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Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted from 2021-01-07 to 2021-02-05 according to OECD 301 B at the test facility.
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:
Water solubility approximately 10 g/L soluble, turbid
pH 9 – 10 (20 °C) concentration: 10 g/L
TOC* 70.3 %
Appearance slightly yellowish clear liquid
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: 2021-01-05
- 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 with CO2 free air for two days before test start. Further treatment see section ‘preparation of the test vessels’. 4.13 mL/L of this mixture were used to initiate inoculation (25.0 mg/L dw).

Duration of test (contact time):
28 d
Initial conc.:
15 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 20.5 – 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 the reference item with silicone oil, 1 for toxicity control (test and reference item), 1 for the toxicity control (test and reference item) with silicone oil, 2 for the control, 2 for the control with silicone oil, 2 for the test item, 2 for the test item with silicone oil
- 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:
The necessary amounts of ultrapure water, mineral salts medium and inoculum was 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.


- 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 were prepared:
- two for the inoculum control (C1, C2)
- two for the inoculum control with silicone oil (CS1, CS2)
- one for the functional control (R1)
- one for the functional control with silicone oil (RS1)
- two for the test item concentration (P1, P2)
- two for the test item concentration with silicone oil (PS1, PS2)
- one for the toxicity control (T1)
- one for the toxicity control with silicone oil (TS1)


The necessary amounts of ultrapure water, mineral salts medium and inoculum was 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.

The test and reference item were weighed out. The test item was weighed out into small beakers. A defined amount of ultrapure water was added to the test item. The test item dispersions and the reference item were transferred to the respective incubation vessels with ultrapure water. The silicone oil stock solution was prepared as describe above. 1 mL silicone oil or silicone oil stock solution was pipetted into 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:
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
- Toxicity control Test item and reference item in test concentration
with Silicone oil



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
Preliminary study:

Optimization of the test item bioavailability / distribution
Pre-tests were carried out to determine the most suitable method of preparation.
1) 25 mg and 50 mg test item were dispersed in 1000 mL ultrapure water. The test item appears to have been dissolved optically in water.
From the preliminary solubility work and following the recommendations of the International Standards Organisation (ISO, 1995) it was concluded that the best testable dispersions were obtained when the test item is applied without preparation.

Toxicity Tests
Two pre-tests have been carried out, one in the test design according to OECD 301 B and one according to OECD 301F.

Preliminary Test acc. to OECD 301 B
A non GLP preliminary test was carried out to check the toxicity of the test item 2,2’(C12-14 evennumbered alkyl imino) diethanol (batch no.: DEG4451870) to the inoculum (i.e. a non-adapted activated sludge) over a test period of 6 days in the Modified Sturm Test. The study was conducted from 2020-12-03 to 2020-12-09 according to OECD 301 B at the test facility. The test item was tested at a concentration of 15 mg/L with 2 replicates containing the test item and reference item sodium benzoate and with 2 replicates each with test item and reference item with silicone oil, with silica gel and with humid acid, corresponding to a carbon content (TOC) of 10.5 mg C/L in the test vessels. The test vessels were incubated at low light conditions and at a temperature of 22 ± 2 °C.
In all toxicity control replicates with and without carrier a biodegradation of more than 25% was determined within 4 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.

Biodegradation of the toxicity control of test item 2,2’(C12-14 evennumbered alkyl imino) diethanol
Biodegradation [%] Study Day [d]
(mean of two replicates) 1 4 6
Test Item 0 34 66
Test Item with silicone oil 2 37 78
Test Item with silica gel 0 38 77
Test Item with humid acid 2 39 83
Test performance:
Definitive study
All test groups (test item, functional control, toxicity control as well as the inoculum control) were tested with and without Silicone oil.
Test Item
Test Item 2,2’(C12-14 evennumbered alkyl imino) diethanol
Replicates Duplicates
Test concentration 15 mg/L
TOC 0.703 mg C/mg
ThCO2 2.58 mg CO2/mg
Carbon content in 10.5 mg C/L
the vessel
Pre-treatment None

