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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
Study period:
1983
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Though the study met basic scientific principles, degradation analysis was conducted with water samples taken directly from the Tamariver, which was contaminated with numerous other pollutants
Qualifier:
no guideline followed
Principles of method if other than guideline:
In this test, biodegradability was measured as the rate of degradation of organic constituents, i.e. total organic carbon (TOC), extractable organic carbon with ethyl acetate (EOC), hydrocarbons, fatty acids and sterols, and the changes in their composition during incubation with a river water sample, using Shimadzu LKB 9000 gas chromatograph-mass spectrometer.



GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
natural water
Duration of test (contact time):
29 d
Parameter followed for biodegradation estimation:
other: rate of degradation of organic constituents, i.e. total organic carbon (TOC), extractable organic carbon with ethyl acetate (EOC), hydrocarbons, fatty acids and sterols
Details on study design:
TEST CONDITIONS
- Test temperature: 5 ± 2°C and 25 ± 3°C
- pH adjusted : No
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 10 L and 1L glass bottles with glass stopper
- Method used to create aerobic conditions: Vigorous shaking

SAMPLING and STORAGE:
- To determine TOC, each 50 mL sample taken in a 100 mL polyethylene bottle was frozen until analysis.
- To determine EOC, each 1 L sample was acidified immediately with 3 mL concentrated hydrochloric acid and extracted three times with ethyl acetate (200, 100 and 100 mL). The ethyl acetate extracts were combined, concentrated to 5.0 mL under reduced pressure at temperatures below 30°C and stored at -20°C until analysis.



Reference substance:
other: 1 L glass bottle with water sample
Key result
Parameter:
% degradation (TOC removal)
Value:
5
Sampling time:
29 d
Remarks on result:
other: TOC decreased from 10.9 to 5.0 mg C/L
Key result
Parameter:
other: EOC
Value:
77
Sampling time:
29 d
Remarks on result:
other: EOC decreased from 2.5 to 0.58 mg C/L
Key result
Parameter:
other: Unsaturated fatty acids (UFA)
Value:
99
Sampling time:
29 d
Remarks on result:
other: UFA decreased from 536 to 3.7 µg/L
Details on results:
- The contents of TOC, EOC, hydrocarbons, fatty acids and sterols in the 10L main bottle decreased considerably during incubation.
- Organic contents for a 1 L glass bottle acidified with concentrated hydrochloric acid (25 + 3°C) decreased reasonably from 56 to 86% of the original organic contents. However, the contents of organic constituents in a 1 L glass bottle acidified (5 ± 2°C) and a 1 L glass bottle chloroform added (25 ± 3°C) were unchanged even after 29 d of incubation. The rinsing of glass bottles with ethyl acetate indicated that organic constituents were degraded by microbial activity rather than abiotical chemical degradation.

TOC and EOC:
During the 29 d of incubation, the contents of TOC and EOC decreased from 10.9 to 5.0 mg C/L and 2.5 to 0.58 mg C/L, respectively. The extent of degradation for the TOC and EOC in this experiment were 54 and 77%, respectively.

Unsaturated Fatty acids (UFA):
- During 29 d of incubation, the contents of UFA decreased from 536 to 3.7 µg/L (i.e., 99% degradation).
- The contents, UFA decreased rapidly within 1 day, gradually decreased during the next 10 d and then very slowly degraded over these periods. Thus the degradation of UFA was considered to occur in three steps:
(i) The UFA decreased rapidly and attained less than a half of the original content within 1 day of incubation. The rate constant (k1) for the first step degradation was 1.4 day -1 with a t1/2 of 0.50 d.
(ii) For the second step, the degradation rate constant (k2) of the UFA was considerably lower than those of the first step.
(iii) For the third step, the rate constant (k3) for the degradation of the UFA was much lower than those of the earlier steps (please refer to the figure 1 in the word document attached under attached backgroud material).
Validity criteria fulfilled:
not specified
Interpretation of results:
readily biodegradable
Conclusions:
Under the test conditions, the constituent fatty acids, C16 to C18 and C18 unsatd. (UFA) was considered to be readily biodegradable.
Executive summary:

