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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April - May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
GLP compliance:
yes
Remarks:
The guidelines of chemical testing good laboratory practices (HJ/T 155)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Activated sludge was obtained from a sewage plant treating predominantly domestic sewage (Nanjing Chengdong Sewage Treatment Plant) (Batch No.: AS201604051).
The sludge was washed with basal mineral salt medium (BSM) by settling the sludge in a graduated cylinder, decanting the supernatant liquid, replacing with fresh BSM, aerating, and settling. The above washing process was repeated 3 times, then the sludge was centrifuged one time (2500 r/min, 4°C, 10 minutes) and the supernatant liquid decanted. Thereafter, a small amount of the washed sludge was weighed and then dried at 100°C until the weight of the dried sludge did not change significantly anymore. Then the dry weight percentage of suspended solids (p) of the activated sludge was calculated to be 10%. Finally 40 g wet sludge after centrifugation was mixed with 1 L BSM (Batch No.: BSM20160408) to obtain an activated sludge with a mixed liquor suspended solids level of 4 g/L. Cell density was determined to be 7.2×108 CFU/L using microscope.
Duration of test (contact time):
28 d
Initial conc.:
50 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Key result
Parameter:
% degradation (O2 consumption)
Value:
81.2
Sampling time:
28 d
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The results show that under the experimental conditions, based on calculation of ThODNO3, biodegradation rate of the test substance (Genamin DMG 75) was 81.2% at the end of the 28-day study, and 62.4% at 10-d window.
Executive summary:

According to “The guidelines for the testing of chemicals”, SEPA (HJ/T 153-2004), “The Guidelines for the Testing of Chemicals, Degradation and Accumulation” (2nd edition),Beijing: China Environmental Press. 2013, “Chemicals -Readybiodegradability - Manometric respirometry test” (GB/T 21801-2008); and with reference to Procedure 301F of the “Guidelines for Testing of Chemicals” of the OECD:“Manometric Respirometry Test” (1992), the ready biodegradability ofGenamin DMG 75was determined in a 28-day oxygen consumption test (OECD 301F) using activated sludge from a domestic waste water treatment plant as the source of the microbial inoculum.

The initial concentration of the test substance in the reaction vessels was approximately50 mg/L. The microbial inoculum concentration (mixed liquor suspended solids) was 30 mg/L.

The study met the validation criteria specified in the above testing guidelines. During the test, the temperature was kept at(22±1)°C.The pHvalueof the content in the test container was maintained between 6.84 and 7.55.The total oxygen uptake in the inoculum blank was38.8mgO2/L at the end of the study, not exceeding 60 mg O2/L. Biodegradation of the reference substance, sodium benzoate, reached80.2%at14 days(>60%). The difference between replicate values was less than 20% during the study.Biodegradation of inhibition control was 75.4% at 14 days(> 25%), and the oxygen consumption by the test substance is great than 60% ofinoculum blankduring the test period, indicating that there was no inhibition effect to inoculum.Thus, the test was considered valid.

Under the valid conditions,based on calculation of ThODNO3,biodegradationrateof the test substance (Genamin DMG 75)was81.2% at the end of the 28-day study, and 62.4% at 10-d window.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2016-02-18 to 2016-04-19
Reliability:
1 (reliable without restriction)
Qualifier:
according to
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 Genamin DMG 75
Batch number RAK-KRS 00022
CAS No. 76326-99-3
Molecular formula C8H19NO5
Molecular weight 209.24 g/mol
Purity (certified) N,N-Dimethyl glucamine 71.2 % (w/w)
N-Methyl glucamine 0.4 % (w/w)
Sorbitol 2.5 % (w/w)
Total of other unspecified constituents 1.1 % (a/a)
Water 24.8 % (w/w)
Water solubility Soluble
TOC* 33.86 %
Appearance Light yellow liquid
Expiry date 2018-04-01
Recommended storage Sensitive to freezing. Keep container tightly closed.

*) The TOC was calculated at the test facility.


