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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:

31.03.2022 - 31.05.2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: State Environmental Protection Administration of China. HJ/T153-2004 The Guidelines for the test of chemical [S]. Beijing: China Environmental Science Press.2004.

Qualifier:

according to guideline

Guideline:

other: Chemical Registration Center of MEP. The Guidelines for the Testing of Chemicals Degradation and Accumulation, 301B CO2 Evolution Test[M]. Second Edition. Beijing: China Environmental Science Press. 2013: 32 - 37.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Enhanced Biodegradation Screening Test, 60 d

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

mixture of sewage, soil and natural water

Details on inoculum:

Name Mixture (non pre-adaptation) of activated sludge, river water, lake water and soil
Source Activated sludge was collected from the aeration tank of a Water Purification Plants which is mainly working on domestic sewage treatment. River water was collected from Pearl River near Zhoutouju park of Guangzhou. Lake water was collected from Donghu park of Guangzhou. Soil was collected from Pogang Nature Trail of South China Botanical Garden.
Batch No. I20220328-1
Selection reason The inoculum is recommended by "The Guidelines for the Testing of Chemicals".
Pre-treatment A pre-treatment was performed as following:
(1) Mixed 100 g of soil with 1000 mL of test medium, and aerated for 24 h under the test temperature, settled down for 1 h to obtain the soil supernatant.
(2) Mixed 500 mL of activated sludge suspension, 500 mL of river water and 500 mL of lake water, and then made this incubated for 24 h under aeration at test temperature. After that, any coarse particles and impurities on the surface was removed from this mixture by sieving with the aperture of 80 meshes, and 500 mL of the above soil supernatant was further mixed to obtain the inoculum containing four sources. Keep this inoculum in aeration under the test temperature until being required.
(3) Determined the solids dry weight and aerobic plate count of the inoculum.
(4) Before the study, adjusted the inoculum initial concentration to 3.6 g/L, and kept in aeration. On the day of the test, a certain volume of the inoculum was added into each test vessel to give a final concentration of 30 mg suspended solids per liter (counted in dry weight).
Aerobic plate count The aerobic plate count in the test system was calculated as 1.4107 CFU/L.

Duration of test (contact time):

60 d

Initial conc.:

10 - 20 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Enhanced Biodegradation Screening Test is based on the prolongation of the test duration to 60 d and testing in larger vessels, and the mixture of four sources was used as inoculum to increase the diversity of microbial community.
Test substance in the test system (within 10 mg - 20 mg TOC/L) as the nominal sole source of organic carbon, is inoculated with non pre-adaptation inoculum and stirred in a closed bottle, aerated by the passage of carbon dioxide-free air at a controlled rate in the dark at 22°C±2°C for up to 60 days. To check the rate of biodegradation in % by determination of the carbon dioxide evolution, and the CO2 produced is trapped in NaOH. The amount of CO2 produced by the subjects is expressed as a percentage of ThCO2 (corrected by a blank control test containing only the inoculum).

Reference substance:

aniline

Key result

Parameter:

% degradation (CO2 evolution)

Value:

71.1

Sampling time:

28 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

92.8

Sampling time:

60 d

Details on results:

On day 14 after starting the study, percentage biodegradation of the procedure control and the toxicity control were 68.8% and 52.3%, which had respectively reached the pass levels of 60% and 25% of the theoretical amount of CO2 produced, and showed that the inoculum activity met the requirement of the test and the test substance was considered not to have a toxic effect on the sewage sludge micro-organisms used in the study. Under the present conditions of the study, percentage biodegradation of the two replicates from test substance at the end of the test over 60 days were 89.5% and 96.2%, respectively, with a calculated mean of 92.8%.

At the beginning of the test, TIC concentration in the test solution was less than 5% of TC.
- After starting the test, the total CO2 evolution in the inoculum blank was 35.0 mg/L, no more than 70 mg/L within 28 days.
- Percentage biodegradation of the procedure control and the toxicity control were 68.8% and 52.3%, which had respectively reached the pass levels of 60% and 25% of the total theoretical amount of CO2 produced within 14 days.
- During the experimental period, the difference of extremes of replicate values of the removal of the test substance was all less than 20%.