A study was conducted to assess the biodegradability of the constituent fatty acids, C16 to C18 and C18 unsatd. The method measured the rate of the degradation of organic constituents, i.e.total organic carbon (TOC), extractable organic carbon (EOC) with ethyl acetate by gas chromatography and mass spectrometry. The contents of organic constituents in a 1 L glass bottle under acidic conditions at 5 ± 2°C and in a 1 L glass bottle with added chloroform at 25 ± 3°C were unchanged even after 29 d of incubation. During the 29 d of incubation, the contents of TOC, EOC and UFA decreased from 10.9 to 5.0 mg C/L (i.e., 54% degradation), 2.5 to 0.58 mg C/L (77% degradation) and 536 to 3.7 µg/L (i.e., 99% degradation) respectively . The rate constant (k1) for the first step degradation of UFA was 1.4 per day with a t1/2 of 0.50 d. Under the test conditions, the constituent fatty acids C16 to C18 and C18 unstad (UFA) was considered to be readily biodegradable (Matsumoto, 1983).

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Estimation done according to Warburg method
Qualifier:
according to guideline
Guideline:
other: Kinetic results were obtained from Warburg data by the method used by Benedict (1969)
Deviations:
not specified
Principles of method if other than guideline:
In this test, biodegradability is measured by calculating the oxygen uptake rates in presence of microorganisms and a final calculation of the specific utllization rate and obtaining the MONOD kinetic constants for the common long chain fatty acids (i.e., palmitic, stearic, oleic and linoleic acid). Different concentrations of the sodium salts of the fatty acids were placed into Warburg flasks and oxygen uptake rates were measured at the temperatures 20 to 30°C. The microorganism concentration placed into each of the flasks was determined as volatile suspended solids according to standard methods.

The rate of O2 uptake as determined from the Warburg data was divided by the initial microorganism concentration placed in the flask to convert the O2 uptake rates to specific utilization rate, U. The rate for each concentration obtained was plotted against the initial substrate concentration in each Warburg flask to yield the Monod type formulation. The constant values, k and K were determined graphically which lead to the establishment of t1/2.
GLP compliance:
no
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): Activated sludge treatment plant in Columbia, Missouri
- Pretreatment: Organisms were washed several times by diluting in tap water, settling and decanting the supernatant
Parameter followed for biodegradation estimation:
other: O2 consumption: the rate of O2 uptake was divided by the initial microorganism concentration to calculate the specific utilization rate, U, which was further used to obtain the MONOD kinetic constants (K and K) and half life.
Key result
Parameter:
% degradation (O2 consumption)
Remarks on result:
other: i.e. maximum rate of substrate degradation per unit weight of microorganism (h-1) was = 0.0027 for stearic acid leading to a half life of 257 h and 0.0546 for linoleic acid.
Key result
Parameter:
% degradation (O2 consumption)
Remarks on result:
other: i.e. maximum rate of substrate degradation per unit weight of microorganism (h-1) was = 0.0546 for linoleic acid leading to a half life of 12.7 h.
Validity criteria fulfilled:
not specified
Interpretation of results:
readily biodegradable
Conclusions:
Under the test conditions,the constituent fatty acids, C16-18 and C18-unsatd. can be considered to be readily biodegradable. The degradation half-time was determined to be less than 1 d to approximately 11 d.
Executive summary:

A study was conducted to assess the biodegradability of the constituent fatty acids, C16 -18 and C-18 unsatd. by Warburg method. The method measured the rate of O2 uptake, which was divided by the initial microorganism concentration to calculate the specific utilization rate U, which was further used to obtain the MONOD kinetic constants (K and K). Further, the rate constant k (h-1) was used for the half life determination using the equation t1/2 = 0.693/k . The degradation half-time ranged from less than 1 d to approximately 11 d for linoleic and stearic acid. Under the test conditions, the constituent fatty acids, C16 -18 and C18 -unsatd. can be considered to be readily biodegradable (Novak and Kraus, 1973).

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:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to guideline
Guideline:
other: By Strum test method (1973), CO2 evolution is measured
Deviations:
yes
Remarks:
no reference substance included
Principles of method if other than guideline:
Biodegradability is measured by Sturm test (1973). It is a method to determine the “ready” ultimate biodegradability of chemicals in aqueous media. In principle, the amount of CO2 produced during the microbial degradation of the organic substance is determined.
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
other: Sewage inoculum
Duration of test (contact time):
21 d
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST SYSTEM
- Culturing apparatus: Sturm appratus (i.e. a glass apparatus of 1.5 L capability, connected to a single CO2 trap. Air circulation is obtained by a peristaltic pump, while agitation of the mineral solution is provided by a magnetic stirrer)
- Number of culture flasks/concentration: One
- Method used to create aerobic conditions: Peristaltic pump and agitation by a magnetic stirrer
- Test performed in closed vessels due to significant volatility of test substance
- Details of trap for CO2 and volatile organics if used: Single CO2 trap, which contains a 100 mL solution of Ba(OH)2


