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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-10-08 to 2015-11-06
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Test material form:
other: liquid
Details on test material:
- Test Item: Licocare RBW 106 FL TP
- Physical state: Slightly yellow flakes
- Purity test date: 2015-04-29
- Lot/batch No.: DEF2105728
- Expiration date of the lot/batch: 2020-01-16
- Storage condition of test material: Room temperature, protected from light in the tightly closed original container.
- Purity (certified): 99.63% (w/w)
- Water solubility: < 1 g/L
- Molecular mass: not specified

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
Municipal sewage treatment plant, D-31137 Hildesheim, Germany
- Receipt: 2015-10-05
- Pretreatment/Concentration of sludge:
The activated sludge was washed twice with chlorine free tap water. After the second washing the settled sludge was resuspended in mineral salts medium and was maintained in an aerobic condition by aeration for 2 hours and 10 minutes. Thereafter the sludge was homogenized with a blender. After sedimentation the supernatant was decanted and maintained in an aerobic condition by aeration with CO2 free air until test start. 20 mL/L of this mixture were used to initiate inoculation.
Colony forming units in the test vessel: approx. 10^7 - 10^8 CFU/L





Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
16 other: mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral salts medium acc. to OECD 301 B / CO2 Evolution Test
- Test temperature: Nominal 22 +/- 2°C (actually measured: 20.0 - 23.5 °C)
- Dispersion treatment: Continuous stirring
- Aeration: 30 - 100 mL/min
- Photoperiod: Low light conditions (brown glass bottles)

TEST SYSTEM
- Culturing apparatus: 5000 mL brown glass flasks
- Number of culture flasks/concentration: 1 for the reference item, 1 for toxicity control (test and reference item), 2 for the control, 2 for the test item
- Method used to create aerobic conditions: Aeration with 30 - 100 mL/min
- Measuring equipment: Visual check of aeration twice per day
- Details of trap for CO2 and volatile organics if used:
CO2 adsorption vessels were connected to the air outlets of the incubation vessels via a series of 3 gas wash bottles, each containing 100 mL of
a 0.0125 mol/L Ba(OH)2 solution.
- Course of the study:
The following incubation vessels were prepared:
- two for the test item concentration (P1, P2)
- one for the functional control (R1)
- two for the inoculum control (C1, C2)
- one for the toxicity control (T1)
The necessary amounts of ultrapure water, mineral salts medium and inoculum were placed in each incubation vessel. The vessels were aerated for 24 h with CO2 free air. After 24 h the CO2 adsorption vessels were connected to the air outlets of the incubation vessels via a series of 3 gas wash bottles, each containing 100 mL of a 0.0125 mol/L Ba(OH)2 solution. Test and reference item were weighed out. The test item was weighed out into small beakers and a defined amount of ultrapure water was added and treated with ultrasound. The test item dispersions and the reference item were transferred to the respective incubation vessels. The vessels were made up to 3 L with ultrapure water and connected to the system for the production of CO2 free air. On day 28, 1 mL 37 % HCl was added to each of the vessels. Aeration was continued for further 24 h and the quantity of CO2 released was determined.


SAMPLING
- Sampling frequency:
Backtitration of the residual Ba(OH)2 with 0.05 N HCL was carried out three times a week during the first ten days and thereafter twice weekly.
- Sampling method:
For each titration the first gas wash bottle was removed and a new bottle was connected to the last one.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Test medium without test and/or reference item
- Abiotic sterile control: No
- Toxicity control: Test item and reference item in test concentration


STATISTICAL METHODS:
- The theoretical production of carbon dioxide (ThCO2) of the test item and functional control was calculated by the carbon content (1) and the
molecular formula (2), respectively.

ThCO2 [mgCO2/mg] = 3.67 * TOC [mgC/mg test item] (1)

ThCO2 [mgCO2/mg] = (C-Atoms *molecular weight of CO2)/molecular weight of reference item) (2)


- The produced CO2 was calculated by: 1 mL HCl (c = 0.05 mol/L) = 1.1 mg CO2

- The net amount of CO2 produced was calculated by correcting the results of the test item and functional control for endogenous CO2 production
of the inoculum controls.

