Polar modified Rice Bran Wax

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03.2020 - 04.2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

Inoculum: activated sludge, from a domestic sewage treatment plant is normally used as the microbial inoculums based on “Ministry of environmental protection of the people's Republic of China, The Guidelines for the Testing of Chemicals–Degradation and Accumulation, 2013, 301B Ready Biodegradability: CO2 Evolution Test” and “OECD Guidelines for the Testing of Chemicals. Ready Biodegradability 301B: CO2 Evolution Test, 1992” Characteristics which make activated sludge suitable for ready biodegradability test are their higher cell densities, no pre-adaped to chemical, and their ease of giving lower scattering of results.
Collection: a fresh sample of activated sludge was collected on Feb 27, 2020 from the aeration tank of the Zhu Yuan Wastewater Treatment Plant in Shanghai which treats predominantly domestic sewage with Anaerobic-Oxic process (A/O). The activated sludge was kept by aeration until used (non-aeration during transport from the wastewater treatment plant to the laboratory was kept to a minimum).
Treatment: When back to the laboratory, the sludge was washed with mineral medium. After centrifugation, the supernatant was decanted. This procedure was repeated three times (4000 rpm, 4°C, centrifuge 20 minutes). Then, 0.401g of washed sludge was weighed and dried by moisture meter at 105°C for 1h to calculate the dry weight which was 6.98%.
Sludge suspension: 12.89g wet sludge was suspended in 1L mineral medium to obtain the concentration of 900mg dry matter/L. The sludge was stirred and aerated until use. 100mL of 900mg dry matter/L sludge was added into the test system, such that the final concentration of sludge in test solution was 30mg dry matter/L

Initial conc.:

24 mg/L

Based on:

test mat.

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (CO2 evolution)

Value:

>= 44

Sampling time:

28 d

Parameter:

% degradation (CO2 evolution)

Value:

>= 27

Sampling time:

19 d

Details on results:

The relative degradation values calculated from the measurements performed during the test period revealed some degradation of the test substance, the mean biodegradation of test substance amounted to 27% during the 10-d window test period (Day 19), and 44% during the 28 days period of the test.
The pass level for ready biodegradability, i.e. biodegradation of at least 60% of the ThCO2 within the 10-d window period of the test was not reached. The test substance was not considered to be readily degradable

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Test substance didn’t meet the criteria of ready biodegradability under the test conditions within 28 days.

Executive summary:

The study was conducted according to “Ministry of environmental protection of the people's Republic of China, The Guidelines for the Testing of Chemicals–Degradation and Accumulation, 2013, 301B Ready Biodegradability: CO₂ Evolution Test” and “OECD Guidelines for the Testing of Chemicals. Ready Biodegradability 301B: CO₂ Evolution Test, 1992”.

Determination of ready biodegradability of the test substance LICOCARE RBW 106 was performed with the carbon dioxide (CO₂) evolution test (modified Sturm test). The duration of the test was 28 days. The study contained further of two inoculum blanks, one procedure control (sodium benzoate), one toxicity control(test substance and sodium benzoate) and two test substance groups. The test substance was tested for its ready biodegradability in duplicates at 72.11mg and 72.04mg per 3 litres separately, and total organic carbon (TOC) analysis of test substance in the test vessels were 14.76mg C/L and 14.74mg C/L at day 0.

The total CO₂ release in the blank at the end of the test was 18.26mg CO₂/L, which did not exceed 70mg CO₂/L. During the test, the difference of duplicate values for degradation of the test substance during 28d testing period was less than 20%. The degradation of reference compound on 14^th day was 73%, more than 60% within 14 days. The degree of biodegradation of the toxicity control by Day 14 was 53%, reached the pass level of toxicity control(≥25% based on total ThCO₂ within 14 days) , indicated there was no toxicity inhibition to the inoculum. Therefore, the test was valid.

Biodegradation rate is calculated according to the output of CO₂ and the theoretical reduction of CO₂ of the sample. The relative biodegradation percentage of the test substance which was calculated based on CO₂ production was 27% at Day 19 (10-d window), indicated the test substance didn’t reach the pass level of ready biodegradability. So test substance didn’t meet the criteria of ready biodegradability under the test conditions within 28 days.

Reference 2

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)

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

GLP compliance:

yes

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 conc.:

16 other: mg/L

Based on:

test mat.

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:

benzoic acid, sodium salt

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.

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.

