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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
Internal standard method B-21
Principles of method if other than guideline:
With this test, the inhibitory concentration (IC50) of test substances is determined that leads to an inhibition of oxygen consumption of aerobic bacteria after 3 hours under defined environmental conditions.
The method was designed according to ETAD method no. 103: “ A screening test for the assessment of the possible inhibitory effect of a chemical substance on aerobic waste-water bacteria” and the OECD method 209: “Activated Sludge, Respiration Inhibition Test”.
With every test series a reference substance has to be tested in parallel, in addition a growth control without test substance has to be performed at the beginning and at the end of each test series.
GLP compliance:
no
Specific details on test material used for the study:
Product Name: UVITEX K/ FAT 65004/E
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
For each test substance, the following concentrations are tested: 1.0; 3.2; 10; 32; and 100 mg/L. In addition with each assay, the reference substance (10; 32; and 100 mg/L) and a growth control (without test and reference substance) has to be tested.
Test organisms (species):
activated sludge
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
not reported
Test temperature:
not reported
pH:
not reported
Dissolved oxygen:
not reported
Salinity:
not reported
Conductivity:
not reported
Nominal and measured concentrations:
not reported
Details on test conditions:
see below
Key result
Duration:
3 h
Dose descriptor:
IC50
Effect conc.:
> 400 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration

Method B-21, Bacterial toxicity

Determination of the inhibitory effect of organic substances on aerobe microorganisms

Introduction

At Ciba Geigy AG acute testing on microorganisms in sewage sludge was performed according to published methods, which were adapted and standardized for the specific needs of the company, well before Test Guidelines for these methods were established by the OECD beginning 1981.

The results of these studies were reported until the early 1980s in summary reports which do not present details of the methods used, but focus on a summary presentation of results.

This statement is intended to describe the methods used and give some guidance on the interpretation of the results described in the above mentioned summary reports.

Principle of the test

Objective:

This method is designed to evaluate the acute toxicity to aerobic bacteria in sewage sludge.

Principle:

With this test, the inhibitory concentration (IC50) of test substances is determined that leads to an inhibition of oxygen consumption of aerobic bacteria after 3 hours under defined environmental conditions.

The method was designed according to ETAD method no. 103: “ A screening test for the assessment of the possible inhibitory effect of a chemical substance on aerobic waste-water bacteria” and the OECD method 209: “Activated Sludge, Respiration Inhibition Test”.

With every test series a reference substance has to be tested in parallel, in addition a growth control without test substance has to be performed at the beginning and at the end of each test series. 

Materials and conditions:

Lab conditions:

The experiment is performed at room temperature (20 ± 2 °C).

 

Materials:

-       BSB5Testing tubes (100 ml)

-       Thermometer

-       pH-meter

-       O2-electrode with graphic plotter

-       Flow-meter

-       Magnetic stirrer

-       Analytical balance

-       Additional standard lab equipment

 


 

Chemicals:

-       Peptone (Caseine

-       Meat extract

-       Urea

-       NaCl

-       CaCl x 2 H2O

-       MgSO4x 7 H2O

-       K2HPO4

-       3,5-dichlorophenol

Water:

-       Tap water / demineralized water

 

Microorganisms:

The required activated sludge might be taken from an active Husman apparatus (aerated compartment).

Controls:

-       Reference substance: 3,5-dichlorophenol (positive control)

-       Growth control: Each experiment contains two growth controls, done at the beginning and at the end of the experiment. The growth control consists of a broth solution without test substance (blank control).

 

Test design:

Routine screening:

For each test substance, the following concentrations are tested: 10; 32; and 100 mg/L. In addition with each assay, the reference substance (10; 32; and 100 mg/L) and a growth control (without test and reference substance) has to be tested.

 

Remark:

If the determined IC50 (3h) below 10 mg/L, the test substance has to be diluted further to 10; 3.2; and 1 mg/L.

 

Extended testing design:

For each test substance, the following concentrations are tested: 1.0; 3.2; 10; 32; and 100 mg/L. In addition with each assay, the reference substance (10; 32; and 100 mg/L) and a growth control (without test and reference substance) has to be tested.