SAMPLING
- Sampling frequency: Days 0, 1, 3, 5, 7, 9, 12, 14, 16, 19 and 21
- Sampling method: Syringe is utilised to withdraw samples from the reactor






Key result
Parameter:
% degradation (CO2 evolution)
Value:
95
Sampling time:
21 d
Details on results:
After an initial lag phase, stearic acid reached the “pass level” of biodegradation, corresponding to 60%, within 10 d and 95% after 21 d. Hence, ready biodegradability was obtained for 'Fatty acids, C16 and C18 and C18-unsatd.' (stearic acid).

Sturm test results for stearic acid:

Days

CO2 produced in mg

% biodegradability

1

1

2

3

5

9

5

14

24

7

21

36

9

31

53

12

40

69

14

44

76

16

48

83

19

52

90

21

55

95

Validity criteria fulfilled:
not specified
Interpretation of results:
readily biodegradable
Conclusions:
Under the test conditions, the constituent 'fatty acids, C16 and C18 and C18-unsatd.' (stearic acid) is considered to be readily biodegradable.
Executive summary:

A study was conducted to assess the biodegradability of the constituent 'fatty acids, C16 and C18 and C18 -unsatd.' (stearic acid) according to the method described in a Sturm test. The test substance reached the “pass level” of biodegradation, corresponding to 60%, within 10 d and 95% after 21 d. Under the test conditions, the constituent 'fatty acids, C16 and C18 and C18-unsatd.' (stearic acid) is considered to be readily biodegradable (Ruffo, 1984).

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
18 Feb, 1988 to 31 Mar, 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to "European Economic Communities, 1984. Method for the determination of ecotoxicity at level 1, Biodegradation, Repetitive Die Away Test. DG XI/400/84. Rev.1".
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
other: European Economic Communities, 1984. Method for the determination of ecotoxicity at level 1, Biodegradation, Repetitive Die Away Test. DG XI/400/84. Rev.1
Deviations:
yes
Remarks:
The test medium did not contain ammonium chloride, and the acclimation of the sludge took place in a 1 liter vessel at 350 mg s.s./L.
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: The inoculum was taken from a municipal waste water treatment plant in Duiven, NL. This plant received only waste water from domestic origin.
- Preparation of inoculum for exposure: The sludge was preconditioned during a week, to reduce high residual respiration rates. The density of the inoculum was 35 mg s.s./L.
- Concentration of sludge: 7.4 mL/L medium; 32.9 mg dry substance/L medium
Duration of test (contact time):
42 d
Initial conc.:
7.5 g/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Test temperature: 20°C
- pH: pH was measured weekly once in each bottle.


TEST SYSTEM
- Culturing apparatus: Dark glass bottles, which were filled at two thirds, to provide a supernatant gas phase as an oxygen reservoir.
- Number of culture flasks/concentration: Three
- Other: The bottles were shaken continously to assure steady state oxygen partitioning between the liquid and gas phase.


SAMPLING
- Sampling frequency: Weekly


CONTROL AND BLANK SYSTEM
- Toxicity control: Yes
Reference substance:
acetic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
> 60
Sampling time:
14 d
Remarks on result:
other: Biodegradation is expressed as the percentage oxidation
Details on results:
The oxidation percentage was 50% after one week, increasing to 62% during the second week. Repetitive additions confirmed that the biodegradation level varied between 30% and 50% per week.
Key result
Parameter:
COD
Value:
2.2 g O2/g test mat.
Results with reference substance:
The biodegradation of sodium acetate was not inhibited in the presence of 7.5 mg oil/L.
Validity criteria fulfilled:
not specified
Interpretation of results:
readily biodegradable
Conclusions:
Under the test conditions, the test substance was found to be readily biodegradable.
Executive summary:

A ready biodegradability study was conduced for the constituent castor oil according to "European Economic Communities, 1984. Method for the determination of ecotoxicity at level 1, Biodegradation, Repetitive Die Away Test. DG XI/400/84. Rev.1". A defined medium was inoculated with activated sludge, preconditioned for a week to reduce high residual respiration rates. The density of the inoculum was 35 mg s.s./L. The initial test substance concentration was 7.5 g/L. The chemical oxygen demand (COD) was determined according to the method of Kelkenberg (1975). The toxicity control was a combination of test substance with sodium acetate. The study was carried out in triplicate. The oxidation percentage was 50% after one week and increased to 62% during the second week. Repetitive additions confirmed that the biodegradation level varied between 30% and 50% per week. Under the test conditions, the test substance was found to be readily biodegradable (Balk and Meuwsen, 1988).

