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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:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study is performed according to the current OECD and GLP guidelines.
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Type of inoculum: mixed population of aquatic organisms (activated sludge)
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): aeration tank of a wastewater plant treating predominantly domestic sewage (Wupper area water authority, WWTP Odenthal)
- Date of collection: 2012-07-02
- Concentration of inoculum: 30 mg/L suspended solids
- Preparation of inoculum for exposure: see pre-treatment
- Pretreatment: (1) sludge was washed twice by adding mineral medium and centrifuging for 10 min at 2000 rpm at 20° and decanting of supernatant, (2) an aliquot of the wet sludge was dried to determine wwt/dwt ratio of the sludge and prepare a stock suspension of 3 g dw/L, (3) the calculated amount of sludge, needed to achieve 300mL of this stock suspension was dissolved in mineral medium & filled up to a defined end volume, (4) inoculum was stored for one day before use (at room temperature under continuous shaking with aeration).
- Concentration of sludge: 3 g dw/L
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Details on study design:
In practice, a suspension of 100 mg/L test item in a mineral medium, equalling to 50-100 mg ThOD or COD/Litre as the nominal sole source of organic carbon, was stirred in a closed flask and inoculated at a constant temperature (22 +/- 1 °C) for up to 28 days under aerobic conditions in the dark. The consumption of oxygen (BOD) was determined by measuring the drop in pressure in the automated respirometer flasks. Evolved carbon dioxide was absorbed in sodium hydroxide. The amount of oxygen taken up by the test item (corrected for uptake by blank inoculum, run in parallel) was expressed as a percentage of chemical oxygen demand (COD). The endogenous activity of the inoculum was checked running parallel blanks with inoculum but without test item. A reference compound (sodium benzoate) was run in parallel to check the operation of the procedures. A toxicity control (test item and reference compound mixed) was run in parallel, to ensure that the chosen concentration of the test item was not inhibitory to microorganisms.

Degradation was followed by the determination of oxygen uptake and measurements were taken at frequent intervals to allow the identification of the beginning and end of biodegradation and the slope of the biodegradation curve. The test lasted for 28 days. Because of the nature of biodegradation and of the mixed bacterial populations used as inoculum, determinations of test item and of reference compound in duplicate and inoculum blank were carried out in triplicate. The oxygen uptake was calculated from the readings taken at regular and frequent intervals, using the method given by the manufacturer of the equipment. At the end of incubation, the pH was measured in the flasks.

The degradation rate of the test item was set in relation to its experimentally determined COD. As the COD implicitly covers the oxygen demand for the nitrification process, there was no need to take additional correction measures into account.

Summary of exposure conditions:
- Test volume: 250 mL
- Test apparatus: OxiTopControl System (WTW)
- Mixing: 1 magnetic stirrer per test vessel
- Incubation time: 28 days
- Incubation temperature: 22+/- 1°C
- Medium: mineral medium was composed from stock solutions of mineral components: mineral salt solution, magnesium sulphate solution, calcium chloride solution and iron (III) chloride solution (for more details see actual study report)

Reference substance:
benzoic acid, sodium salt
Remarks:
Sigma-Aldrich, Batch no 1438955, purity 99.7%, concentration: 100mg/L
Preliminary study:
no preliminary study was performed
Test performance:
- pH values after 28 days: 7.2 to 7.8
- Chemical oxygen demand (analytically determined): 2380 mg O2/mg
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
7 d
Parameter:
% degradation (O2 consumption)
Value:
19.7
Sampling time:
14 d
Parameter:
% degradation (O2 consumption)
Value:
52
Sampling time:
21 d
Parameter:
% degradation (O2 consumption)
Value:
60.9
Sampling time:
28 d
Results with reference substance:
- Chemical oxygen demand (analytically determined): 1665 mg O2/mg
- The reference compound sodium benzoate showed 85.1% degradation after 14 days.
Validity criteria fulfilled:
yes
Remarks:
all criteria were fulfilled
Interpretation of results:
readily biodegradable, but failing 10-day window
Conclusions:
Within 28 days, a degradation rate of 60.9% was determined for the test substance of concern. In conclusion, DBPP is considered to be readily biodegradable without reaching the 10-day window.
Executive summary:

The study was conducted in accordance with the Council Regulation (EC) No 440/2008, Method C.4-D “Manometric Respirometry Test“(2008) and OECD301F (adopted July 1992).

