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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:
supporting study
Study period:
8th May 1990 to 19th June 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Study performed according to guidelines comparable to internationally accepted guidelines and to GLP.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 301 E (Ready biodegradability: Modified OECD Screening Test)
Deviations:
not specified
Principles of method if other than guideline:
Test conducted "essentially" to TG 301E, however deviations not specified.
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Sourced from an oxidation ditch situated on the premises of TNO, Delft, The Netherlands, used to treat domestic sewage
- Concentration of sludge: 5.0 g of solid substance per litre
- Initial cell/biomass concentration: 120 ml of the mixed sludge used to inoculate 20 litres of medium yielding about 30 mg of solid substance/litre.
Duration of test (contact time):
28 d
Initial conc.:
24.5 mg/L
Based on:
test mat.
Initial conc.:
48.9 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Composition of medium: KH2PO4, K2HPO4, Na2HPO4.2H20, NH4Cl, MgSO4.7H20, CaCl2.2H20, FeCl3, MnSo4.H20, H3BO3, ZnSO4.7H20,
(NH4)6Mo7)24, EDTA, yeast extract
- Test temperature: 20 +/- 2°C
- pH: 7.6 to 7.3+/-1.0
- Aeration of dilution water: not specified
- Continuous darkness: yes


TEST SYSTEM
- Number of culture flasks/concentration: 2
- Test performed in closed vessels


SAMPLING
- Sampling frequency: 0, 7, 14, 21 and 28 days
- Sampling method: not specified
- Sterility check if applicable: not specified
- Sample storage before analysis: not specified


CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: yes
- Toxicity control: yes



STATISTICAL METHODS: No statistical analyses were performed
Reference substance:
acetic acid, sodium salt
Parameter:
% degradation (DOC removal)
Value:
99
Sampling time:
28 d
Remarks on result:
other: initial concentration = 24.5 mg/L
Parameter:
% degradation (DOC removal)
Value:
95
Sampling time:
28 d
Remarks on result:
other: initial concentration = 48.9 mg/L

After an initial lag phase (to the the 14 day point) biodegradation of the test substance (Pentaerythrit R) proceeded rapidly. Between the 14 and 21 day time points of the test over 90% of the DOC was removed for both concentrations tested.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test substance (Pentaerythrit R), at initial concentrations of 24.5 and 48.9 mg/L, was readily biodegradable under the test conditions up to 99% removal of DOC after 28 days. The 10 day window criterium was fulfilled for both concentrations.
Executive summary:

Using removal of DOC (Dissolved Organic Carbon) as test criterion, the biodegradability of Pentaerythrit R was determined essentially by the method described in the OECD Guideline 301 E; Ready Biodegradability: Modified OECD Screening Test. The control test showed that the activity of the inoculum was sufficient and that no significant effects of the test substance on this activity were found. The test substance was readily biodegradable in this test.

At both concentrations tested after an inital lag phase (which lasted up to the 14 day point) removal of DOC was rapid and progrssed beyond the 90% mark by day 21 of the test.

Endpoint:
biodegradation in water: screening test, other
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1981
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Documentation is not sufficient for assessment or expert judgement and the Study or data does not give sufficient experimental details and which are only listed in short abstracts or secondary literature (books, reviews etc).
Qualifier:
equivalent or similar to guideline
Guideline:
other: numerous. see list in principles of method
Deviations:
not applicable
Principles of method if other than guideline:
AFNOR, EPA Activated Sludge, Trickling Filter, Closed Bottle Test, Modified OECD Screening Test, Sturm Test.
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
None Specified
Duration of test (contact time):
14 - 28 d
Parameter followed for biodegradation estimation:
DOC removal
Parameter followed for biodegradation estimation:
O2 consumption
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
None specified
Reference substance:
aniline
Parameter:
% degradation (DOC removal)
Value:
96
Sampling time:
28 d
Remarks on result:
other: OECD Confirmatory Test (coupled units test)
Parameter:
% degradation (DOC removal)
Value:
93
Sampling time:
28 d
Remarks on result:
other: Trickling Filter-coupled units test
Parameter:
% degradation (O2 consumption)
Value:
109
Sampling time:
30 d
Remarks on result:
other: Closed Bottle Test
Parameter:
% degradation (DOC removal)
Value:
92
Sampling time:
19 d
Remarks on result:
other: Modified OECD Screening Test-preacclimated Zahn-Wellens
Parameter:
% degradation (CO2 evolution)
Value:
83
Sampling time:
28 d
Remarks on result:
other: Sturm Test
Results with reference substance:
Fully biodegradable

