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Environmental fate & pathways

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:
2018-06-14 to 2018-11-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
adopted July 17, 1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
May 2008
Deviations:
no
Principles of method if other than guideline:
The test item was investigated for its ready biodegradability in a manometric respirometry test over a period of 60 days. The mean biodegradation after 28 days was 54% (ThODNH4, ThODNO3). Since the test item showed biodegradation closely below 60% after 28 days, and it seemed that the plateau of biodegradation was not reached, the test was prolonged to 60 days.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: AH17.001
- Expiration date of the lot/batch: 2019-06-30
- Aggregate state at room temperature: Liquid
- Color: Brown-Orange
- Storage conditions at Test Facility: 4°C +/- 4°C
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): Aerobic activated sludge, microorganisms from a domestic waste water treatment plant was supplied by the sewage treatment plant Rossdorf, Germany.
- Pretreatment: The aerobic activated sludge used for this study was deposited for 60 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 and aerated overnight. This suspension was used for the experiment.
- Concentration of sludge: 3.5 g dry material per litre (final sludge concentration in test flasks: 28.7 mg sludge/L)
- Water filtered: Reconstituted water was prepared with pure water
Duration of test (contact time):
60 d
Initial conc.:
102.7 mg/L
Based on:
test mat.
Remarks:
102.7 mg/L corresponding to an oxygen demand of about 284.6 mg/L (ThODNH4) and 286.0 mg/L (ThODN03)
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium:
For preparation of the mineral medium 50 mL of solution (A) and 5 mL of each stock solution (B), (C), and (D) were added and filled up to the final volume of 5000 mL with pure water. Before use, the mineral medium is aerated for about one hour and the pH is adjusted to 7.4 ± 0.2.

A: Potassium dihydrogenphosphate (KH2PO4): 8.50 g,
Dipotassium hydrogenphosphate (K2HPO4): 21.75 g,
Disodium hydrogenphosphate dihydrate (Na2HPO4 * 2 H2O): 33.40 g, and
Ammonium chloride (NH4Cl): 0.50 g are dissolved in demineralised water and made up to 1 litre.
B: Magnesium sulfate heptahydrate (MgSO4 * 7H2O): 11.25 g is dissolved in demineralised water and made up to 0.5 litre.
C: Calcium chloride dihydrate (CaCl2 * 2H2O): 18.2 g is dissolved in demineralised water and made up to 0.5 litre.
D: Iron (III) chloride hexahydrate (FeCl3 * 6H2O): 0.125 g is dissolved in demineralised water, stabilised with one drop of concentrated HCl and made up to 0.5 litre.

- Test temperature: 22°C ± 1°C
- pH: 7.4 (measured at the start of the test), 7.4 (measured at the end of the test)
- pH adjusted: yes
- Continuous darkness: yes
- Additional substrate: no
- Solubilising agent (type and concentration if used): no
- Suspended solids concentration: Stock suspension of 3.5 g/L on dry matter base (final sludge concentration in test flasks: 28.7 mg sludge/L)


TEST SYSTEM
- Culturing apparatus: BSB/BOD-Sensor-System, Aqualytic Dortmund, Germany; Manometric Test System with test flasks containing a volume of approximately 500 mL.
- Number of culture flasks/concentration: 7 flasks: 2 test item, 2 Inoculum control, 1 Procedure control, 1 Abiotic control, 1 toxicity control
- Preparation of Test Flasks: The amounts of test item and reference item were directly weighed into the test flasks. No emulsifiers or solvents were used, but the solutions were dispersed by stirring to achieve a homogeneous solution of the test item.
- Incubation: The closed test flasks were incubated in a climatized chamber under continuous stirring. The consumption of oxygen was determined by measuring the change of pressure in the flasks. Evolved carbon dioxide was absorbed in an aqueous solution (45%) of potassium hydroxide.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 replicates
- Abiotic sterile control: 1 replicate
- Toxicity control: 1 replicate
- Other: Procedure control – 1 replicate
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
54
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
65
Sampling time:
60 d
Details on results:
The mean biodegradation of 10% of Jasmine Absolute was reached at day 1 (ThODNH4, ThODNO3). At the end of the 10-day window at day 10, the mean degradation of Jasmine Absolute was 37% (ThODNH4, ThODNO3). The mean biodegradation after 28 days was 54% (ThODNH4, ThODNO3). Since the test item showed biodegradation closely below 60% after 28 days, and it seemed that the plateau of biodegradation was not reached, the test was prolonged to 60 days. At test end after 60 days the mean biodegradation was 66% (ThODNH4) and 65% (ThODNO3).

Table 3: Validity criteria

Inoculum Control:

The oxygen demand of the inoculum control (medium and inoculum) was 7.5 mg O2/L and thus not greater than 60 mg O2/L within 28 days as required by the test guideline.

pH-Value:

The pH-value of the test item flasks at the end of the test was 7.4 and therefore within the range of pH 6.0 to 8.5 as required by the test guideline.

