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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-07-07 to 2016-08-05
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Test item A130 6-Methoxy-2-methylamino-3-Aminopyridin HCl
Trade name 3-Amino-2-methylamino-6-methoxypyridine HCl
Batch number 20151207
CAS No. 83732-72-3
Purity (certified) 93.36/ 93.69 % (Photometric)
99.63% (HPLC area-%)
Sum formula C7H13Cl2N3O
Water solubility >100 g/L
TOC* 37.0%
Appearance Grey, red, brown powder
Expiry date 2017-12-07
Recommended storage Keep containers tightly closed in a cool and well-ventilated place


* The TOC was calculated at the test facility.

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/ activated sludge (e.g. location, sampling depth, contamination history, procedure):
Municipal sewage treatment plant, D-31137 Hildesheim, Germany
- Receipt: 2016-06-30
- 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 ½ 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). 10 mL/L of this mixture were used to initiate inoculation.
Colony forming units in the test vessel: approx. 10^7 - 10^8 CFU/L
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
29 other: mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral salts medium acc. to OECD 301 B / CO2 Evolution Test
- Test temperature: Nominal 22 ± 2 °C, actually measured 20 – 24 °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 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.

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. The test item dispersions and the reference item were transferred to the respective incubation vessels. The vessels were made up to 3 L with ultrapure water and connected to the system for the production of CO2 free air.

On day 28, 1 mL 37 % HCl was added to each of the vessels. Aeration was continued for further 24 h and the quantity of CO2 released was determined.



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

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


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

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

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


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

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

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

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

Results and discussion

% Degradation
Key result
Parameter:
% degradation (CO2 evolution)
Value:
8
Sampling time:
28 d
Details on results:
Based on the carbon content a ThCO2 of 1.35 mg CO2/mg test item was calculated. A test concentration of 29 mg/L, corresponding to a carbon content of 10.7 mg C/L in the test vessels was selected.


Colony Forming Units of the Inoculum
Colony forming units (CFU) of the inoculum for the Modified Sturm Test were determined prior to test start by standard dilution plate count: approx. 1.97  109 CFU/L, corresponding to approx. 1.97  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 85% on day 28. The validity criterion degradation  60% after 14 days is fulfilled.

In the toxicity control containing both test and reference item a biodegradation of 24% was determined within 14 days and it came to 28% after 28 days. The biodegradation of the reference item was slightly inhibited by the test item in the toxicity control, the validity criterion was marginally missed. Since the test item replicates showed a tendency towards the end of the study to show degradation, a major toxicity seems unlikely.

The biodegradation of the test item is shown in comparison to the readily degradable functional control and the toxicity control. Both test item replicates did not reach the 10% level (beginning of biodegradation) until test end. The mean biodegradation on day 28 was 8%.

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 26.3 mg CO2/L after 28 days.

BOD5 / COD results

Results with reference substance:
In the toxicity control containing both test and reference item a biodegradation of 24% was determined within 14 days and it came to 28% after 28 days. The biodegradation of the reference item was slightly inhibited by the test item in the toxicity control, the validity criterion was marginally missed. Since the test item replicates showed a tendency towards the end of the study to show degradation, a major toxicity seems unlikely.

Applicant's summary and conclusion

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 A130 6 -Methoxy-2 -methylamino-3 -Aminopyridin HCl (batch no.: 20151207) 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 2016 -07 -07 to 2016 -08 -05 according to OECD 301 B at the test facility.The test item was tested at a concentration of 29 mg/L with 2 replicates corresponding to a carbon content (TOC) of 10.7 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 85% on day 28.

 

In the toxicity control containing both test and reference item a biodegradation of 24% was determined within 14 days and it came to 28% after 28 days. The biodegradation of the reference item was slightly inhibited by the test itemin the toxicity control, the validity criterion was marginally missed. Since the test item replicates showed a tendency towards the end of the study to show degradation, a major toxicity seems unlikely.

 

The biodegradation of the test item is shown in comparison to the readily degradable functional control and the toxicity control. Both test item replicates did not reach the 10 % level (beginning of biodegradation) until test end. The mean biodegradation on day 28 was 8%.

 

 

 

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

 

Biodegradation of the Test Item A130 6 -Methoxy-2 -methylamino-3 -Aminopyridin HCl in Comparison to the Functional Control and Toxicity Control

 

Biodegradation [%]

 

Study Day [d]

 

6

14

21

28

Test Item, 1stReplicate

4

4

4

6

Test Item, 2ndReplicate

3

3

5

9

Functional Control

55

81

84

85

Toxicity Control
test item + reference item

22

24

26

28