- The biodegradation was calculated from the ratio theoretical CO2 production to net CO2 production:

Degradation [%] = (net CO2 * 100)/(THCO2 [mg CO2/3L])
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
25
Sampling time:
28 d
Details on results:
Based on the carbon content a ThCO2 of 2.47 mg CO2/mg test item was calculated. A test concentration of 16 mg/L, corresponding to a carbon content of 10.8 mg C/L in the test vessels was selected.

Colony forming units (CFU) of the inoculum for the Modified Sturm Test were determined prior
to test start by standard dilution plate count: approx. 1.04 x 10^9 CFU/L, corresponding to
approx. 1.04 x 10^7 CFU/L in the test vessel.



The adaptation phase of the functional control changed after 2 days into the degradation phase (degradation > 10 %). The course of the degradation was rapid and the functional control reached the pass level of 60 % within 8 days and a biodegradation of 72 % after 28 days. The validity criterion degradation > 60 % after 14 days is fulfilled.

In the toxicity control containing both test and reference item a biodegradation of 29 % was determined within 14 days and it came to 42 % after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.

The biodegradation of the test item is shown graphically in comparison to the readily degradable functional control and the toxicity control. The 1st test item replicate reached the 10 % level (beginning of biodegradation) on day 25 and the 2nd test item replicate on day 14. The 60 % pass level was not reached by both test item replicates until test end. The 1st test item replicate reached a maximum of 16 % within 28 days and the 2nd test item replicate reached a maximum of 34 % biodegradation until test end. The mean biodegradation on day 28 was 25 %. A plateau was not reached.

Under the test conditions the test item is classified as not readily biodegradable within the 28 day period of the study.

In the inoculum control the total CO2 production was 33.3 mg CO2/L after 28 days.

BOD5 / COD results

Results with reference substance:
In the toxicity control containing both test and reference item a biodegradation of 29 % was determined within 14 days and it came to 42 % after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
other: not readily biodegradable
Conclusions:
Under the test conditions the test item is classified as not readily biodegradable within the 28 day period of the study.
Executive summary:

The ready biodegradability of the test item LICOCARE RBW 106 FL TP was determined with a non-adapted activated sludge over a test period of 28 days in the Modified Sturm Test. The study was conducted from 2015-10-08 to 2015-11-06 according to OECD 301 B at the test facility. The test item was tested at a concentration of 16 mg/L with 2 replicates corresponding to a carbon content (TOC) of 10.8 mg C/L in the test vessels.The test vessels were incubated at low light conditions and at a temperature of 22 ± 2 °C.

The biodegradation of the test item was followed by titrimetric analysis of the quantity of CO2produced by the respiration of bacteria. The degradation was stopped on day 28 by acidification of the test solutions. The last titration was made on day 29 after residual CO2had been purged from the test solutions over a period of 24 hours. The percentage CO2production was calculated in relation to the theoretical CO2production (ThCO2) of the test item. The biodegradation was calculated for each titration time.

 

To check the activity of the test system sodium benzoate was used as functional control. The percentage degradation of the functional control reached the pass level of 60 % within 8 days and a maximum biodegradation of 72 % after 18 days.

 

In the toxicity control containing both test and reference item a biodegradation of 29 % was determined within 14 days and it came to 42 % after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.

 

The biodegradation of the test item is shown graphically in comparison to the readily degradable functional control and the toxicity control. The 1st test item replicate reached the 10 % level (beginning of biodegradation) on day 25 and the 2nd test item replicate on day 14. The 60 % pass level was not reached by both replicates until test end. The 1st test item replicate reached a maximum of 16 % within 28 days and the 2nd test item replicate reached a maximum of 34 % biodegradation until test end. The mean biodegradation on day 28 was 25 %. A plateau was not reached.

 

 

 

The test item is classified as
not readily biodegradable
within the 28 day period of the study.

 

 

Biodegradation of the Test ItemDisperse Blue 1092in Comparison to the Functional Control and Toxicity Control

 

Biodegradation [%]

 

Study Day [d]

 

6

14

21

28

Test Item, 1st Replicate

2

9

9

16

Test Item, 2nd Replicate

5

12

19

34

Functional Control

57

71

72

72

Toxicity Control
test item + reference item

22

29

34

42