Test Item with Silicone oil
Test Item 2,2’(C12-14 evennumbered alkyl imino) diethanol
Replicates Duplicates
Test concentration 15 mg/L
TOC 0.703 mg C/mg
ThCO2 2.58 mg CO2/mg
Carbon content in 10.5 mg C/L
the vessel
Pre-treatment The test item was applied via a stock solution in silicone oil. The stock solution was prepared by weighing out of 45 g/L test item in a measuring flask and adding the necessary amount of silicone oil. The stock solution was shaken until the test item was fully dissolved. 1 mL was pipetted into the brown glass bottles.
Silicone oil stock solution 450 mg test item / 10 mL silicone oil
Volume of stock solution 1 mL / 3L

Functional control
Reference item Sodium benzoate (SIGMA-ALDRICH)
Test facility ID 70025_1
Batch number BCBQ5652V
CAS No. 532-32-1
Purity 99.6 %
Expiry date 2021-11-15
Replicates Single
Test concentration 20 mg/L
ThTOC 0.58 mg C/mg
ThCO2 2.13 mg CO2/mg
Carbon content 11.6 mg C/L
in the vessel

Toxicity control Test item and reference item in test concentration
Replicates Single

Toxicity control Test item and reference item in test concentration
with Silicone oil
Replicates Single
Silicone oil 1 mL / 3L


Inoculum control Test medium without test and/or reference item
Replicates Duplicates

Inoculum control Test medium without test and/or reference item
with Silicone oil
Replicates Duplicates
Silicone oil 1 mL / 3L

Key result
Parameter:
% degradation (CO2 evolution)
Value:
100
Sampling time:
28 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
48
Remarks on result:
other: with silicone oil
Details on results:
Based on the carbon content a ThCO2 of 2.58 mg CO2/mg test item was calculated. A test concentration of 15 mg/L, corresponding to a carbon content of 10.5 mg C/L in the test vessels was selected.
The total amount of CO2 produced in 28 days was analysed by titration in 11 measurements. The 28 d-values are shown in comparison to the readily degradable functional control in summarized form.
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 6 days and a maximum biodegradation of 100 % on day 28. The adaptation phase of the functional control with silicone oil changed within 6 days into the degradation phase (degradation  10 %). The course of the degradation was fast and the functional control with silicone oil reached the pass level of 60 % within 11 days and a maximum biodegradation of 82 % on day 60. The validity criterion degradation  60 % after 14 days was fulfilled.

In the toxicity control containing both test item and reference item a biodegradation of 57 % was determined within 14 days, with 89 % after 28 days. In the toxicity control containing both test item with silicone oil and reference item a biodegradation of 57 % was determined within 14 days, with 75 % 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 replicates reached the 10 % level (beginning of biodegradation) within 6 days and reached the 60 % pass level within 18 days. The 2nd test item replicate reached the 10 % level (beginning of biodegradation) within 4 days and reached the 60 % pass level within 21 days. The mean biodegradation on day 28 was 100* %.
The 1st test item replicates with silicone oil reached the 10 % level (beginning of biodegradation) within 6 days. The 2nd test item replicate with silicone oil reached the 10 % level (beginning of biodegradation) within 4 days. Both test item replicates with silicone oil did not reach the 60 % pass level within 28 days. The mean biodegradation on day 28 was 48 %.

*values above 100% were set as 100%

Results with reference substance:
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 6 days and a maximum biodegradation of 100 % on day 28. The adaptation phase of the functional control with silicone oil changed within 6 days into the degradation phase (degradation  10 %). The course of the degradation was fast and the functional control with silicone oil reached the pass level of 60 % within 11 days and a maximum biodegradation of 82 % on day 60. The validity criterion degradation  60 % after 14 days was fulfilled.

 


Preliminary Test acc. to OECD 301F


A non GLP preliminary test to check the toxicity of the test item 2,2’(C12-14 evennumbered alkyl imino) diethanol to the inoculum was determined with non-adapted activated sludge in the Manometric Respirometry Test for a period of 14 days. The study was conducted from 2020-12-03 to 2020-12-17, according to OECD guideline 301 F at the test facility. The test item concentration selected as appropriate was 18.5 mg/L, corresponding to a ThOD of 50.1 mg O2/L per test vessel. The test item was tested with reference item sodium benzoate and with test item and silicone oil, silica gel and humid acid as carrier. The oxygen was depleted by the respiration of bacteria and the degradation was followed by measuring the oxygen concentration. The biodegradation rate is therefore expressed as the percentage BOD (biological oxygen demand) and was calculated for each study day.


