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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:
07 May - 04 Jun 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
EEC Publication No. L 142/496, May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
adopted July 17, 1992
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Aerobic activated sludge, microorganisms from a domestic waste water treatment plant was supplied by the sewage treatment plant Rossdorf, Germany.
- Storage conditions: 22°C ± 1°C, Darkness
- Storage length: overnight
- Preparation of inoculum for exposure: The aerobic activated sludge used for this study was deposited for 15 min, washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in test water and centrifuged again. This procedure was done three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 3.5 g dry material per litre were mixed with test water (see 6.5) and aerated overnight. This suspension was used for the experiment.

- Concentration of sludge: 3.5 g dry material per litre
- Water filtered: no
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
171 mg/L
Based on:
ThOD
Remarks:
(ThODNO3)
Initial conc.:
150.2 mg/L
Based on:
ThOD
Remarks:
(ThODNH4)
Initial conc.:
102.7 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Remarks:
The change of pressure in the test flasks was measured by means of a manometric method (BSB-Sensomat-System©).
Details on study design:
TEST CONDITIONS
- Composition of medium: Reconstituted Test Water
- Test temperature: 22 °C ± 1 °C
- pH: 7.5 (measured at the start of the test), 7.3 to 7.6 (measured at the end of the test)
- pH adjusted: The pH of the test water was 7.7 and was adjusted to pH 7.5 with 1 M HCl solution.
- Aeration of dilution water: no
- Suspended solids concentration: 3.5 g dry material per litre
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: BSB-Sensomat-System©
- Number of culture flasks/concentration: 2 per test item treatment and inoculum control, 1 per procedure control, abiotic control and toxicity control
- Method used to create aerobic conditions: aerated of the prepared sludge suspension overnight before test start
- Measuring equipment: The change of pressure in the test flasks was measured by means of a manometric method (BSB-Sensomat-System©).
- Test performed in closed vessels: The test flasks were closed gas-tight by a measuring head.
- Details of trap for CO2 and volatile organics if used: The pressure decrease in the reaction vessels was measured over complete experimental phase of 28 days using the BSB-Sensomat-System©. Potassium hydroxide solution (45%) 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.

CONTROL AND BLANK SYSTEM
- Inoculum blank: contaning activated sludge and test water (2 replicates)
- Abiotic sterile control: containing test item, CuSO4 solution and test water (1 replicate)
- Toxicity control: containing test item, reference item, activated sludge and test water (1 replicate)

STATISTICAL METHODS: No
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Preliminary study:
none
% Degradationopen allclose all
Key result
Parameter:
% degradation (O2 consumption)
Value:
36.5
Sampling time:
28 d
Remarks on result:
other: based on ThODNO3
Key result
Parameter:
% degradation (O2 consumption)
Value:
41.5
Sampling time:
28 d
Remarks on result:
other: based on ThODNH4
Details on results:
Nitrogen is part of the molecular structure of the test item; therefore nitrification (ThODNO3) was considered for the evaluation of the results. The criterion for ready biodegradability under the conditions of a manometric respirometry test is the degradation of the test item of at least 60%, reached within a 10-day window; the 10-day window starts when the degradation of the test item reaches at least 10% degradation.
The 10-day window started at day 2, the mean degradation of DFPP malat was 17% and 15% at day 2 based on ThODNH4 and ThODNO3, respectively. At the end of the 10-day window at day 12, the mean degradation of DFPP malat was 30% and 27% based on ThODNH4 and ThODNO3, respectively. The degradation at test end at day 28 was 41.5% based on ThODNH4 and 36.5% ThODNO3.

BOD5 / COD results

Results with reference substance:
The reference item sodium benzoate was sufficiently degraded to 82% after 14 days and to 93% after 28 days of incubation.
The percentage biodegradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum.

Any other information on results incl. tables

Results:


In the toxicity control containing both, the test item and the reference item sodium benzoate, 48% (ThODNH4) biodegradation was noted within 14 days and 53% (ThODNH4) biodegradation after 28 days of incubation (45% and 50% based on ThODNO3).























































































































































































































Time
% biodegradation


% biodegradation


% biodegradation


% biodegradation

daystest item 1test item 2sodium benzoatetoxicity control
1

9



9



24



16


2

14



15



38



23


3

16



16



46



27


4

17



18



53



32


5

17



18



59



33


6

20



21



65



36


7

20



21



68



41


8

20



24



71



42


9

23



24



74



44


10

23



24



74



44


11

23



24



76



44


12

26



27



76



45


13

26



27



79



45


14

26



27



82



45


15

29



30



82



45


16

29



30



85



47


17

29



30



85



47


18

29



30



88



47


19

32



33



88



48


20

32



33



88



48


21

32



33



91



48


22

32



35



91



49


23

35



35



91



49


24

35



35



94



49


25

35



35



94



49


26

33



37



93



50


27

36



37



93



50


28

36



37



93



50



 


Validity Criteria: 









































Validity criteriavalue required by guidelineobtained valuecriteria fulfilled
oxygen demand of the inoculum control60 mg O2/L within 28 days7.5 mg O2/Lyes
pH-value of the test item flasks at the endpH 6.0 to 8.57.5 and 7.6yes
degradation of reference item sodium benzoate> 60% within 14 days60% after 6 days of incubation and to 82% after 14 days.yes
difference of duplicate values for the degradation of the test item at test end< 20%1%yes
 toxicity test, containing both the test item and a reference item < 25% biodegradation (based on ThODNH4) occurred within 14 daysThe biodegradation was 48% at day 14the test item was not inhibitory.

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
see above
Interpretation of results:
not readily biodegradable
Conclusions:
The degradation rate of the test item did not reach 60% within the 10-day window or after 28 days. Therefore, the test item is considered to be not readily
biodegradable based on ThODNH4 and ThODNO3.
Executive summary:

Test Design: The test item was investigated for its ready biodegradability in a manometric respirometry test over a period of 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.


Endpoints: Degradation rate of test item calculated by the oxygen consumption of the aerobic activated sludge microorganisms after 28 days of incubation.


Test Item Loading Rate (initial concentration in medium C0): 102.7 mg/L corresponding to an oxygen demand of about 150.2 mg/L (ThODNH4) and 171.0 mg/L (ThODNO3)


Reference Item: Sodium benzoate, 102.0 mg/L corresponding to an oxygen demand of about 170.0 mg/L (ThODNH4


Test Conditions: 22 °C ± 1 °C, darkness


Results: Nitrogen is part of the molecular structure of the test item; therefore the evaluation of biodegradation has to be based on ThODNH4 and ThODNO3. The criterion for ready biodegradability under the conditions of a manometric respirometry test is the degradation of the test item of at least 60%, reached within a 10-day window; the 10-day window starts when the degradation of the test item reaches at least 10% degradation. The 10-day window started at day 2, the mean degradation of DFPP malat was 17% and 15% at day 2 based on ThODNH4 and ThODNO3, respectively. At the end of the 10-day window at day 12, the mean degradation of DFPP malat was 30% and 27% based on ThODNH4 and ThODNO3, respectively. The degradation at test end at day 28 was 41.5% based on ThODNH4 and 36.5% ThODNO3.


Therefore, the test item is considered to be not readily biodegradable based on ThODNH4 and ThODNO3.