OECD Confirmatory Test (coupled units test)

 

Closed Bottle Test

Modified OECD Screening Test

Sturm Test

Continuous (% DOC removal )

Trickling Filter (coupled units test) (% DOC removal)

% BODT

Test used as preacclimation step

%DOC removal after 19 days

Test used as preacclimation step

% CO2 production (28 days)

% DOC removal (28 days)

Test used as preacclimation step.

96

93

109

Zahn-Wellens

92

Zahn-Wellens

53

88

Zahn-Wellens

Validity criteria fulfilled:
not specified
Interpretation of results:
readily biodegradable
Conclusions:
Pentaerythritol is biodegradable under some test conditions.
Executive summary:

Pentaerythritol had not been degraded in any of the previously emplyed screening tests (MITI, AFNOR, OECD Screening, Sturm, Closed Bottle).  It was degraded highly in the coupled units test version of the OECD Confirmatory Test after a working-in period of 4 weeks. When an inoculum was drawn from this test the compound was degraded in the Closed Bottle and the Modified OECD Screening Test proving that its degradation is just a matter of acclimatization. The compound had previously also been degraded in the coupled units test in an independent experiment without preacclimation. Using a positive Zahn-Wellens Test run as a preacclimation step for the screening tests, a high degradation was achieved in these also. This shows that the degradation of pentaerythritol does not require special conditions such as the presence of carbon dioxide. As a-matter of fact, only the activation of the Entner-Doudoroff metabolic pathway seems to be required.  As such it can be deduced that after an initial lag phase the test substance is expected to be rapidly removed from the STP with the 10 day window and 28 day study period of most ready biodegradability studies. Given this Pentaerythritol should be considered to be readily biodegradable.

Endpoint:
biodegradation in water: screening test, other
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1979
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Klimisch not assignable as paper is a short publication reporting the biodegradability results for a number of test substances. The study was conducted prior to widespread GLP certification and minimal details are provided regarding the test substance, concentrations used, analytical methods, and other important information. The report is submitted as supporting data.
Principles of method if other than guideline:
AFNOR, EPA Activated Sludge, Trickling Filter, Closed Bottle Test, Modified OECD Screening Test, Sturm Test
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
None Specified
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
Not specified
Reference substance:
aniline
Preliminary study:
Pentaerythritol has been discussed previously in several literature sources : Koppe et al. (1974a, b) found it to be degradable under favorable conditions, Wolters (1978) linked its degradation to nitrification, and Ilic (1978a, b) found that the presence of carbon dioxide is necessary for its metabolization. In the coupled units test, which nitrifies almost completely, it was highly degraded after a working-in period of 21 days.
Interpretation of results:
inherently biodegradable
Conclusions:
Biodegradation of pentaerythritol is dependant on test conditions. In the coupled units test and the Zahn-Wallens test removal of DOC was greater than 90% during the course of the investigations.
Executive summary:

Forty-four compounds covering a range of physical, chemical, and biological properties were tested for their biodegradabilities in seven tests, the coupled units test, i.e., the modification of the OECD Confirmatory Test to the use of the carbon analysis, the Zahn-Wellens test, the Japanese MITI test, the French AFNOR T 90·302 test, the carbon dioxide evolution test according to Sturm, the OECD Screening Test modified, for the use of the carbon analysis, and the closed bottle test. Conclusions regarding the individual tests are drawn from comparisons with the other test results.