Results with reference substance:

On day 14 after starting the study, percentage biodegradation of the procedure control and the toxicity control were 68.8% and 52.3%, which had respectively reached the pass levels of 60% and 25% of the theoretical amount of CO2 produced, and showed that the inoculum activity met the requirement of the test and the test substance was considered not to have a toxic effect on the sewage sludge micro-organisms used in the study.

Table 1 Composition of the test system solution in flasks

Test vessel	Test suspension	Inoculum blank	Procedure control	Toxicity control
	Flasks 1, 2	Flasks 3, 4, A	Flask 5	Flask 6
Test medium (mL)	2975	2975	2917	2917
Inoculum initial concentration (g/L)	3.6	3.6	3.6	3.6
Inoculum(mL)	25.0	25.0	25.0	25.0
Test substance (mg)	46.2	―	―	46.2
Reference substance stock solution (mL)	―	―	58	58
Final volume (mL)	3000	3000	3000	3000
Test substance final concentration (mg/L)	15.4	―	―	15.4
TOC concentration from the test substance (mg/L)	12.0	―	―	12.0
Reference substance final concentration (mg/L)	―	―	15.5	15.5
TOC concentration from the reference substance (mg/L)	―	―	12.0	12.0
Inoculum final concentration (mg/L)	30	30	30	30
pH value at the start	―	7.43 / 7.44	7.43	―
pH value at the end	7.45 / 7.41	7.38 / 7.36	7.59	7.59

 

 

Table 2 Amount of CO₂ produced (mg)

Time (d)	Test suspension		Inoculum blank			Procedure control	Toxicity control
	Flask 1	Flask 2	Flask 3	Flask 4	Mean	Flask 5	Flask 6
2	11.242	9.920	2.645	0.882	1.764	6.393	9.258
7	39.679	49.379	23.146	17.415	20.280	72.966	87.515
11	43.978	41.773	20.170	18.627	19.399	49.048	66.683
14	17.084	19.950	8.487	8.487	8.487	12.455	24.579
18	25.681	26.563	15.982	13.998	14.990	20.391	24.359
21	21.162	18.517	10.581	13.667	12.124	12.786	18.076
25	22.264	21.603	14.549	18.517	16.533	20.280	16.313
28	15.210	14.108	11.904	11.242	11.573	12.786	15.210
30	9.258	9.699	7.054	8.377	7.715	8.597	10.802
35	18.076	17.856	12.345	15.872	14.108	14.329	18.076
40	18.737	20.721	9.699	12.124	10.912	14.329	16.533
46	29.759	27.775	21.824	20.060	20.942	25.571	26.894
50	15.541	18.186	12.014	17.966	14.990	15.761	17.745
55	20.611	23.256	19.509	19.729	19.619	20.170	19.729
60	25.461	23.256	21.052	23.036	22.044	22.595	24.800

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 3 Percentage biodegradation during the test (%)

Time (d)	Test suspension				Procedure control	Toxicity control
	Flask 1	Flask 2	Mean	Difference	Flask 5	Flask 6
2	7.2	6.2	6.7	1.0	3.5	2.8
7	21.9	28.2	25.0	6.3	43.4	28.3
11	40.5	45.1	42.8	4.7	65.8	46.2
14	47.0	53.8	50.4	6.8	68.8	52.3
18	55.1	62.6	58.8	7.5	72.9	55.8
21	61.9	67.4	64.6	5.5	73.4	58.1
25	66.2	71.2	68.7	5.0	76.2	58.0
28	69.0	73.2	71.1	4.2	77.2	59.4
30	70.2	74.7	72.4	4.5	77.8	60.5
35	73.2	77.5	75.3	4.3	78.0	62.0
40	79.1	84.9	82.0	5.8	80.6	64.2
46	85.8	90.1	87.9	4.3	84.1	66.4
50	86.2	92.5	89.4	6.3	84.7	67.4
55	86.9	95.3	91.1	8.3	85.1	67.5
60	89.5	96.2	92.8	6.7	85.5	68.5

 

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable, but failing 10-day window

Conclusions:

Under the conditions of the study, percentage biodegradation of the test substance at the end of the test over 60 days was 92.8%, greater than 60%, therefore the test substance can be considered not to be persistent.

Executive summary:

On day 14 after starting the study, percentage biodegradation of the reference substance (aniline) and the toxicity control were 68.8% and 52.3%, which had respectively reached the pass levels of 60% and 25% of the theoretical amount of CO₂ produced.