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 9 October, 2009 to 11 December, 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: Municipal sewage treatment plant (Rossdorf, Germany)
- Preparation of inoculum for exposure: The activated sludge was washed by repeated centrifugation and re-suspension in tap water. The sediment of the last washing re-suspended in test medium and aerated until use.
- Pretreatment: The washed activated sludge was pre-conditioned in test medium for a maximum of 7 d
- Concentration of sludge: 1.5 g dry material per L of test medium
Duration of test (contact time):
ca. 28 d
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: 85 mg KH2PO4, 217.5 mg K2HPO4, 334 mg Na2HPO4 x 2 H2O, 5 mg NH4Cl, 22.5 mg MgSO4 x 7 H2O, 36.4 mg CaCl2 x 2 H2O and deionised water up to 1000 mL volume
- Test temperature: 22 ± 1°C
- pH: 7.4 ± 0.2
- Continuous darkness: Yes
- Other: Continuous stirring of test flasks in climatic chamber


TEST SYSTEM
- Culturing apparatus: Manometric test system with test flasks containing a volume of approximately 500 mL
- Number of culture flasks/concentration: Duplicate
- Measuring equipment: Pressure decrease (to determine oxygen consumption) measured using BSB Sensomat system, Aqualytic Dortmund
- Test performed in closed vessels: Yes (closed gas-tight by a measuring head)
- Details of trap for CO2 if used: Evolved carbon dioxide was absorbed in an aqueous solution (45%) of potassium hydroxide.

SAMPLING
- Sampling frequency: Days 0 and 28 (for analysis of nitrate and nitrite using ion chromatography)
- Sampling method: Sufficient aliquot was withdrawn from the bottles containing the test item and inoculum, from the inoculum control and from the toxicity control (after measurement of oxygen concentration).
- Sample storage before analysis: Below -10°C

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes (duplicate)
- Abiotic sterile control: Yes (single)
- Toxicity control: Yes (single)
- Other: Procedure control (single)


Reference substance:
benzoic acid, sodium salt
Remarks:
ThOD: 1.666 mg oxygen per mg
Key result
Parameter:
% degradation (O2 consumption)
Value:
65
Sampling time:
1 d
Remarks on result:
other: 10-d window failed
Details on results:
- The 10-d window failed.

- The difference of the duplicate values for the degradation of the test substance and the plateau, at the end of the test and at the end of the 10 d window, was less than 20%.

- The test substance contains little amounts of nitrogen, therefore the evaluation of biodegradation has to be based on the assumption that nitrification occurred, expressed as ThODNO3.

However, the nitrate concentration in the test substance treated vessels was below LOQ. Therefore, no nitrification was found to occur.

If no nitrification is considered, the mean biodegradation was 65% after 28 d of incubation (ThODNH4), still failing the 10-d window.
Results with reference substance:
The reference substance sodium benzoate was sufficiently degraded to 90% after 14 d and to 98% after 28 d of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.

In the toxicity control containing both the test substance and the reference substance sodium benzoate, 52% biodegradation was noted within 14 d based on ThODNH4and ThODNO3respectively. After 28 d of incubation a degradation of 51% were measured.

Thus, the test substance can be considered to be non-inhibitory to the aerobic activated sludge microorganisms.

For more details on the results, see table 1 - 4 and figure 1 - 2 under the field 'attached background material'.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable, but failing 10-day window
Conclusions:
Under the test conditions, the test substance was determined to be readily biodegradable, but failed the 10-d window.
Executive summary:

A study was conducted to determine the ready biodegradability of the constituent fully hydrogenated coconut oil according to OECD Guideline 301F and EU Method C4 -D. Fully hydrogenated coconut oil was exposed to aerobic activated sludge from the aeration tank of a domestic waste water treatment plant for 28 d. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference substance sodium benzoate was tested simultaneously under the same conditions as the test substance, and functioned as a procedure control. Considering that fully hydrogenated coconut oil contains little amount of nitrogen, the nitrate concentration was measured. However, the nitrate concentration in the test substance treated vessels was below LOQ. Therefore, no nitrification was found to occur. The reference substance degraded to more than 60% after 3 d of incubation. Under the test conditions, fully hydrogenated coconut oil was determined to be readily biodegradable, but failed the 10 -d window (Feil, 2010c).