A suspension of Dibutyl phenyl phosphate in a mineral medium was inoculated and incubated for 28 d under aerobic conditions. During this period, degradation was followed by continuous automated BOD determinations.

Dibutyl phenyl phosphate showed:

- 0.0 % degradation after 7 days

- 19.7 % degradation after 14 days

- 52.0 % degradation after 21 days

- 60.9 % degradation after 28 days

Therefore, Dibutyl phenyl phosphate is considered to be “Readily Biodegradable without reaching the ten-day-window“.

The reference compound sodium benzoate showed 85.1 % degradation after 14 days.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was carried out according to guideline and under GLP. Detailed report available.
Qualifier:
according to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Qualifier:
according to
Guideline:
other: The Test Method Relating To New Chemical Substances (Yakushokuhatso No. 1121002, Heisei 15.11.13 Seiyoku No. 2, Kanpokihatsu No. 031121002, November 21, 2003; the latest revision)
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
Mixed liquor supsended solid (MLSS): 3250 mg/L (BOD measurement); 2900 mg/L (recovery test)
Source: Chemicals Evaluation and Research Institute, Japan
Duration of test (contact time):
ca. 28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
The test substance was exposed to activated sludge in a closed-system oxygen consumption measuring apparatus. The biochemical oxygen demand (BOD) was measured over a 28-day period. After this period, the amount of the dissolved organic carbon (DOC) and the residual amount of the test substance were measured. The biodegradability of the test substance was evaluated from these results.
Reference substance:
aniline
Remarks:
100 mg/L
Parameter:
% degradation (O2 consumption)
Value:
ca. 11
Sampling time:
28 d
Remarks on result:
other: DBPP (multi-constituent)
Parameter:
% degradation (test mat. analysis)
Value:
ca. 9
Sampling time:
28 d
Remarks on result:
other: DBPP (multi-constituent)
Parameter:
% degradation (test mat. analysis)
Value:
ca. 0
Sampling time:
28 d
Remarks on result:
other: TBP (mono-constituent)
Parameter:
% degradation (test mat. analysis)
Value:
ca. 2
Sampling time:
28 d
Remarks on result:
other: DBPP (mono-constituent)
Parameter:
% degradation (test mat. analysis)
Value:
> 98
Sampling time:
28 d
Remarks on result:
other: BDPP (mono-constituent)
Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
Based on the results of this test, the substance cannot be regarded as readily biodegradable under the tested conditions.
Executive summary:

Ready biodegradability was assessed based on Biochemical Oxygen Demand (BOD) and measurement of the residual test substance amounts. The degradability based on the BOD measurement was ca. 11%, while that based on residual test substance amount was ca. 9%.

The degradability of each component of the test substance was calculated by each peak on the LC/MS chromatograms for measurement of the residual test substance amount. Degradability of tri-butyl phosphate ( TBP) was 0%, that of dibutyl phenyl phosphate (DBPP) was 0 -4% and that of butyl diphenyl phosphate (BDPP) was over 98%.

All of TBP and DBPP remained. All of BDPP disappeared.