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: 2016-02-10
- 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. 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 (7 days). 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):
60 d
Initial conc.:
30 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 - 24.0 °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 test item stock solution of 9000 mg/L was prepared by weighing out the test item and filling up with ultrapure water in a measuring flask. The concentration of the test item, 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 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 reference item was weighed out. The reference item was transferred to the respective incubation vessels. 10 mL of the test item 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 60, 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
Key result
Parameter:
% degradation (CO2 evolution)
Value:
11
Sampling time:
60 d
Details on results:
Based on the carbon content a ThCO2 of 1.24 mg CO2/mg test item was calculated. A test concentration of 30 mg/L, corresponding to a carbon content of 10.2 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. 0.83 x 109 CFU/L, corresponding to
approx. 0.83 x 107 CFU/L in the test vessel.



The adaptation phase of the functional control changed within 4 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 maximum biodegradation of 83 % on day 60. The validity criterion degradation  60 % after 14 days is fulfilled.

In the toxicity control containing both test and reference item a biodegradation of 45 % was determined within 14 days and it came to 45 % after 28 days and 56 % after 60 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 Figure 1 in comparison to the readily degradable functional control and the toxicity control. The 1st test item replicate did not reach the 10 % level (beginning of biodegradation) until test end and the 2nd test item replicate reached the 10 % level on day 14. Both test item replicates did not reach the 60 % pass level until test end. The mean biodegradation on day 28 was 8 % and on day 60, 11 %.

Under the test conditions the test item is classified as not readily biodegradable within the 60 day period of the study.



In the inoculum control the total CO2 production was 48.5 mg CO2/L after 28 days and
84.1 mg CO2/L after 60 days.
Results with reference substance:
In the toxicity control containing both test and reference item a biodegradation of 45 % was determined within 14 days and it came to 45 % after 28 days and 56 % after 60 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 60 day period of the study.
Executive summary:

The ready biodegradability of the test item Genamin DMG 75 was determined with a non-adapted activated sludge over a test period of 60 days in the Modified Sturm Test. The study was conducted from 2016-02-18 to 2016-04-19 according to OECD 301 B at the test facility. The test item was tested at a concentration of 30 mg/L with 2 replicates corresponding to a carbon content (TOC) of 10.2 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 60 by acidification of the test solutions. The last titration was made on day 61 after residual CO2had 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 83 % on day 60.

 

In the toxicity control containing both test and reference item a biodegradation of 45 % was determined within 14 days and it came to 45 % after 28 days and 56 % after 60 days. The biodegradation of the reference item was not inhibited by the test itemin 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 1sttest item replicate did not reach the 10 % level (beginning of biodegradation) until test end and the 2nd test item replicate reached the 10 % level on day 14. Both test item replicates did not reach the 60 % pass level until test end. The mean biodegradation on day 28 was 8 % and on day 60, 11 %.

Under the test conditions the test item is classified as
not readily biodegradable
within the 60 day period of the study.

 

Biodegradation of the Test Item Genamin DMG 75 in Comparison to the Functional Control and Toxicity Control

 

Biodegradation [%]

 

 

Study Day [d]

 

6

14

21

28

40

49

60

Test Item, 1stReplicate

2

2

2

2

6

6

6

Test Item, 2ndReplicate

6

10

14

14

14

14

16

Functional Control

54

76

78

79

80

80

83

Toxicity Control
test item + reference item

30

45

45

45

45

48

56

Description of key information

There are two tests available. In a manometric respirometry test according OECD 301 F the ready biodegradability of Genamin DMG 75was determined in a 28-day oxygen consumption test using activated sludge from a domestic waste water treatment plant as the source of the microbial inoculum. Under the valid conditions,based on calculation of ThODNO3,biodegradationrateof the test substance (Genamin DMG 75)was81.2% at the end of the 28-day study, and 62.4% at 10-d window. In a second test accordiing OECD 301 B a mean degradation on day 28 was 8 % and on day 60, 11%.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Both tests are valid. According ECHA Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7b: Endpoint specific guidance, R.7.9.5. Conclusion for degradation / biodegradation, Version 3.0, February 2016 it can be concluded that the substance is readily biodegradable.

 

“When conflicting results in ready biodegradability tests are obtained the positive results could be considered valid irrespective of negative results, when the scientific quality of the former is good and the positive test results are well documented, including assurance of the use of non-pre-exposed (non-adapted) inoculum.”