Validity criteria fulfilled:

yes

Interpretation of results:

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 1^st test item replicate reached the 10 % level (beginning of biodegradation) on day 25 and the 2^nd test item replicate on day 14. The 60 % pass level was not reached by both replicates until test end. The 1^st test item replicate reached a maximum of 16 % within 28 days and the 2^nd 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, 1^stReplicate	2	9	9	16
Test Item, 2^ndReplicate	5	12	19	34
Functional Control	57	71	72	72
Toxicity Control test item + reference item	22	29	34	42

Reference 3

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12.08.2020 - 04.02.2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))

Deviations:

yes

Remarks:

Activated Sludge: According to the guideline OECD 302 C, the inoculum should be sampled in Japan at not less than 10 places throughout the country (waste water treatment plants, rivers, lakes sea). The inoculum should be prepared from the different sample

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: Licocare RBW 106 FL VITA
Synonym: LICOCARE RBW 106 TP; Licocare RS
Batch No.: DEF2105728
Aggregate State at Room Temperature: Solid
Color: Yellow
Chemical Composition: 77.4% carbon, 13.0% hydrogen and 9.6% oxygen
Theoretical Oxygen Demand: ThODNH4: 2.996 mg oxygen per mg test item
Certificate of Analysis Date: December 12, 2019
Minimum Shelf Life Date: January 16, 2022
Storage Conditions at Test Facility: At 20 +-5 °C, in the dark

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

Species / Origin:
Activated sludge, microorganisms from a domestic waste water treatment plant was supplied by the sewage plant Roßdorf, Germany.

Conditioning:
The activated sludge used for this study was deposited for 15 minutes, washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in test water and again centrifuged. This procedure was done three times. The sediment of the last washing was re-suspended in test water. An aliquot of this final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 2 g dry material per litre were mixed with test water (see 6.5) and then aerated overnight until use. The concentration of the sludge in the test flasks was 100 mg/L. This suspension was used for the experiment.

Duration of test (contact time):

28 d

Initial conc.:

30 mg/L

Based on:

ThOD

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

The purpose of this study was to determine the biodegradability of the test item Licocare RBW 106 FL VITA. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. The grade of primary degradation was assessed by measurement of the dissolved organic carbon content in the test and control vessels by DOC analysis.
A reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.
This study is recognized by the OECD guideline and provides a rational basis to assess the inherent biodegradation properties of the test item when incubated with activated sludge.

Type and Size:
Manometric test system with test flasks of 500 mL volume containing 300 mL test liquid (see 6.8).
Apparatus:
BSB-Sensomat-System©
Principle:
The test flasks were prepared according paragraph 6.6 and were incubated at 25°C * 2°C. The pressure decrease in the reaction vessels was measured over complete experimental phase using the BSB-Sensomat-System©. The test flasks were closed gas-tight by a measuring head. An appropriate CO2-absorber (KOH) was used for trapping the produced carbon dioxide. The amount of O2 consumed by the activated sludge was calculated from the decrease of pressure in the reaction vessel.
Identification:
Each test unit was uniquely identified with the study number, treatment and replicate number.

Surrounding Type:
Climatic chamber
Temperature:
25°C ± 2°C
Light Conditions: Darkness
pH-Value of Test Item Vessels: 7.4 (measured at the start of the test)
6.8–6.9 (measured at the end of the test)
Recording: Test conditions (temperature) were recorded continuously with suitable instruments, documented in the raw data and reported in the final report. (Short-term deviations (≤ 2 hours) from the recommended temperature range do normally not result in major disturbances of the test performance and are not reported.)

Basal Culture Medium:
Analytical grade salts were added to ultrapure water to prepare the following stock solutions:
a) 4.25 g KH2PO4, 10.875 g K2HPO4, 11.1 g Na2HPO4 x 2 H2O, 0.85 g NH4Cl filled up with ultrapure water to 500 mL volume.
b) 11.25 g MgSO4 x 7 H2O filled up with ultrapure water to 500 mL volume
c) 18.21 g CaCl2 dihydrate filled up with ultrapure water to 500 mL volume
d) 0.125 g FeCl3 x 6 H2O filled up with ultrapure water to 500 mL volume
In order to avoid precipitation of iron hydroxide in the stock solution d) immediately before use, one drop of concentrated HCl per litre was added.
15 mL each of stock solutions a) to d) were combined and filled up to a final volume of 5000 mL with ultrapure water.

Equipment:
The change of pressure in the test flasks was measured automatically by means of a manometric method (BSB-Sensomat-System©) about 12 times a day for a 28 day incubation period. The device consists of reaction vessels with CO2-absorber, measuring heads and a controller (infrared communication).


Principle:
The sealed measuring system is not affected by barometric air-pressure fluctuations. The carbon dioxide which is released as a result of the metabolic processes of the microorganisms enters the gaseous atmosphere and is absorbed by a CO2 absorber. CO2 is produced and absorbed while O2 is consumed and therefore pressure decreases in the vessels and this decrease is recorded by the measuring heads.
CO2 produced during test item breakdown can be calculated from the consumed O2; based on the stoichiometry of O2 consumption and CO2 production during respiration (1 mg of consumed O2 corresponds to 1.375 mg of respired CO2).