Reference Item:

The percentage degradation of the reference item should reach the level for ready biodegradability (>60%) within 14 days as required by the test guideline.

The reference item sodium benzoate was degraded to more than 60% after 5 days of incubation.

Test Item:

The difference of duplicate values for the degradation of the test item at the end of the 10-day window was less than 20%. The difference of duplicate values at day 28 differed by 0%.

The validity criterion was fulfilled.

Toxicity Control:

If in a toxicity test, containing both the test item and a reference item less than 25% biodegradation (based on total ThOD) occurred within 14 days, the test item can be assumed to be inhibitory. The biodegradation was 46% at day 14; the test item was not inhibitory.

 

Table 4. Biodegradation in the Toxicity Control

Percentage Biodegradation:

In the toxicity control containing both, the test item and the reference item sodium benzoate, 54% (ThODNH4) biodegradation was noted within 14 days and 58% (ThODNH4) biodegradation after 28 and 60 days

Conclusion:

According to the test guidelines the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days.

 

 

Abiotic Control

Oxygen Demand:

The oxygen demand in the abiotic control was 0 mg/L during the test duration.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The degradation rate of Jasmine Absolute didn’t reach 60% within the 10-day window or after 28 days. Therefore, the test item cannot be considered as readily biodegradable according to OECD 301 F guideline. The mean biodegradation after 28 days was 54% of ThOD. Since the test item showed biodegradation closely below 60% after 28 days, and it seemed that the plateau of biodegradation was not reached, the test was prolonged to 60 days. At test end after 60 days the mean biodegradation was 66% (ThODNH4) and 65% (ThODNO3). As a consequence, Jasmine Absolute is considered to be not readily biodegradable based on ThODNH4 and ThODNO3. However, Jasmine Absolute shows 66% (ThODNH4) and 65% (ThODNO3) biodegradation after 60 days and could therefore be considered as partially (inherent) biodegradable.
Executive summary:

The ready biodegradability of Jasmine Absolute was assessed in a Manometric Respirometry Test according to OECD 301F and under GLP. The ready biodegradability was evaluated over a period of 60 days since the pass level curve for degradation showed that biodegradation has started but that the plateau has not been reached by day 28.

The biodegradation of the test item was studied in reconstituted water inoculated with activated sludge from a municipal wastewater treatment plant with a concentration corresponding to approx. 30 mg dry solids per litre. The manometric respirometry test determines the ready biodegradability of test items following their oxygen consumption in closed flasks at a constant temperature of 22 ± 1°C. The consumption of oxygen is determined by measuring the negative pressure in the flasks after absorption of the evolved carbon dioxide in potassium hydroxide. A solution or suspension of the test item in a mineral medium, corresponding to 102.7 mg/L test item giving approx. 285 mg/L of theoretical oxygen demand (ThOD), is inoculated with activated sludge (28.7 mg d.s./L) for 60 days.

The degradation rate of the test item is calculated by the oxygen consumption of the aerobic activated sludge (non-adapted domestic sewage) microorganisms after 28 days of incubation. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.

The mean biodegradation of 13% of Jasmine Absolute was reached at day 1 (ThODNH4, ThODNO3). At the end of the 10-day window at day 10, the mean degradation of Jasmine Absolute was 37% (ThODNH4, ThODNO3). The mean biodegradation after 28 days was 54% (ThODNH4, ThODNO3). Since the test item showed biodegradation closely below 60% after 28 days, and it seemed that the plateau of biodegradation was not reached, the test was prolonged to 60 days. At test end after 60 days the mean biodegradation was 66% (ThODNH4) and 65% (ThODNO3). As a consequence, Jasmine Absolute is considered to be not readily biodegradable based on ThODNH4 and ThODNO3. Still Jasmine Absolute shows 66% (ThODNH4) and 65% (ThODNO3) biodegradation after 60 days and could therefore be considered as partially (inherent) biodegradable. In the toxicity control containing both, the test item and the reference item sodium benzoate, 54% (ThODNH4 / ThODNO3) biodegradation was noted within 14 days and 58% (ThODNH4 / ThODNO3) after 28 and 60 days. According to the test guidelines, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days. The test is valid according to OECD Test Guideline 301F (July 1992).

Description of key information

The mean biodegradation of Jasmine Absolute determined according to OECD guideline 301 F within 28 days was 54 % of ThOD.The test item did not reach the criteria for ready biodegradability (60 % ThOD within a 10-d window).

Since the test item showed biodegradation closely below 60% after 28 days, and it seemed that the plateau of biodegradation was not reached, the test was prolonged to 60 days. At test end after 60 days the mean biodegradation was 66% (ThODNH4) and 65% (ThODNO3). As a consequence, Jasmine Absolute is considered to be not readily biodegradable based on ThODNH4 and ThODNO3. However, Jasmine Absolute shows 66% (ThODNH4) and 65% (ThODNO3) biodegradation after 60 days and could therefore be considered as partially (inherent) biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable
Type of water:
freshwater

Additional information