 

Remark:

If the determined IC50 (3h) below 1.0 mg/L,the test substance has to be diluted further to 1.0; 0.32; 0.1 and 0.032 mg/L.

 


 

Media:

Modified OECD Broth:

The Nutrient Broth (Stock 100-fold) consists of:

16.0 g peptone from casein (Merck No. 7213)

11.0 g meet extraction (Liebig)

3.0 g urea (sterilized by filtration and added after thermal sterilization process)

0.7 g NaCl

0.4 g CaCl2x 2 H2O

0.2 g MgSO4x 7 H2O

2.8 g K2HPO4

500 ml of Tap water

Remark:

The modification is the addition of K2HPO4. The final preparationof the broth is prepared from two stock solutions:

Stock solution A:

16.0 g peptone from casein (Merck No. 7213)

11.0 g meet extraction (Liebig)

Filled up with 500 ml of Tap water and heated up until boiling occurs

 

Stock solution B:

3.0 g urea (sterilized by filtration and added after thermal sterilization process)

0.7 g NaCl

0.4 g CaCl2x 2 H2O

0.2 g MgSO4x 7 H2O

2.8 g K2HPO4

500 ml of Tap water

To be dissolved at room temperature

 

The final broth is prepared mixing both stock solutions together.

 

Test substance:

Stock solution:

500 g of the test substance are dissolved in 1 liter of demineralized water. Further dilutions are prepared as follows:

20 ml of stock solution plus 100 ml of water ≈ 100 mg/L = ppm

6.4 ml of stock solution plus 100 ml of water ≈ 32 mg/L = ppm

2.0 ml of stock solution plus 100 ml of water ≈ 10 mg/L = ppm

0.64 ml of stock solution plus 100 ml of water ≈ 3.2 mg/L = ppm

0.2 ml of stock solution plus 100 ml of water ≈ 1 mg/L = ppm

 

Remark:

If the stock solution can’t be prepared because of solubility of the test substance, a stock solution of a lower concentration (e.g. 100 mg/L) has to be prepared.

 

Reference substance:

Stock solution:

500 mg 3,5-dichlorophenol is dissolved in 10 ml of 1 N NaOH and subsequently diluted with 30 ml of demineralized water. 1N H2SO4(about 8 ml) is added until the flocculation is dissolved completely. After that the solution is filled up with water to a final volume of 1 liter. The pH-value should be in the range of pH 6.5 – 8.

 

Further dilutions are equivalent to the dilutions for the test substance:

20 ml of stock solution plus 100 ml of water ≈ 100 mg/L = ppm

6.4 ml of stock solution plus 100 ml of water ≈ 32 mg/L = ppm

2.0 ml of stock solution plus 100 ml of water ≈ 10 mg/L = ppm

 

Preparation of the inoculum:

Inoculation suspension:

In case of active sewage sludge testing, sludge is directly used from an Husmann apparatus and further aerated immediately. The sludge is washed 3 times with tap water and subsequently the sludge is allowed to sediment. The supernatant is discarded. A sample for the determination of the dry weight is taken (compare methods (A-25, A-26). Subsequently the sludge content is adjusted to 5 g/L

 

e.g. 8 g/L dry weight; 500 ml sludge: (8 g/L/5 g/L)*500 ml = 800 ml

 

The sludge is aliquoted in 500 ml plastic bottles and frozen. Frozen sludge has to be used within 3 months.

 

Prior to use the sludge is thawed overnight. Subsequently for each of the 500 ml batches, 25 ml of the 100-fold concentrated stock solution of broth is added. After that the sludge is aerated at 20 ± 2 °C overnight. After that the test vials can be inoculated with 40 ml of sludge.

Testing conditions:

One test unit consists of one BSB5-bottle connected to an aeration device. Aeration is mediated via a Pasteur-pipette closed with a wad and the aeration rate is regulated to 1 liter per minute. 

Procedure:

The operation of the testing unit has to be started and oxygen amount is measured (duration about 10 minutes).

