PIGMENT ROT 5021 A

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study according to OECD 209

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Details on test material:

- Physical state: solid
- Storage condition of test material: At room temperature at about 20 °C, away from direct sunlight

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)
A pre-test was performed to determine the solubility of the test item and to select suitable methods for the preparation of the stock solution and for dosage of the test item into the test media.
This pre-test was not performed in compliance with GLP Regulations and it is excluded from the Statement of Compliance. Raw data are archived at RCC Ltd., CH-4452 Itingen.
According to the results of this pre-test, the test item was not soluble at a concentration of 100 mg/L.
Therefore, 50.03 mg of the test item were weighed by means of an analytical balance and transferred to a test flask with 284 mL of tap water. The test item was mixed into the tap water using ultrasonic treatment over 15 minutes and intense stirring over 24 hours at room temperature in the dark to dissolve a maximum amount of the test item and/or disperse it as homogeneously as possible. No emulsiflers or solvents were used. The stirring period of 24 hours was chosen according to the results of a pre-test (without GLP) which showed that during this time no signiflcant loss of the test item was to be expected.
After the stirring period, 16 mL of synthetic wastewater and 200 mL of the activated sludge inoculum were added.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Fullinsdorf, Switzerland) treating predominanfly domestic wastewater. To eliminate possible inhibitory material, the sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.
Based on this ratio, an aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 3 g dry material per liter. During the holding period of four days prior to use, the sludge was fed daily with 50 mL synthefic wastewater per liter and was kept at room temperature under continuous aeration until use. Before use, the dry weight of the activated sludge was measured again in the inoculum used in the test. The pH of the activated sludge inoculum was adjusted from 8.3 to 7.4 with a diluted sulfuric acid solution.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test conditions

Test temperature:

20 °C at the start and 19 °C at the end of the incubation period

pH:

7.4

Dissolved oxygen:

8.6 - 9 mg O2/L

Nominal and measured concentrations:

Nominal: 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 1000-mL glass flasks
- Experimental Conditions: At the start of the test (after the stirring period of 24 h), synthetic wastewater and activated sludge inoculum were added. The inoculum had a sludge concentration of 3.6 g/L dry weight (corresponding to about 1.44 g dry material per liter test medium). The sludge was added in time intervals of 15 minutes (an arbitrary but convenient interval) firsfly to a control, secondly to the test solutions of the reference item, thirdly to the test solution of the test item, and finally to the second control.
During the incubation period of exactly 3 hours the test medium and the controls were continuously aerated with compressed air at a flow of approximately one liter per minute.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Synthetic wastewater
- Culture medium different from test medium:
- Intervals of water quality measurement: The pH-values and the dissolved oxygen concentrations were determined after addition of synthetic wastewater 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 end of the incubation period, and was continuously monitored in all test media and the controls during measurement of the respiration rate. Before the addition of activated sludge and synthetic wastewater, the appearance of the test media was recorded.

OTHER TEST CONDITIONS
- Adjustment of pH: yes, the activated sludge inoculum was adjusted from 8.3 to 7.4

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Effect concentrations 2

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Effect concentrations 3

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 100 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 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 22 mg/L (the 95% confidence limits were not calculable). The 3-hour EC50 is within the guideline-recommended range of 5-30 mg/L, confirming the suitability of the activated sludge used.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The inhibition of the degradation activity of activated sludge is not anticipated when introduced in appropriate low concentrations.