Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 7 sept 2009 to 23 nov 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Based on the information provided by the Sponsor, the test item is soluble at a concentration of about 2 g/L in water. Based on the results of a pre-test (without GLP), the test item was not soluble at a concentration of 1 g/L in tap water under the pre-test conditions.
Therefore, test item amounts of 5.05, 16.08, 50.22, 160.42 and 500.42 mg were weighed by means of an analytical balance and transferred to the designated test vessels with 284 mL tap water. The test item was mixed into the tap water by intense stirring for 30 minutes at room temperature to dissolve a maximum amount of the test item and/or disperse it as homogeneously as possible. No emulsifiers or solvents were used.
After a stirring period of 30 minutes, the appearance of the test media was evaluated, and in all test concentrations (except for the lowest test concentration of 10 mg/L) the test item
4-Tert-Butylpyrocatechol (4-TBC) was not completely dissolved in the test media under the test conditions. Then, 16 mL synthetic sewage feed and 200 mL activated sludge inoculum were added.
- Controls: two controls containing only tap water, synthetic sewage feed and inoculum were tested in parallel to the five concentrations of the test item under identical test conditions.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: no, aerobic activated sludge came from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf / Switzerland).
- Method of cultivation: During the holding period of three days prior to use, the sludge was fed daily with 50 mL synthetic sewage feed per liter and was kept at room temperature under continuous aeration until use. The pH of the activated sludge inoculum was 7.7.
- Preparation of inoculum for exposure: The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. An aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 3 g dry material per liter.
- Pretreatment: no
- Initial biomass concentration: 3 g dry material per liter

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
none

Test conditions

Hardness:
no data
Test temperature:
The temperature in the test media, measured in one control, was 20°C at the start and at the end of the incubation period
pH:
At the beginning and at the end of the 3-hour incubation, the pH value was in the range of 7.6 to 8.4.
Dissolved oxygen:
At the beginning and at the end of the 3-hour incubation, the oxygen concentration value was in the range of 8.0 to 8.9 mg O2/L.
Salinity:
Not applicable
Nominal and measured concentrations:
nominal concentrations: 10, 32, 100, 320 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: open
- Material, size, headspace, fill volume: The test was performed in 2000 mL glass beakers.
- Aeration: During the incubation period of 3 hours all test media and the controls were continuously aerated by intense stirring on magnetic stirrers to avoid possible foaming and/or stripping of the test item.
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Biomass loading rate: sludge concentration = 1.6 g dry material / L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water
- Total organic carbon, Particulate matter, Metals, Pesticides, Chlorine, Alkalinity, Ca/mg ratio, Conductivity: no data
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH values and dissolved oxygen concentrations were determined after the addition of synthetic sewage feed and activated sludge in all test media and the controls at the start and at the end of the 3-hour incubation period. The water temperature was measured in one control at the start and at the end of the incubation period. Before the addition of activated sludge and synthetic sewage feed, the appearance of the test media was recorded.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: no data
- Light intensity: no data


EFFECT PARAMETERS MEASURED: For measurement of the respiration rate a well-mixed sample of each test medium was poured into a BOD-flask after three hours incubation time, and was not further aerated. Then the dissolved oxygen concentration was measured with an oxygen electrode (WTW TriOxmatic® 300 and an oxygen meter WTW Oxi 539, Wissenschaftlich-Technische Werkstaetten WTW, Weilheim/Germany), and was continuously recorded. During measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen consumption rate (in mg O2 L-1 minute-1) was determined from the linear part of the respiration curve in the range 6.5–2.5 mg O2/L. In case of very rapid oxygen consumption, the range used was below the limits indicated above but always within the linear part of the respiration curve. In case of low oxygen consumption the rate was determined over a period of at least ten minutes.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.1 / 3.2
- Range finding study: none
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol tested at 5, 16 and 50 mg/L

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
0.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 0.0 - 3.0 mg/L
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
16 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 3.5 - 32 mg/L
Details on results:
At the lowest test concentration of 10 mg/L, the test item 4-Tert-Butylpyrocatechol (4-TBC) had a significant inhibitory effect of 41% on the respiration rate of activated sludge after the incubation period of three hours. At the next higher test item concentration of 32 mg/L, the inhibitory effect increased to 61%. At the following test item concentrations of 100, 320 and 1000 mg/L, a plateau was reached with inhibitory effects of 82, 89 and 87%, respectively.

The oxygen consumption rates of the two controls (run at the start and at the end of the test) differed by 9% (guideline-recommendation: the two control respiration rates should be within 15% of each other).
Results with reference substance (positive control):
The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 11 mg/L with 95% confidence limits from 0.9 to 55 mg/L. The 3-hour EC50 is within the guideline-recommended range of 5–30 mg/L, confirming suitability of the activated sludge used.

Any other information on results incl. tables

Table 1: Influence of 4-tert-butylpyrocatechol (4 -TBC) and 3,5-dichlorophenol on the oxygen consumption of activated sludge

Nominal concentration of test chemical

Oxygen consumption rate

Inhibition

(mg/L)

(mg O2L-1min-1)

(%)

Control

 

 

0

0.891

 

0

0.972

 

Mean 

0.932

 

Deviation (%)

9.0

 

Test item: 4-TBC

 

 

10

0.552

40.7

32

0.366

60.7

100

0.172

81.5

320

0.106

88.6

1000

0.126

86.5

Reference item: 3,5-dichlorophenol

 

 

5

0.718

22.9

16

0.313

66.4

50

0.084

91.0

% inhibition: increased oxygen consumption rate relative to control

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
The EC50 of the reference item is in the acceptance range and the oxygen consumption rates of the 2 controls differed by less than 15%.
Conclusions:
The concentration of the test item 4-tert-butylpyrocatechol (4-TBC) leading to an inhibitory effect of 50% on the respiration rate of activated sludge after the incubation period of three hours, compared with the mean respiration rate of two controls, is calculated to be 16 mg/L (with 95% confidence limits of 3.5 – 32 mg/L).
Executive summary:

In a study (Harlan, 2009), the inhibitory effect of the substance 4-tert-butylpyrocatechol on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3-hour respiration inhibition test according to OECD guideline for Testing of Chemicals, n°209 (1984).

The nominal concentrations tested were 10, 32, 100, 320 and 1000 mg/L.

In addition, two controls and three different concentrations of the reference item 3,5-dichlorophenol (5, 16 and 50 mg/L) were tested in parallel. The results of these reference treatments (EC50 = 11 mg/L within the guideline-recommended range of 5 - 30 mg/L) confirmed suitability of the activated sludge.

EC50 = 16 mg/L (95% C.L.: 3.5 - 32 mg/L)

NOEC = 0.6 mg/L (95% C.L.: 0.0 - 3.0 mg/L)

Based on the results of this study, 4 -tert-butylpyrocatechol would be considered as harmful for microorganisms.

This study is classified as acceptable and satisfies the guideline requirements for toxicity study to activated sludge in a respiration inhibition test.