Humic acids and fulvic acids extracted from leonardite, reaction product with formaldehyde, potassium hydrogen sulfite, sodium hydrogen sulfite and potassium hydroxide

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

from October 22, 2007 to November 14, 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was carried out in accordance with internationally valid GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Humic acids, potassium salts

EC Number:

271-030-1

EC Name:

Humic acids, potassium salts

Cas Number:

68514-28-3

IUPAC Name:

68514-28-3

Details on test material:

- Name of test material (as cited in study report): Humic acids, potassium salts
- Molecular formula: not known - UVCB substance
- Molecular weight: not known - UVCB substance
- Substance type: technical product
- Physical state: solid
- Lot/batch No.: 16. 5. 2007/R
- Expiration date of the lot/batch: 05/2022
- Stability under test conditions: stable
- Storage condition of test material: dry conditions

Sampling and analysis

Analytical monitoring:

not required

Test solutions

Details on test solutions:

The test substance is soluble in water in concentrations needed for the test.
The stock solution was prepared by dissolution of 2.5 g of test substance in 500 mL deionised water on the day of testing.
In the preliminary test the concentrations were in the range of 46 - 1000 mg.L-1 with a geometric progression factor of 2.2.
In the main test the concentrations were in the range of 100 - 1000 mg.L-1 with a geometric progression factor of 1.8.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

The activated sludge was collected two days before the day of testing.
After the sample collection the sludge was washed with potable water for 0.5 hour and subsequently decanted for 0.5 hour. This procedure was repeated three times in total.
Further the sludge was modified by addition of 50 mL of cultivation medium per 2 L of diluted sludge suspension at permanent aeration till the day of test.
The dry weight was determined from 10 mL of sludge suspension after 0.5-hour sedimentation.
Before the test the sludge was suspended in water up to concentration about 4000 mg of sludge dry weight per litre. The pH adjustment to 6.0 was carried out. In that way modified sludge suspension was aerated until the use.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test conditions

Test temperature:

20 +/- 2°C

pH:

The pH value during the main test was in the range of 7.1 to 7.9.

Nominal and measured concentrations:

The preliminary test was performed using 5 concentrations in range 46 - 1000 mg.L-1 with a geometric factor of 2.2 to determine the concentrations to be used in the main test.
In the main test, 5 concentrations of the test substance in geometric progression with factor of 1.8 (100, 180, 320, 560, 1000 mg.L-1) were used together with two control experiments (without the test substance), one at the beginning and one at the end of the test.

Details on test conditions:

Temperature: 20 +/- 2°C
Lighting: daily
Stirring and aeration: with filtered pressured air, airflow rate 0.8 L/min.
The pH value of reaction mixtures during the main test: approx. 7 – 8.

The deionised water was used for the preparation of solutions. Analytical grade reagents were used.
The stock solution of cultivation medium (synthetic nutrient medium):
Component Mass
peptone 16 g
meat extract 11 g
urea (NH2)2CO 3 g
sodium chloride NaCl 0.7 g
calcium chloride dihydrate 0.4 g
magnesium sulphate heptahydrate MgSO4.7H2O 0.2 g
anhydrous dipotassium hydrogen phosphate K2HPO4 2.8 g
water up to volume of 1000 mL
The pH value of this solution was 7.0-8.0.
The cultivation medium for sludge adaptation was always prepared fresh on the day of the use.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

Under the current experimental conditions, all tested concentrations in the main test provided no significant inhibition of respiration rate. Therefore it was not possible to calculate the EC50 value.

Results with reference substance (positive control):

- Results with reference substance valid? yes
The 3h-EC50 value of reference substance, 3,5-dichlorophenol: EC50 = 11.4 mg.L-1 (95 % confidence interval: 10.6 – 12.3 mg.L-1)

Reported statistics and error estimates:

Calculation of EC values
The values of EC50, EC20 and EC80 for the reference substance were calculated from equation of regression line prepared by commercial software Microsoft® Excel 2000.
The confidence intervals of EC50, EC20 and EC80 for the reference substance were calculated by the software QC.Expert 2.5 © 1998-2000 (product of TriloByte Ltd., Czech Republic).

Any other information on results incl. tables

Validity of test

The test met the following validity criteria:

- the respiration rates of two control experiments should not differ from each other by more than 15 %

- the EC50 value of the reference substance should be in the range of 5 – 30 mg.L-1

The difference of two respiration rates of control experiments at the beginning and the end of the main test from each other was:

1.74 mg.L-1.h-1 = 6.58 % (if the value of lower control 26.46 mg.L-1.h-1 = 100 %)

The 3h-EC50 value of reference substance, 3,5-dichlorophenol: EC50 = 11.4 mg.L-1 (95 % confidence interval: 10.6 – 12.3 mg.L-1)

Since all criteria for acceptability of the test were met, this study is considered to be valid.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Executive summary:

The influence of the test substance on the respiration rate of activated sludge was investigated after a contact time of 3 hours.

Test was performed according to:

Method C.11 - Activated Sludge Respiration Inhibition Test, Directive 88/302/EEC. Published in OJ L 133 1988.

The preliminary test was performed using 5 concentrations in range 46 - 1000 mg.L^-1 with a geometric factor of 2.2 to determine the concentrations to be used in the main test.

In the main test, 5 concentrations of the test substance in geometric progression with factor of 1.8 (100, 180, 320, 560, 1000 mg.L^-1) were used together with two control experiments (without the test substance), one at the beginning and one at the end of the test.

3,5-Dichlorophenol was used as reference substance. Five concentrations in geometric progression with a factor of 1.4 (5.2, 7.2, 10, 14 and 19 mg.L^-1) were selected.

The abiotic decomposition was not detected even at the highest concentration of the test substance.

The pH value during the main test was in the range of 7.1 to 7.9.

On the basis of the effective concentrations describing the respiration inhibition of activated sludge caused by the test substance the EC₅₀was found to be:

                                                           EC₅₀> 1000 mg.L^-1

Under the current experimental conditions, all tested concentrations in the main test provided no significant inhibition of respiration rate Therefore it was not possible to calculate the EC₅₀ value.