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: CEC L-33-T-82 (1982) (Method published by the Coordinating European Council, now listed as CEC L-33-A-934)
Deviations:
not specified
Principles of method if other than guideline:
In this test, biodegradability was measured as disappearance of CH3-CH2 units, using an infrared spectrophotometer.

Mineral test medium, test substance diluted in 1,1,2-trichlorofluorethane and microorganisms from municipal wastewater treatment plant sludge were pooled in an Erlenmeyer flask which was then closed and incubated for 21 d at 25 +/- 1°C in the dark.

On Days 0, 7 and 21, the CO2 content was verified. There were three replicates for each analysis day. Also, a toxicity control was included (1 mL of 1% methylmercury was added to inhibit bacterial growth).

Di-isotridecyl-adipate (DIPA) was used as a positive control.

The % biodegradation was calculated using the following formula: % degradation = ((P-T)/P )* 100
(P = average % CO2 from the toxicity controls; T = % CO2 from the test substance; % CO2 calculated based on extinction values from the spectrophotometer)
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Duration of test (contact time):
21 d
Parameter followed for biodegradation estimation:
other: Disappearance of CH3-CH2 (ethyl) units
Reference substance:
other: di-isotridecyl-adipate
Key result
Parameter:
other: Disappearance of CH3-CH2 units
Value:
>= 87.5 - <= 96.8
Sampling time:
7 d
Key result
Parameter:
other: Disappearance of CH3-CH2 units
Value:
>= 91.8 - <= 97.5
Sampling time:
21 d
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Under the test conditions, the test substance was found to be readily biodegradable.
Executive summary:

A study was conducted to assess the biodegradability of the constituent low erucic acid rapeseed oil according to the Coordinating European Council Method CEC L-33 -T-82 (now called CEC L-33 -A-934). The method measured disappearance of CH3 -CH2 units by spectrophotometry. The test substance proved to degrade by more than 87.5% within 7 d and more than 91.8% within 21 d. Under the test conditions, the constituent low erucic acid rapeseed oil was found to fulfill the criteria for ready biodegradability (Fabig, 1989).

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
From 01 March, 2010 to 14 April, 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
KL2 due to RA
Justification for type of information:
Refer to the section 13 for details on the read across justification. The ready biodegradability study with the read across substance is used as a supporting study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Municipal sewage treatment plant (Darmstadt, Germany)
- Preparation of inoculum for exposure: The activated sludge was washed by repeated centrifugation and re-suspension in tap water. The sediment of the last washing was re-suspended in test medium and aerated overnight.
- Concentration of sludge: 1.5 g dry material per L of test medium
Duration of test (contact time):
28 d
Initial conc.:
102 mg/L
Based on:
test mat.
Initial conc.:
312 mg/L
Based on:
other: ThODNH4
Initial conc.:
313 mg/L
Based on:
other: ThODN03
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: 85 mg KH2PO4, 217.5 mg K2HPO4, 334 mg Na2HPO4 x 2 H2O, 5 mg NH4Cl, 22.5 mg MgSO4 x 7 H2O, 36.4 mg CaCl2 x 2 H2O, 0.25 mg FeCl3 x 6H2O and deionised water up to 1000 mL volume
- Test temperature: 22 ± 1°C
- pH: 7.4 ± 0.2
- Continuous darkness: Yes
- Other: Continuous stirring of test flasks in climatic chamber


TEST SYSTEM
- Culturing apparatus: Manometric Test System with test flasks containing a volume of approximately 500 mL
- Number of culture flasks/concentration: Duplicate
- Measuring equipment: Pressure decrease (to determine oxygen consumption) measured using BSB Sensomat system, Aqualytic Dortmund
- Test performed in closed vessels : Yes (closed gas-tight by a measuring head)
- Details of trap for CO2 and volatile organics if used: Evolved carbon dioxide was absorbed in an aqueous solution (45%) of potassium hydroxide.