Pentaerythritol was found to be readily biodegradable under the conditions of coupled units test and the Zahn-Wallens test with removal of DOC greater than 90% during the course of the investigations. The test substance should be considered to be readily biodegradable and dependant on test conditions.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Study not performed to GLP and test parameters are similar to internationally accepted guidelines. Parameters are sufficient to accept the data and investigations are well documented and scientifically acceptable.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Deviations:
not specified
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): secondary effluent (activated sludge) from domestic sewage treatment plant
- Pretreatment: sparging with carbon dioxide-free air for about one hour
- Initial cell/biomass concentration: 1-2 mg/litre.
- Type and size of filter used, if any: coarse filter, Whatman No.1
Duration of test (contact time):
28 d
Initial conc.:
10 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Stock solutions (a)-(d) were prepared by dissolving the following salts in 1 litre of deionised water: (a) 8.50 g KH2P04, 21.75 g K2HP04, 33.40 g Na2HP04.2H20, 0.5 g NH4CI; (b) 36.4 g CaCI2.2H20; (c) 22.50 g MgS04.7H20; (d) 0.2 g FeCls.6H20, 0.4 g EDTA disodium salt. The test medium was prepared by diluting 10 ml of solution (a) and 1 ml of solutions (b)-(d) in 1 litre aliquots of water.
- pH: 6.5
- pH adjusted: yes
- Aeration of dilution water: not specified
- Suspended solids concentration: 0.5 to 10% by volume of inoculum and 2 to 10 mg/litre of test substance as organic carbon


TEST SYSTEM
- Number of culture flasks/concentration: 12
- Method used to create aerobic conditions: inoculum sparged with CO2 free air for 1 hour prior to combining with medium and test substance. The vials are placed on an orbital shaker after prepared and sealed
- Details of trap for CO2: vials are sealed with butyl rubber septa and aluminium crimp seals


SAMPLING
- Sampling frequency: every fourth day
- Sampling method: gas syringe
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (CO2 evolution)
Value:
83.7
St. dev.:
3.87
Sampling time:
28 d
Remarks on result:
other: inoculum concentration = 5%
Details on results:
Degradation was dependant on initial inoculum concentration. But ulitmately, the test substance is readily biodegradable at inoculum concentrations within the range specified in the test guidelines.
Results with reference substance:
The biodegradation of sodium benzoate was investigated at an initial concentration of 2 mg/litre DOC and an inoculum concentration of 1% vtv of secondary effluent to limit the background response produced from the controls. The mean % biodegradation was 100.9% (SD 2.5, 95% CI: 96.9-104.9).
Validity criteria fulfilled:
not specified
Interpretation of results:
readily biodegradable
Conclusions:
Pentaerythritol is readily biodegradable with inoculum concentration at least 10^5 viable cells per litre.
Executive summary:

The recommended inoculum concentration for ready biodegradability is in the range of 10^4 to 10^8 cells per litre. When given at least 0.05% vol/vol of secondary effluent (~ 3 x 10^ 5 viable cells/litre), within the recommended range, pentaerythritol will biodegrade by >70% after 27 days and with 5.0% vol/vol secondary effluent, the biodegradation is up to 86% after 27 days, indicating that pentaerythritol is readily biodegradable.

From the results of the study it is clear that after an initial lag period (of around 10 days) biodegradation of the test substance occured rapidly and within the 10 day window from day 10 to day 20. During this time biodegration rates progressed well beyond the 80% level. As such the substance meets the classification criteria for ready biodegradability.

Endpoint:
biodegradation in water: screening test, other
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1983 - 1984
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
The study was conducted in numerous labs and therefore GLP could not be assessed. Additionally, only minimal details on substance are available.
Principles of method if other than guideline:
This ring-test programme was carried out by 26 participants accustomed to the technique of respirometry; hence the results may be accepted with confidence.
The EEC manometric respirometric method, similar to the MITI I test, was found suitable for reliably assessing ready biodegradability.