 


In all toxicity control replicates with and without carrier a biodegradation of more than 25% was determined within 4 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.


 


Biodegradation of the Test Item 2,2’(C12-14 evennumbered alkyl imino) diethanol


      Biodegradation [%]


      Study Day [d]


(mean of two replicates)       1       4       6       12       14


Test Item       1       39       44       44       46


Test Item with silicone oil       3       39       39       39       39


Test Item with silica gel       0       34       40       46       47


Test Item with humid acid       2       33       38       40       40


 


Conclusion


From the preliminary solubility work and the preliminary toxicity tests it was concluded that the best study design is the OECD 301 B with test item with and without silicone oil. Details are given below.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Under the test conditions the test item is classified as readily biodegradable within the
28 day period of the study.
Executive summary:

 


The ready biodegradability of the test item 2,2’(C12-14 evennumbered alkyl imino) diethanol  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 2021-01-07 to 2021-02-05 according to OECD 301 B at the test facility. The test item was tested at a concentration of 15 mg/L with 2 replicates with and without silicone oil corresponding to a carbon content (TOC) of 10.5 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 with and without silicone oil was used as functional control. The percentage degradation of the functional control reached the pass level of 60 % within 6 days and a maximum biodegradation of 100* % on day 28. The percentage degradation of the functional control with silicone oil reached the pass level of 60 % within 11 days and a maximum biodegradation of 82 % on day 28.


 


In the toxicity control containing both test and reference item a biodegradation of 57 % was determined within 14 days, with 89 % after 28 days. In the toxicity control containing both test and reference item with silicone oil a biodegradation of 57 % was determined within 14 days, with 75 % after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.


 


The 1st test item replicates reached the 10 % level (beginning of biodegradation) within 6 days and reached the 60 % pass level within 18 days. The 2nd test item replicate reached the 10 % level (beginning of biodegradation) within 4 days and reached the 60 % pass level within 21 days. The mean biodegradation on day 28 was 100* %.


The 1st test item replicates with silicone oil reached the 10 % level (beginning of biodegradation) within 6 days. The 2nd test item replicate with silicone oil reached the 10 % level (beginning of biodegradation) within 4 days. Both test item replicates with silicone oil did not reach the 60 % pass level within 28 days. The mean biodegradation on day 28 was 48 %.


 


*values above 100% were set as 100%


 


 


Biodegradation of the Test Item 2,2’(C12-14 evennumbered alkyl imino) diethanol in Comparison to the Functional Control and Toxicity Control


      Biodegradation [%]


      Study Day [d]


      6       14       21       28


Test Item, 1st Replicate*       15       53       93       100


Test Item, 2nd Replicate       25       45       82       97


Test Item with silicone oil, 1st Replicate       15       31       36       47


Test Item with silicone oil, 2nd Replicate       21       30       38       48


Functional Control*       61       89       98       100


Functional Control with silicone oil       21       65       72       82


Toxicity Control


Test item + Reference item       40       57       69       89


Toxicity Control with silicone oil


Test item with silicone oil+ Reference item       38       57       65       75


*values above 100% were set as 100%

Description of key information

The registration substance is readily biodegradable according to a study performed according to OECD301B.


A primary fatty amine ethoxylate is a chemical consisting of a hydrophilic group linked to a hydrophobic moiety. Biodegradation of both moieties of surfactants requires the concerted action of at least two micro-organisms as a single organism usually lacks the full complement of enzymatic capabilities (Ginkel, CG van ; Complete degradation of xenobiotic surfactans by consortia of aerobic microorganisms ; 1996 ; Biodegradation 7; 151-164). In ready biodegradability tests, the two moieties of this fatty amine derivative are therefore degraded sequentially.The degradation curve will therefore be the sum of two growth curves. Furthermore, the same is true for UVCBs and multi-constituent substances, as the single constituents will degrade following different curves, too. The biodegradation of the two moieties and for the different constituents may be fully in line with the time-day window criterion when judged as separate chemicals. The time window criterion was developed on the assumption that a compound is degraded according to the “standard” growth curve in ready biodegradability tests and the time-window should therefore be ignored as a pass fail criterion for these surfactants.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information