At the beginning of the test, total inorganic carbon (TIC) concentration of the test solution was less than 5% of the total carbon (TC). Within 28 days after starting the test, the total CO₂ evolution in the inoculum blank was 35.0 mg/L, no more than 70 mg/L. During the test, the difference of extremes of replicate values of the removal of the test substance was all less than 20%.

The above results all met the requirement of the test, which revealed that the test results were valid.

Under the present conditions of the study, percentage biodegradation of the two replicates from the test substance at the end of the test over 60 days were 89.5% and 96.2%, respectively, with a calculated mean of 92.8%.

Reference 2

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09.2021 - 10.2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: China National standardizing committee. GB/T 27849-2011 Chemicals-Degradation screening test - Chemical oxygen demand. China Standards Press, 2011

Version / remarks:

2011

Qualifier:

according to guideline

Guideline:

other: State Environmental Protection Administration of the People's Republic of China. HJ 828-2017 Water quality-Determination of Chemical oxygen demand (CODcr). Ministry of Environmental Protection, 2017.

Version / remarks:

2017

Qualifier:

according to guideline

Guideline:

other: State Environmental Protection Administration of China. HJ 84-2016 Water quality-Determination of inorganic anions . Beijing: China Environmental Science Press. 2016.

Version / remarks:

2016

Qualifier:

according to guideline

Guideline:

other: State Environmental Protection Administration of China. HJ/T 153-2004 The Guidelines for the test of chemical. Beijing: China Environmental Science Press. 2004.

Qualifier:

according to guideline

Guideline:

other: The national hazardous chemical management standardization technical committee. GB/T 21818-2008 Chemical Inherent Biodegradability: Modified MITI Test (Ⅱ) [S]. Beijing: China Standards Press. 2008.

Version / remarks:

2008

Qualifier:

according to guideline

Guideline:

other: Chemical Registration Center of MEP. The Guidelines for the Testing of Chemicals Degradation and Accumulation, 302C Inherent Biodegradability: Modified MITI Test (Ⅱ). Second Edition. Beijing: China Environmental Science Press. 2013: 74 - 81.

Version / remarks:

2013

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1981

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name LICOCARE RBW 300 FL TP
Chemical Name Waxes and Waxy substances, rice bran, oxidized,
calcium salts
CAS No. 1850357-57-1

Since the test substance is insoluble in water, the method of directly addition was adopted for the study.
Test substance was sampled after being taken out several times in a small amount and grinded it in a clean and dry mortar.
14.80 mg - 15.25 mg of the test substance was respectively weighed into bottles 1, A, 2, 3, 4, B, 8, D, and dispersed in 200 mL of deionized water or test medium using ultrasonic treatment method for 10 minutes.
The purity of the test substance was calculated as 100%.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

Fresh samples from the ten sites, mainly in areas where varieties of chemicals were used and discharged were collected. From sites such as sewage treatment works, industrial waste-water treatment works, rivers, lakes, seas, etc. Each 1L samples of sludge or water were collected and mixed thoroughly together. After removing the floating matter and allowing to standing, the supernatant and the original sludge supernatant were taken equal amount into the sludge incubation flask to mix and adjust to pH 7.0±1.0, then incubated at 25°C±2°C, The above liquid is aerated for about 23.5h, and 30 minutes after ceasing, one third of the whole volume of the supernatant is discarded and an equal volume of a solution containing 0.1% each of glucose, peptone and monopotassium phosphate is added to the remaining portion of the supernatant and aeration re-commenced, followed by adjusting to pH 7.0±1.0 with dilute phosphoric acid. This procedure is repeated once per day. The unit is maintained in a good operation; and the final obtained sludge should settle well and be aerated continuously. The sludge can be used as an inoculum for one month after cultivation. The sludge can be used as an inoculum within 18h - 24h after the unit had been fed.
Before starting the test, an appropriate volume of the inoculum was taken out to settle down for 30 minutes, the supernatant was then discarded, and the sludge was further re-suspended in the test medium, follow by aerating until being required.

Duration of test (contact time):

28 d

Initial conc.:

30 mg/L

Based on:

test mat.