SAMPLING
- Sampling frequency: Day 0 and 28 (for analysis of nitrate and nitrite using ion chromatography)
- Sampling method: Sufficient aliquot was withdrawn from the bottles containing the test item and inoculum, from the inoculum control and from the toxicity control (after measurement of oxygen concentration).
- Sample storage before analysis: ≤-10°C

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes (Duplicate)
- Abiotic sterile control: Yes (single)
- Toxicity control: Yes (single)
- Other: Procedure control (single)
Reference substance:
benzoic acid, sodium salt
Remarks:
ThOD(NH4): 1.666 mg oxygen per mg
Key result
Parameter:
% degradation (O2 consumption)
Value:
65
Sampling time:
28 d
Remarks on result:
other: 10 d window failed
Details on results:
- For the test substance, 10% level (beginning of biodegradation) was reached by one replicate after 2 d and the pass level of 60% was reached after 25 d. The other replicate reached the 10% level after 3 d and 60% after 25 d. The mean biodegradation of the test substance was calculated as 65% within 28 d indicating that the test substance is readily biodegradable but does not fulfil the 10-d window.

- The oxygen demand of the inoculum control (medium and inoculum) was 30 mg O2/L and thus not greater than 60 mg 02/L within 28 d.

- The difference of the duplicate values for the degradation of the test substance and the plateau, at the end of the test and at the end of the 10 d window, was less than 20%

- The test substance contains little amounts of nitrogen, therefore the evaluation of biodegradation has to be based on the assumption that nitrification occurred and expressed as ThODNO3. However, the nitrate concentration in the test substance treated vessels was below limit of quantification (LOQ). Therefore, no nitrification occurred. If no nitrification is considered, the mean biodegradation was also 65% after 28 d of incubation (ThODNH4), still failing the 10-d window.
Results with reference substance:
The reference substance sodium benzoate was sufficiently degraded to 87% after 14 d and to 90% after 28 d of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.

In the toxicity control containing the test substance and the reference substance sodium benzoate, 45% biodegradation was noted within 14 d and 51 % biodegradation after 28 d of incubation. Thus, the test substance can be assumed to be non-inhibitory to the aerobic activated sludge micro organisms.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable, but failing 10-day window
Conclusions:
Based on the results of the read across study, the test substance, Soaps, stocks, vegetable oil, acidulated, is considered to be readily biodegradable.
Executive summary:

A study was conducted to determine the ready biodegradability of the read across substance 'oils, vegetable, deodorizer distillates' according to OECD Guideline No. 301F and EU Method C4 -D. The read across substance was exposed to aerobic activated sludge from the aeration tank of a domestic waste water treatment plant for 28 d. The biodegradation was followed by the oxygen uptake of the micro-organisms during exposure. Sodium benzoate was used as a functional control to check the activity of the test system. Considering that the read across substance contains little nitrogen, the nitrate concentration was measured. However, the nitrate concentration in the read across substance treated vessels was below the limit of quantification (LOQ). Therefore, no nitrification was assumed to occur. The functional control reached a biodegradation rate of more than 60% after 3 d. In the toxicity control containing both the test and the reference substance, a biodegradation rate of 45% was reached within 14 d; which is greater than the 25% criteria. With the read across substance, the 10% level (beginning of biodegradation) was reached by one replicate after 2 d and the pass level of 60% was reached after 25 d. The other replicate reached the 10% level after 3 d and 60% after 25 d. The mean biodegradation of the read across substance was calculated as 65% within 28 d. Hence, under the test conditions, the read across substance was considered to be readily biodegradable, but failed the 10-d window (Feil, 2010a). Based on the results of the read across study, the test substance, Soaps, stocks, vegetable oil, acidulated, is considered to be readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
From 08 March, 2010 to 14 April, 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
KL2 due to RA
Justification for type of information:
Refer to the section 13 for details on the read across justification. The ready biodegradability study with the read across substance is used as a supporting study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Municipal sewage treatment plant (Darmstadt, Germany)
- Preparation of inoculum for exposure: The activated sludge was washed by repeated centrifugation and re-suspension in tap water. The sediment of the last washing was re-suspended in test medium and aerated overnight.
- Concentration of sludge: 1.5 g dry material per L of test medium
Duration of test (contact time):
28 d
Initial conc.:
>= 25 - < 25.5 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: 85 mg KH2PO4, 217.5 mg K2HPO4, 334 mg Na2HPO4 x 2 H2O, 5 mg NH4Cl, 22.5 mg MgSO4 x 7 H2O, 36.4 mg CaCl2 x 2 H2O, 0.25 mg FeCl3 x 6H2O and deionised water up to 1000 mL volume
- Test temperature: 22 ± 1°C
- pH: 7.4 ± 0.2
- Continuous darkness: Yes
- Other: Continuous stirring of test flasks in climatic chamber


