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Ecotoxicological information

Toxicity to microorganisms

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Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
2010
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): 2,4,6-trimethylphenol (TMP)
- Physical state: White crystalline powder
- Storage condition of test material: Room temperature
- Analytical purity: 99.9 % constituent TMP in the product
- Lot/batch No.: 6829K

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
yes
Details on test solutions:
PREPARATION OF THE BOTTLES
The test was performed at 20°C ± 2°C and all diluitions were done using deionized water.
24 bottles were prepared as follows:
-3 different concentrations for the test item at 3 replica/concentration (9 bottles)
-3 bottles for the blank control
-3 different concentration of the test reference (3,5 dichlorophenol) at 3 replica/concentrations (9 bottles)
-3 bottles for abiotic control tested at the highest test item concentration
PREPARATION OF TEST ITEM
A stock solution of the test item was prepared at concentration of 10 g/l in deionized water under stirring: this concentration corresponds to 10 g/l of active ingredient, concentration required by the guideline. Bottles were prepared with deionized water, synthetic sewage feed and 3 different concentration of the test item for the range finding test (1 g/l, 100 mg/l and 10 mg/l).
The pH of the stock solution was checked.
The mud was added before starting the test.
PREPARATION OF THE REFERENCE SUBSTANCE
A stock solution of the reference substance 3,5 dichlorophenol was prepared at concentration of 1g/l in deionized water. 3 different concentrations
(50 mg/l, 10 mg/l and 1 mg/l) were prepared in the same way of the test items.
The pH of the stock solution was checked.
The mud was added before starting the test.
BLANK CONTROLS PREPARATION
As blank was used water containing an equal volume of activated sludge and synthetic medium wthout reference substance and test item.
ABIOTIC CONTROL
Abiotico control was prepared with the highest concentration amount of the test item, with the same amount of the synthetic sewage feed without the inoculum.
Abiotic control was used to measure abiotic oxygen consumption.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
For the preparation of the inocula a sample of aerobic mud was selected by the mixed treatment plant of urban (about 66%) and industrial (about 34%)liquid sewage.
Activated sludge was collected and preferably used on day of collection, if is not possible the mud was used no later than two days feeding it with medium.
If in the sludge there was present coarse particles they were removed by setting for a period of 10 minutes. Then the mud samples, before theri use, were washed and analyzed to prepare the inoculum. The sludge was continuosly aerated at the test temperature. The suspended solids in the mud solution was 3 g/l to have in the final mixture a concentration of 1.5 g/l.

Study design

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

Test conditions

Test temperature:
20°C±2°C
Nominal and measured concentrations:
Nominal concentrations of test item for range finding test: 1 g/l, 100 mg/l and 10 mg/l.
Details on test conditions:
Conditions of assay execution
The incubation starts with the initial contact of the activated sludges with the other constituent of the final mixture. Each incubation was started in turn at intervals of about 10 minutes.
Then the vessels were aerated to keep the dissolved oxygen concentration above 60-70% saturation and to mantain the sludge flocs in suspension
The bottles prepared were incubated at 20°C±2°C for three hours.
After the exposure period each sample was transferred from the aeration vessel to a bottle fitted with a magnetic stirrer. An oxygen probe was inserted into the neck of the bottle to measure the concentration of dissolved oxygen.
The concentration of dissolved oxygen was recorded in turn continuously for a periodo of 10 minutes or until concentration falls below 2 mg/l.
The probel was washed with deionized water between measurements.
Reference substance (positive control):
yes
Remarks:
3,5 Dichlorophenol

Results and discussion

Effect concentrations
Key result
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
Reported statistics and error estimates:
Probit analysis.

Any other information on results incl. tables

EXPRESSION OF RESULTS

Calculate the oxygen consumption rate (R) in mg/l h according to equation below:

R = (Q1 -Q2)/ ∆t x 60

where:

Q1= is the oxygen concentration at the beginning of the linear phase (mg/l)

Q2= is the oxygen concentration at the end of the linear phase (mg/l)

∆t= is the time interval between these two measurements (min)

The specific respiration rate (Rs) is expressed as the amount of oxygen consumed per g dry weight of sludges per hour (mg/gh) according to the equations below:

Rs=R/SS

SS= is the concentration of suspended solids in the mixture (g/l)

Different indices of R which may be combinated are:

S=specific rate

T=total respiration rate

A=rate due to abiotic processes

B=rate based on blank assays control (mean)

To calculate the percentage inhibition It (%) of total consumption at each concentration of the test item will be used the following equation:

It = [1 -(Rt-Rta)/Rtb]x100%

Rta is equal to 0 if there will be not oxygen uptake in the preliminary test in the abiotic control or if the test item don't have strong reducing properties.

VALIDITY CRITERIA

- the blank controls oxygen uptake rate should not be less than 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour.

- the coefficient of variation of oxygen uptake rate in control replicates should not be more than 30% at the end of definitive test.

- EC50 of 3,5 Dichlorophenol must be lie in the range 2 mg/l and 25 mg/l for total respiration.

INTERPRETATION OF RESULTS

EC50 values including their associated 95% confidence limit were calculated by EPA PROBIT program version 1.5.

RESULTS

Quality criteria of the test were satisfied:

-the blank controls oxygen uptake rate: 30.28 mg oxygen per one gram of activated sludge in an hour.

-the coefficient of variation of oxygen uptake rate in control replicates: 25%

-EC50 of the reference substance 3,5 -Dichlorophenol: 14.3 mg/l for total respiration.

-pH synthetic sewage feed: 7.57

-pH reference substance stock solution: 7.28

-oxygen consumption rate for abiotic control: 0,50 mg/l h

Mean percent inhibition total respiration rate of the test item 2,4,6 -trimethylphenol is detailed in the following table:

 Concentration  % of inhibition total respiration rate of the test item

2,4,6 -trimethylphenol

10 mg/l 19.88 
100 mg/l 30.25
1000 mg/l 49.34 

 

Mean percent inhibition total respiration rate of the reference substance is detailed in the following table:

 Concentration  % of inhibition total respiration rate of the reference substance

2,4,6 -trimethylphenol

1 mg/l 9.97 
10 mg/l 45.18
50 mg/l 71.20 

The absence of inhibition of oxygen consumption by the Test item, tested in the range finding test, demonstrate that a definitive test is unnecessary.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The obtained results, in compliance with assay validity criteria, interpreted in accordance to OECD 209:2010, show that the EC50 value of the test item 2,4,6 trimethylphenol after 3 hours results higher than 100 mg/l.
Executive summary:

The aim of this study is to determine the effects of the test item 2,4,6 trimethylphenol on microorganism from activated sludge by measuring their respiration rate, accordin to OECD 209:2010.

The respiration rates of activated sludge both in control, reference and treatment groups have been measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours at 20°C.

The obtained results, in compliance with assay validity criteria, interpreted in accordance to OECD 209:2010, show that the EC50 value of the test item 2,4,6 trimethylphenol after 3 hours results higher than 100 mg/l.