, 120 and 300 mg/L.
Details on test conditions:
Negative controls were run before the first and after the last test substance sample. After incubation the respiration rates were determined in closed bottles using an oxygen sensitive electrode. The inhibition of respiration was calculated from the respiration rates using the mean value of the negativecontrols as 100 %.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (Aldrich, Art. No. D 7,060-0, Lot 15809KI, purity according to the certificate of analysis: 99.7 %).

Results and discussion

Effect concentrations
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 300 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Results with reference substance (positive control):
The EC50 of 3,5-dichlorophenol was in the accepted range of 5 to 30 mg/L. The actual values of the EC20, EC50 and EC80 for 3,5-dichlorophenol were: EC20 = 6.4 mg/L EC50 = 13.7 mg/L EC80 = 29.4 mg/L
Reported statistics and error estimates:
The concentrations of the test substance around 50 % inhibition (if such data exist) and the concentrations of the positive control are converted to log10. These values and the corresponding percentage of inhibition are subjected to linear regression analysis. These analyses are used to obtain the regression coefficients and the respective EC20, EC50 and EC80 values.

Any other information on results incl. tables

Respiration rates and inhibition of samples

Sample

Nominal Conc. (mg/L)

mg O2/L at start

mg O2/L at end

Respiration rate (mg O2/L/h)

% inhibition

Control 1

0

6.39

4.61

16.90

6.11

 

Succinic acid

7.7

7.41

5.23

17.68

1.79

19.3

7.29

5.04

18.05

-0.28

48.2

6.93

4.36

21.54

-19.66

120.6

6.74

3.97

21.53

-19.61

301.5

7.17

4.49

20.83

-15.73

Control 2

0

6.74

4.41

19.10

-6.11


3,5-DCP

5.1

7.21

4.99

16.82

6.56

12.3

8.21

7.27

8.25

54.19

30.3

8.57

8.07

4.14

76.98

Negative values indicate enhanced respiration compared to the control cultures.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The 3-hour EC50 based on respiration inhibition was >300 mg succinic acid/L.
Executive summary:

Activated sludge obtained from a sewage treatment plant was exposed in a 3-hour respiration inhibition test to nominal concentrations of 0 (control), 7.7, 19.3, 48.2, 120.6 and 301.5 mg succinic acid/L. The test design included duplicate samples and followed OECD 209 guidelines. The 3-hour EC50 based on respiration inhibition was > 300 mg succinic acid/L.