At the beginning of the test (t=0) control vials (K1) have to be filled with 5 ml of OECD broth solution and 40 ml of bacteria suspension (concentration: 2 g/l dry weight). Subsequently the volume is filled up to 100 ml with tap water and the system is aerated for 180 minutes.

At the end of the 180 min period, aeration is stopped and the vial is placed on a magnetic stirrer. The O2-electrode is placed in the vial and the decrease of oxygen is measured over a period of 3-6 minutes.

 

10 minutes after the control vial is prepared, the first test vial is prepared with 5 ml of OECD broth solution, 40 ml of bacteria suspension (concentration: 2 g/l dry weight) and 0.2 ml stock solution (1 mg/L). Subsequently the volume is filled up to 100 ml with tap water and the system is aerated for 180 minutes.

At the end of the 180 min period, aeration is stopped and the vial is placed on a magnetic stirrer. The O2-electrode is placed in the vial and the decrease of oxygen is measured over a period of 3-6 minutes.

 

The procedure is repeated until all vials are incubated. As soon as all five concentrations of the test substance are measured the concentrations of the reference substance are measured. The last sample measured is again a blank control without test and reference substance (K2).

Interpretation of results:

The inhibitory effect is given as the difference between the values of the oxygen consumption with the test substance and mean value of the controls K1 and K2.

 

Inhibition [%] =

 

O2consumption [mg O2/L* min]

TS: Test substance

K1: Blanc control 1

K2: Blanc control 2

 

The oxygen consumption is determined based on the recorded plot, resulting from oxygen consumption of the microorganisms. A flat curve shows a strong oxygen consumption while a steep curve shows a low oxygen consumption or respiration inhibition.

 

Remark:

The difference between initial oxygen content and final oxygen content is calculated and may not be greater than 15 %, otherwise the test is not valid. The difference has to be calculated for each individual test concentration.

 

Validity:

The test is valid if

-       The IC50(3h) of the reference substance (3,5-dichlorophenol) is between 10 and 25 mg/L

-       The difference of the oxygen consumption between blanc control K1 and K2 is smaller than 15 %.


 

General remarks

From the above it is obvious that the results in the studies performed at Ciba Geigy AG before test guidelines for the respective endpoint were established by a standardized procedure comparable to those presently in use and thus are valid for characterizing the (eco)toxicity of the substances tested.

The form of reporting the results, however, does not meet today’s requirements. But in some cases it may be possible that the details of the procedures, information additionally needed for an evaluation or open questions can be clarified by consulting the (hand written) raw data in German. Some information can only be obtained through the interpretation of raw data by senior experts. In cases when the results of such studies are needed for an assessment, all necessary information should be integrated into a (robust) study summary by Huntsman toxicologists. Taking the study results reported and this general method description into account, such studies might be rated with “2” (Reliable with restrictions) based on the scoring system published by Klimisch et al. in 1997.

References:

Klimisch HJ, Andreae M, Tillmann U. A systematic approach for evaluating the quality of experimental toxicological and ecotoxicological data.Regul. Toxicol. Pharmacol. 1997 Feb;25(1):1-5.

 

 

Langweid, 27.05.2015

Huntsman Textile Effects (Germany) GmbH

 

 

Dr. rer. nat. M. Kresse, ERT

 

 

 

Annex:

Original method description used by Ciba Geigy AG (in German).

Validity criteria fulfilled:
yes
Conclusions:
IC50 Value: > 400 mg/l
Executive summary:

A study was performed following internal standard procedure B-21, Bacterial toxicity which is similar to OECD method 209: “Activated Sludge, Respiration Inhibition Test".

IC50 (3h) was determined to be > 400 mg/l.

Description of key information

IC50 (3h) was determined to be >400 mg/l.

Key value for chemical safety assessment

EC50 for microorganisms:
400 mg/L

Additional information

Both the available studies were performed following internal standard procedure B-21, Bacterial toxicity which is similar toOECD method 209: “Activated Sludge, Respiration Inhibition Test".

IC50 (3h) was determined to be >400 mg/l.