Details on study design:

Test conditions
Test substance LICOCARE RBW 300 FL TP
Test groups Abiotic control (bottles 1 and A)
Test suspension (bottles 2, 3, 4 and B)
Procedure control (bottle 5)
Inoculum blank 1 (bottles 6 and C)
Inoculum blank 2 (bottle 7)
Toxicity control (bottles 8, D)
Test substance concentration 30 mg/L
Reference substance concentration 100 mg/L
Sterilant concentration 500 mg/L
Inoculum concentration 100 mg/L (test suspension, inoculum blank 1 and toxicity control)
30 mg/L (procedure control and inoculum blank 2)
Total volume 500 mL
Test temperature 25.3°C - 25.7°C (within 25°C±2°C)
Test period 28 d (dark and stir in airtight)
pH values 7.45 - 7.47 (at the start, 5.46 in abiotic control)
7.19 - 8.05 (at the end, 4.15 in abiotic control)

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (DOC removal)

Value:

83.5

Sampling time:

28 d

Parameter:

BOD5

Value:

83.5

Results with reference substance:

Percentage biodegradation of the reference substance (sodium benzoate) was 82.1% (>40%) on day 7 and was 85.3% on day 14 (>65%).

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

Under the present conditions of the study, percentage biodegradation of the test substance calculated by BOD on day 28 was 83.5%, therefore the test substance can be considered to be ultimate inherent biodegradable.

Executive summary:

Percentage biodegradation of the reference substance (sodium benzoate) was 82.1% (>40%)

on day 7 and was 85.3% on day 14 (>65%).

Percentage biodegradation of three replicates from the test substance on day 28 calculated by

BOD were respectively 85.4%, 85.0% and 80.1%, with a calculation average of 83.5%. Percentage biodegradation of the toxicity control on day 14 and 28 were respectively 60.1%

and 69.7%.

Test substance concentrations had not been analyzed in this study, therefore the primary biodegradation rate was not calculated, and the final test result was only referenced as BOD percentage biodegradation, as the followings:

(1) LICOCARE RBW 300 FL TP cannot be dissolved in water and regular solvent, such as methanol, acetonitrile, tetrahydrofuran, isopropanol, etc, so HPLC (High Performance Liquid Chromatography) and LC-MS/MS (Liquid Chromatography-Mass Spectrometer) are not suitable to use for analysis.

(2) The GC (Gas Chromatography) analysis method used in Certificate of Analysis that sponsor provided, which reveal that the test substance should be derived firstly, and derivation must be conducted under non-aqua system, is also not suitable for this water solution system and the predicted limit of quantification (LOQ) of the analysis method would be high.

(3) The carbon chain of some component from the test substance is much more than 40, so it’s not guaranteed that all constituents of test substance would be peaked because of the high boiling point, and it would cause residual and further pollute the apparatus.

(4) The biodegradation rate could not be accurately characterized by the determination of dissolved organic carbon (DOC) in the test solution, for the test substance could not be completely dissolved in the test concentration of 30 mg/L.

 

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 300 FL VITA
Synonym: LICOCARE RBW 300 FL TP
Batch No.: DEF2114527
Aggregate State at Room Temperature: Solid
Color: Yellow
Chemical Composition: 78.5% carbon, 13.3% hydrogen and 5.9% oxygen
Theoretical Oxygen Demand: ThODNH4: 3.086 mg oxygen per mg test item
Certificate of Analysis Date: April 06, 2015
Minimum Shelf Life Date: November 05, 2020
Storage Conditions at Test Facility: At 20  5 °C, in the dark

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Species / Origin:
Activated sludge, microorganisms from a domestic waste water treatment plant was supplied by thesewage 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 resuspended 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:

Details on study design:
The purpose of this study was to determine the biodegradability of the test item Licocare RBW 300 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 reactionvessel.
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 perform ance 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 ultrap
ure 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 manomet ric 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 O2is consumed and therefore pressure decreases in the vessels and this decrease is recorded by themeasuring 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 300 FL VITA was 41% and 64% after 14 and 28 days of incubation related to ThODNH4, respectively. The degradation rates of the three single replicates were 23%, 68% and 32% at day 14 and 45%, 88% and 61% at day 28.