TEST SYSTEM
- Culturing apparatus: Manometric Test System with test flasks containing a volume of approximately 500 mL
- Number of culture flasks/concentration: Duplicate
- Measuring equipment: Pressure decrease (to determine oxygen consumption) measured using BSB Sensomat system, Aqualytic Dortmund
- Test performed in closed vessels: Yes (closed gas-tight by a measuring head)
- Details of trap for CO2 and volatile organics if used: Evolved carbon dioxide was absorbed in an aqueous solution (45%) of potassium hydroxide.


SAMPLING
- Sampling frequency: Days 0 and 28 (for analysis of nitrate and nitrite using ion chromatography)
- Sampling method: Sufficient aliquot was withdrawn from the bottles containing the test substance and inoculum, from the inoculum control and from the toxicity control (after measurement of oxygen concentration).
- Sample storage before analysis: Below -10°C

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes (Duplicate)
- Abiotic sterile control: Yes (single)
- Toxicity control: Yes (single)
- Other: Procedure control (single)
Reference substance:
benzoic acid, sodium salt
Remarks:
ThOD(NH4): 1.666 mg oxygen per mg
Key result
Parameter:
% degradation (O2 consumption)
Value:
54
Sampling time:
28 d
Remarks on result:
other: 10 d window failed
Details on results:
- The oxygen demand of the inoculum control (medium and inoculum) was 30 mg O2/L and thus not greater than 60 mg 02/L within 28 d.

- The difference of the duplicate values for the degradation of the test substance and the plateau, at the end of the test and at the end of the 10 d window, was less than 20%.

- The test substance contained small amounts of nitrogen, therefore the evaluation of biodegradation was additionally performed based on the assumption that nitrification occurred and expressed as ThODNO3. However, the nitrate concentration in the test substance treated vessels was below the limit of quantification (LOQ). Therefore, no nitrification was assumed to occur. If no nitrification is considered, the mean biodegradation was also 65% after 28 d of incubation, still failing the 10-d window.
Results with reference substance:
The reference substance sodium benzoate was sufficiently degraded to 87% after 14 d and to 90% after 28 d of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Based on the results of the read across study, the test substance, Soaps, stocks, vegetable oil, acidulated, is considered to be inheretnly biodegradable.
Executive summary:

A study was conducted to determine the ready biodegradability of the read across substance 'soybean oil, deodorizer distillates' according to OECD Guideline No. 301F and EU Method C4 -D. The substance was exposed to aerobic activated sludge from the aeration tank of a domestic waste water treatment plant for 28 d. The biodegradation was followed by the oxygen uptake of the micro-organisms during exposure. Sodium benzoate was used as a functional or reference control to check the activity of the test system. Considering that the test substance contained small amount of nitrogen, the nitrate concentration was measured. However, the nitrate concentration in the test substance treated vessels was below LOQ. Therefore, no nitrification was assumed to occur. The reference substance was degraded to more than 60% after 3 d of incubation. In the toxicity control containing both the test and the reference substance, a biodegradation rate of 40% was reached within 14 d. Under the test conditions, the substance degraded by 54% within 28 d and was therefore not considered to be readily biodegradable. However an inherent potential for biodegradation was evidenced (Feil, 2010b). Based on the results of the read across study, the test substance, Soaps, stocks, vegetable oil, acidulated, is considered to be inherently biodegradable.

Description of key information

Overall, the weight of evidence suggests that ‘soaps, stocks, vegetable oil, acidulated’ will be readily biodegradable and is therefore unlikely to persist in the aquatic or terrestrial environments.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

There is no information on the biodegradability of ‘soaps, stocks, vegetable oil, acidulated’. However, considering that its composition is comparable to that of various types of deodorizer distillates produced from vegetable oils, its biodegradability potential has been evaluated on the basis of available studies with major constituents as well as by read-across to the similar deodorizer distillates.