Reference substance:

benzoic acid, sodium salt

Test performance:

Degradation of Test Item by BOD Measurement:

Based on the BOD measurement, the mean biodegradation of Licocare RBW 106 FL VITA was 56% and 73% after 14 and 28 days of incubation related to ThODNH4, respectively. As replicate 1 has clearly lower respiration rates and therefore lower degradation values, it might be meaningful to use only replicate 2 and 3 for calculation rates. In that case, the mean degradation of Licocare RBW 106 FL VITA was 79% and 101% after 14 and 28 days of incubation related to ThODNH4, respectively.

Degradation of Test Item by DOC Measurement:

The measured DOC of the test item has to be corrected by the DOC of the control flasks. After correction, the DOC values of Licocare RBW 106 FL VITA at day 0 and 28 were very low. At day 0, the DOC was 0.314 mg/L, corresponding to less than 2% of the Carbon of the test item. Therefore, the results of the DOC determination cannot be used for calculation of degradation.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

101

Sampling time:

28 d

Remarks on result:

other: inherently biodegradable fulfilling specific criteria

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable, fulfilling specific criteria

Conclusions:

After 28 days of incubation, the test item Licocare RBW 106 FL VITA was completely degradable (100%, mean value of two replicates) based on BOD-measurement. Therefore the test item is classified as inherently biodegradable fulfilling specific criteria.

Executive summary:

Title:	Licocare RBW 106 FL VITA: Inherent Biodegradability in a Modified MITI Test
Guidelines/Recommendations:	OECD Guideline for Testing of Chemicals No. 302 C: "Inherent Biodegradability: Modified MITI Test (II)", adopted May 12, 1981, corrected September 08, 2009.
Material and Methods:
Test Item:	Licocare RBW 106 FL VITA; Batch No.: DEF2105728
Test Species:	Aerobic activated sludge (microorganisms from a domestic waste­water treatment plant) was supplied by the sewage treatment plant of Rossdorf, Germany.
Test Design:	The test item Licocare RBW 106 FL VITA was investigated for its inherent biodegradability in a Modified MITI Test (II) over a period of 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. The grade of primary degradation was assessed by measurement of the dissolved organic carbon content in the test and control vessels by DOC analysis. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.
Endpoints:	The degradation rate of test item calculated by the pressure decrease in the reaction vessels was measured over the complete experimental phase of 28 days using the BSB-Sensomat-System^©.
Test Item Loading Rate (initial concentration in medium C0):	30 mg/L
Reference Item:	Sodium Benzoate
Reference Item Loading Rate:	100 mg/L
Test Conditions:	25°C ± 2°C, darkness
Results:
Biodegradation of Licocare RBW 106 FL VITA:	Based on the BOD measurement, the mean biodegradation of Licocare RBW 106 FL VITA was 56% and 73% after 14 and 28 days of incubation related to ThODNH4, respectively. As replicate 1 has clearly lower respiration rates and therefore lower degradation values, it might be meaningful to use only replicate 2 and 3 for calculation rates. In that case, the mean degradation of Licocare RBW 106 FL VITA was 79% and 101% after 14 and 28 days of incubation related to ThODNH4, respectively. The DOC values of Licocare RBW 106 FL VITA at day 0 and 28 were very low. At day 0, the DOC was 0.314 mg/L, corresponding to less than 2% of the Carbon of the test item. Therefore, the results of the DOC determination cannot be used for calculation of degradation.
Biodegradation of Abiotic Control:	Based on BOD measurement in the abiotic control without addition of activated sludge, no important biodegradation was found after 28 days of treatment.
Biodegradation of Reference Item:	The reference item sodium benzoate was degraded to more than 100% after 14 days and after 28 days of incubation. Degradation rates above 100% result from utilisation of retained resources in the microorganisms or by devouring themselves. The validity criterion of more than 65% degradation within 14 days was fulfilled.
Biodegradation of Toxicity Control:	In the toxicity control, containing both the test item and the reference item sodium benzoate, the degradation was 71% after 14 days and 76% after 28 days of incubation. Thus, the test item can be assumed not to be inhibitory on the activated sludge microorganisms.
Conclusion:	After 28 days of incubation, the test item Licocare RBW 106 FL VITA was completely degradable (100%, mean value of two replicates) based on BOD-measurement. Therefore the test item is classified as inherently biodegradable fulfilling specific criteria.

Description of key information

Under the test conditions of two OECD 301 B Studies the test item is classified as not readily biodegradable within the  28 day period of the studies. In an inherent test according OECD 302 C, after 28 days of incubation, the test item was completely degradable (100%, mean value of two replicates) based on BOD-measurement. Therefore the test item is classified as inherently biodegradable fulfilling specific criteria.

 

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable, fulfilling specific criteria

Type of water:

freshwater