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 300 FL VITA at day 0 and 28 were very low. At day 0, the DOC was 0.633 mg/L, corresponding to less than 5% 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:

64

Sampling time:

28 d

Remarks on result:

other: partially biodegradable

Validity criteria fulfilled:

yes

Interpretation of results:

other: Partially biodegradable

Conclusions:

After 28 days of incubation, the test item Licocare RBW 300 FL VITA was degradable to 64% (mean value) based on BOD-measurement. Therefore the test item is classified as partially biodegradable.

Executive summary:

Title:	Licocare RBW 300 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 300 FL VITA; Batch No.: DEF2114527
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 300 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 300 FL VITA:	Based on the BOD measurement, the biodegradation of Licocare RBW 300 FL VITA was 41% and 64% after 14 and 28 days of incubation related to ThODNH4, respectively. The DOC values of Licocare RBW 300 FL VITA at day 0 and 28 were very low. At day 0, the DOC was 0.633 mg/L, corresponding to less than 5% 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 75% 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 300 FL VITA was degradable to 64% (mean value) based on BOD-measurement. Therefore the test item is classified as partially biodegradable.

Reference 4

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2019-05 to 2019-06

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)

Qualifier:

according to guideline

Guideline:

other: Miniytry of environmental protectio of the people's Republic of China, The Guidelines for the Testing of chicals-Degradation and Accumulation, 2013, 301b 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 OECD Guideline 301B Characteristics which make activated sludge suitable for ready biodegradability test are their higher cell densities, no pre-adapted to chemical, and their ease of giving lower scattering of results.
Collection: a fresh sample of activated sludge was collected an My 8th, 2019 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 aminimum).

Duration of test (contact time):

28 d

Initial conc.:

20.01 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Parameter:

% degradation (CO2 evolution)

Value:

6

Sampling time:

28 d

1      Conclusions

1.1 Theoretical CO₂production

The ThCO₂per test vessel was163.80mg CO₂/3L, 163.83mg CO₂/3L for the test substance(TS1 and TS2), 160.51mg CO₂/3L for procedure control, and 324.39mg CO₂/3L for the toxicity control

1.2 Biodegradation of test substance

The results presented in Section 19.3 and 19.4.

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 to6% during the 28 days period of the test.

The pass level for ready biodegradability, i.e. biodegradation of at least 60% of the ThCO₂within the 10-d window period of the test was not reached. The test substance was not considered to be readily degradable.

Average amount of cumulatiove CO₂and biodegradation of Test substance group

Days	Test substance group	Corrected CO2produced (mg) *	Cumulative CO2(mg)	Biodegradation (%)	Average Biodegradation (%)
28d	TS1	10.162	12.196	7	6
	TS2	9.689	7.595	5

1.3  Biodegradation of toxicity control

In the toxicity control more than 25% degradation occurred within 14 days (40% on 14th day). Therefore, the test substance was assumed not to inhibit microbial activity (Section 19.5).

1.4     Sample TOC determination result

TOC in sample is 743.5 g/kg which measured by TOC analyser

1.5     pH values

The pH measured ranged from7.43to7.72in all vessels during the test(Section19.6).

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The 28d biodegradation test indicates that LICOCARE RBW 300 FL TP (KLEC20181026-2692) was not readily biodegradable under present test conditions.

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: CO₂Evolution Test” and “OECD Guidelines for the Testing of Chemicals. Ready Biodegradability 301B: CO₂Evolution Test, 1992”.

Determination of ready biodegradability of the test substanceLICOCARE RBW 300 FL TP(KLEC20181026-2692) 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 duplicatesat60.03mg and60.04mg per 3 litres separately, and total organic carbon (TOC) analysis of test substance in the test vessels were163.80mg C/L and163.83mg C/L on day 0.

The total CO₂release in the blank at the end of the testwas 47.28mgCO₂/3L, that is 15.76mg CO₂/L, whichdid 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 compoundon 14^thdaywas84%, more than 60% within 14 days. The degree of biodegradation of the toxicity control by Day 14was 40%,reached the pass level of toxicity control(≥25%based on totalThCO₂within 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 CO₂and the theoretical reduction of CO₂of the sample. Results showed that under the experimental conditions, carbon dioxide (CO₂) evolution of test substance was no significant increase, and biodegradation percentage of test substance was 6% from test start until test termination after 28d, so didn’t reach the 10-d window. So test substance didn’t meet the criteria of ready biodegradability under the test conditions within 28 days.