Studies with similar distillates:

A study was conducted to determine the ready biodegradability of the read across substance 'oils, vegetable, deodorizer distillates' according to OECD Guideline No. 301F and EU Method C4 -D. The read across substance was exposed to aerobic activated sludge from the aeration tank of a domestic waste water treatment plant for 28 d. The biodegradation was followed by the oxygen uptake of the micro-organisms during exposure. Sodium benzoate was used as a functional control to check the activity of the test system. Considering that the read across substance contains little nitrogen, the nitrate concentration was measured. However, the nitrate concentration in the read across substance treated vessels was below the limit of quantification (LOQ). Therefore, no nitrification was assumed to occur. The functional control reached a biodegradation rate of more than 60% after 3 d. In the toxicity control containing both the test and the reference substance, a biodegradation rate of 45% was reached within 14 d; which is greater than the 25% criteria. With the read across substance, the 10% level (beginning of biodegradation) was reached by one replicate after 2 d and the pass level of 60% was reached after 25 d. The other replicate reached the 10% level after 3 d and 60% after 25 d. The mean biodegradation of the read across substance was calculated as 65% within 28 d. Hence, under the test conditions, the read across substance was considered to be readily biodegradable, but failed the 10-d window (Feil, 2010a). Based on the results of the read across study, the test substance, Soaps, stocks, vegetable oil, acidulated, is considered to be readily biodegradable.

A study was conducted to determine the ready biodegradability of the read across substance 'soybean oil, deodorizer distillates' according to OECD Guideline No. 301F and EU Method C4 -D.The substance was exposed to aerobic activated sludge from the aeration tank of a domestic waste water treatment plant for 28 d. The biodegradation was followed by the oxygen uptake of the micro-organisms during exposure. Sodium benzoate was used as a functional or reference control to check the activity of the test system. Considering that the test substance contained small amount of nitrogen, the nitrate concentration was measured. However, the nitrate concentration in the test substance treated vessels was below LOQ. Therefore, no nitrification was assumed to occur. The reference substance was degraded to more than 60% after 3 d of incubation. In the toxicity control containing both the test and the reference substance, a biodegradation rate of 40% was reached within 14 d. Under the test conditions, the substance degraded by 54% within 28 d and was therefore not considered to be readily biodegradable. However an inherent potential for biodegradation was evidenced (Feil, 2010b).Based on the results of the read across study, the test substance, Soaps, stocks, vegetable oil, acidulated, is considered to be inherently biodegradable.

Studies with the major constituents:

‘Glycerides, C8-18 and C18-unsatd.’, in the form of fully hydrogenated coconut oil, was tested for biodegradability according to OECD Guideline 301F (Feil, 2010a). Under the conditions of the study, the substance was found to be readily biodegradable. This is in line with results obtained for other substances containing triglycerides with unbranched fatty acids of overall longer chain lengths in the range of C16-18 (i.e. ‘glycerides, C16 and C18-unsatd. and C18-unsatd. hydroxy’ and ‘glycerides, C16-18 and C18-unsatd.’), as shown in the Table above (Balk and Meuwsen, 1988; Fabig, Hund and Gross, 1989).

A wealth of published data indicates that fatty acids with chain lengths up to and including C18 are metabolised rapidly under aerobic conditions and can be considered readily biodegradable (BKH, 1994; HERA, 2003). Selected studies are summarised below:

During a 29-day incubation period of freshwater from the Tamara River, Japan, the concentration of unsaturated fatty acids (C16 and C18) decreased from 536 to 3.7 µg/L, suggesting a half-life of 0.5 days (Matsumoto, 1983). Using the Warburg method with unacclimated activated sludge inoculum, the degradation half-life of palmitic (C16:0), stearic (C18:0), oleic (C18:1) and linoleic (C18:2) acid at 20 – 30°C ranged from less than 1 day to ca. 11 days (Novak and Kraus, 1973, as reported in the HSDB database). In an aerobic screening test, stearic acid was determined to be 95% degraded in the presence of sewage inoculum (Ruffo et al., 1984).

Generally, unbranched fatty acids and the corresponding triglycerides are easily broken down by a range of microorganisms such as gram-positive or gram-negative bacteria, a number of fungi and yeasts as well as several types of algae (Fabiget al., 1989), regardless of their functional groups and chain length (Moucawi, 1981; Hita et al., 1996). The shorter the triglyceride chain, the faster the degradation. As such, they are not expected to persist in the environment.

Overall, the weight of evidence suggests that ‘soaps, stocks, vegetable oil, acidulated’ will be readily biodegradable.