From these results, it was concluded that the test substance is not readily biodegradable and that no transformation products were generated under the conditions of this test.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: RSS is based on the original study report, although the study was also published. Suffient info was available to determine the quality of the study.
Justification for type of information:
The original study report is available and owned by the data submitter. Additionally, the study was published in Environmental Science and Technology with enough detail for assessment. The study discussed different phosphate esters and different endpoints. The current RSS focusses on dibutyl phenyl phoshate. The different studied endpoints are spread over different IUCLID entries (CO2 evolution, SCAS, River Die Away).
Qualifier:
no guideline followed
Principles of method if other than guideline:
The method used is the Thompson-Duthie Carbon Dioxide evaluation test. This test is based on the premise that in the utilization of an organic molecule as a carbon and an energy source by aerobic bacteria, carbon dioxide, water, inorganic salts, and cellular material are produced. By measuring the CO2 produced and relating it to the theoretical yield based on the molecular structure and weight of the material, a parameter related to the ultimate biodegradability of the material is obtained.
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
The inoculum was obtained from a local domestic sewage treatment plant.
Duration of test (contact time):
42 d
Initial conc.:
20 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Acclimated bacterial seed was prepared using a 14-day Bunch-Chambers die-away with no transfer. In this procedure a 2L flask containing 20mg of appropriate ester, 50mg of yeast extract, 100 mL of settled SCAS supernatant, and 900 mL of standard BOD water was incubated in the dark for 14 days at ambient temperature. At the end of the incubation, equal aliquots from each phosphate ester flask were mixed to form a composite seed. A 9L bottle containing 500 ML of composite seed and 5500 mL BOD water was prepared for DBPP and a control. To each of the phosphate ester bottles, a weighed quantity of approximately 120 mg appropriate ester was added. The control bottles received no test material. During the test, CO2 free air was bubbled through the test bottles and the effluent air passed through a set of three CO2 scrubbers each containing 100 mL of 0,05N Ba(OH)2. The evolved CO2 was trapped as barium carbonate and quantified by back-titrating the remaining Ba(OH)2 with 0,1N HCl. Carbon dioxide production from the control bottles was in the range of 10 to 15% of the total CO2 evolved from the phosphate ester bottles. Final values were corrected for the CO2 production of the control.
Preliminary study:
no data
Parameter:
% degradation (CO2 evolution)
Value:
61.5
Sampling time:
7 d
Remarks on result:
other: test 1
Parameter:
% degradation (CO2 evolution)
Value:
84.4
Sampling time:
28 d
Remarks on result:
other: test 1
Parameter:
% degradation (CO2 evolution)
Value:
11.1
Sampling time:
7 d
Remarks on result:
other: test 2
Parameter:
% degradation (CO2 evolution)
Value:
75.6
Sampling time:
28 d
Remarks on result:
other: test 2
Parameter:
% degradation (CO2 evolution)
Value:
84.5
Sampling time:
48 d
Remarks on result:
other: test 2
Details on results:
The same rate trends observed in the river die-away and activated sludge test are apparent in the CO2 evolution data.

It is suggested that the degradation pathway for the phosphate esters most likely involves a stepwise enzymatic hydrolysis to orthophosphate and the phenolic or alcohol moieties according to Barret et al (1969). The alcohol and phenol would then be expected to undergo further degradation.

Validity criteria fulfilled:
not specified
Interpretation of results:
inherently biodegradable
Conclusions:
The study showed that all phosphate esters, including DBPP are inherently biodegradable. The degradation rate of a phosphate ester depends on its chemical composition. In the current CO2 evolution test (with activated sludge) DBPP showed on average 36% degradation after 7 days (lowest 11%, highest 61%), and 80% degradation after 28 days (lowest 76%, highest 84%) and 85% degradation after 48 days.
Executive summary:

The current study describes a CO2 evolution/biodegradability test of different phosphate esters, among which DBPP. Ultimate biodegradation (expressed as complete conversion to CO2, H2O inorganic salts and normal cellular products of bacteria) and primary biodegradation (expressed as disappearance of the original material due to bacterial action) were measured, however this summary describes only the test results of the ultimate biodegradation.

The study showed that DBPP is definitely inherently biodegradable. The degradation rate of a phosphate ester depends on its chemical composition. In the current CO2 evolution test (with activated sludge) DBPP showed on average 36% degradation after 7 days (lowest 11%, highest 61%), and 80% degradation after 28 days (lowest 76%, highest 84%) and 85% degradation after 48 days.

Description of key information

Summary on biodegradation in water: screening test is provided on the overarching level "biodegradation" (section 4.1.3 of the CSR).

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information