Reference 5

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2020-06-04 to 2020-07-03

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)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Test item Licocare RBW 300 FL VITA
Batch number DEF2114527
Purity (certified) 99.75% (w/w)
Active ingredient LICOCARE RBW 300 FL TP / partly saponified natural wax
Water solubility < 1 g/L
TOC* 74.35 %
Appearance yellow / pale yellow solid, flakes and partly coarse powder
Expiry date 2020-11-05
Recommended storage Store container tightly closed in a cool, well-ventilated place


* The TOC based on the information provided by the sponsor.

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: 2020-06-02
- 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 with CO2 free air for two days before test start. Further treatment see section ‘preparation of the test vessels’. 3.65 mL/ L of this mixture were used to initiate inoculation (25.0 mg/L dw).

Duration of test (contact time):

28 d

Initial conc.:

14.5 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 absorption 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 concentration of the test item and the theoretical CO2 production (ThCO2) were calculated based on the carbon content.

The following incubation vessels will be prepared:
- two for the inoculum control (C1, C2)
- one for the functional control (R1)
- two for the test item concentration (P1, P2)
- 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 absorption 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.

The test and reference item were weighed out. The test item was pounded in a mortar and weighed out into small beakers. A defined amount of ultrapure water was added to the test item and treated with ultrasound for ten minutes. The test item dispersions and the reference item were transferred to the respective incubation vessels with ultrapure water. 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:
Back titration 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

Key result

Parameter:

% degradation (CO2 evolution)

Value:

16

Sampling time:

28 d

Details on results:

Based on the carbon content a ThCO2 of 2.73 mg CO2/mg test item was calculated. A test concentration of 14.5 mg/L, corresponding to a carbon content of 10.8 mg C/L in the test vessels was selected.

The adaptation phase of the functional control changed within 4 days into the degradation phase (degradation  10 %). The course of the degradation was fast and the functional control reached the pass level of 60 % within 8 days and a maximum biodegradation of 92 % on day 28. The validity criterion degradation  60 % after 14 days was fulfilled.

In the toxicity control containing both test item and reference item a biodegradation of 46 % was determined within 14 days, with 54 % 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 in comparison to the readily degradable functional control and the toxicity control. Both replicates reached the 10 % level (beginning of biodegradation) within 21 days. Both test item replicates did not reach the 60 % pass level within 28 days. The mean biodegradation on day 28 was 16 %.


In the inoculum control the total CO2 production was 44.3 mg CO2/L after 28 days.

Results with reference substance:

In the toxicity control containing both test item and reference item a biodegradation of 46 % was determined within 14 days, with 54 % 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 300 FL VITA (batch no.: DEF2114527) 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 2020-06-04 to 2020-07-03 according to OECD 301 B at the test facility. The test item was tested at a concentration of 14.5 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 CO2 produced 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 CO2 had been purged from the test solutions over a period of 24 hours. The percentage CO2 production was calculated in relation to the theoretical CO2 production (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 92 % on day 28.

 

In the toxicity control containing both test and reference item a biodegradation of 46 % was determined within 14 days, with 54 % 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 in comparison to the readily degradable functional control and the toxicity control. Both replicates reached the 10 % level (beginning of biodegradation) within 21 days. Both test item replicates did not reach the 60 % pass level within 28 days. The mean biodegradation on day 28 was 16 %.

 

 

Biodegradation of the Test ItemLicocare RBW 300 FL VITAin
  Comparison to the Functional Control and Toxicity Control

	Biodegradation [%]
	Study Day [d]
	6	14	21	28
Test Item, 1^stReplicate	4	8	10	12
Test Item, 2^ndReplicate	2	8	16	19
Functional Control	59	85	90	92
Toxicity Control Test item + Reference item	32	46	48	54

Description of key information

In an enhanced CO2 Evolu6tion test according to OECD 301 B it could be shown that the test substance is readily biodegradable but failed the 10 day windows. After 28 days the biodegradation was 71.1 % and after 60 days 92.8 %

Under the conditions of the study, percentage biodegradation of the test substance at the end of the test over 60 days was 92.8%, greater than 60%, therefore the test substance can be considered not to be persistent.

In two inherent test according OECD 302 C, after 28 days of incubation, the test item was degradable to 83.5% (mean value) respectively 64 % based on BOD-measurement. Therefore the test item is also inherently biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable but failing 10-day window

Type of water:

freshwater