Fourteen chemicals, reported to behave erratically in some way or other in respirometric tests, were assessed by between 9 and 25 participants.

Aniline, used as the control substance, behaved as expected on over 97% of occasions.

The importance of allowing for nitrification in calculating the %ThOD of nitrogen- containing test substance is stressed. It is also concluded that
changes in the concentration of ammonia and oxidised nitrogen should be determined, even with test substances not containing nitrogen. The
effects of nitrification may be lessened by using less ammonium chloride in the medium.

Evidence was obtained to show that the 'pass' level could be reduced to 50% or even 40% ThOD, with concomitant high removal of DOC.

The inocula were ranked in order of activity by three methods and it was concluded that some sludges were more generally metabolically active than
others. No significant correlation was found between the sludge ages of the activated sludge plants from which the inocula were obtained and
metabolic activity. Further investigation of the effect of the source and pre-treatment of the inoculum is necessary to obtain lower and more
consistent endogenous oxygen uptake of the control and, perhaps, more consistent metabolic activity between laboratories.

A better kinetic method of expressing the results of biodegradability tests is needed: one such proposed method is presented but not thoroughly
examined.

A cursory comparison was made between the results obtained in this ring test and with those reported by three laboratories using Repetitive Die-Away (RDA) test.
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): various sources
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
This ring-test programme was carried out by 26 participants accustomed to the technique of respirometry; hence the results may be accepted with confidence.
The EEC manometric respirometric method, similar to the MITI I test, was found suitable for reliably assessing ready biodegradability.

Fourteen chemicals, reported to behave erratically in some way or other in respirometric tests, were assessed by between 9 and 25 participants.

Aniline, used as the control substance, behaved as expected on over 97% of occasions.

The importance of allowing for nitrification in calculating the %ThOD of nitrogen- containing test substance is stressed. It is also concluded that changes in the concentration of ammonia and oxidised nitrogen should be determined, even with test substances not containing nitrogen.
The effects of nitrification may be lessened by using less ammonium chloride in the medium.

Evidence was obtained to show that the 'pass' level could be reduced to 50% or even 40% ThOD, with concomitant high removal of DOC.

The inocula were ranked in order of activity by three methods and it was concluded that some sludges were more generally metabolically active than others. No significant correlation was found between the sludge ages of the activated sludge plants from which the inocula were obtained and metabolic activity. Further investigation of the effect of the source and pre-treatment of the inoculum is necessary to obtain lower and more consistent endogenous oxygen uptake of the control and, perhaps, more consistent metabolic activity between laboratories.

A better kinetic method of expressing the results of biodegradability tests is needed: one such proposed method is presented but not thoroughly examined.

A cursory comparison was made between the results obtained in this ring test and with those reported by three laboratories using Repetitive Die-Away (RDA) test.
Reference substance:
aniline
Parameter:
% degradation (O2 consumption)
Value:
> 60
Sampling time:
28 d
Parameter:
% degradation (DOC removal)
Value:
83
Sampling time:
28 d

Chemical

no. of labs.

Delay time(td days)

After t d+10 days

Mean % ThaD

After t d+10 days

%Labs >60%

After 28 days

Mean%ThaD

After 28 days

% Labs, 60%

After 28 days

DOC

removed (% )

Pentaerythritol

25

13

56

52(64)

64

64(76)

83

Validity criteria fulfilled:
not applicable
Interpretation of results:
readily biodegradable
Conclusions:
Based on the results of 25 labs, the test substance (Pentaerythritol) was considered biodegradable. Based on biological oxygen demand, 52% of the testing labs reported a greater than 60% of the theoretical oxygen demand taken up however the mean value across the labs was 56%. Additionally, it was reported that labs meauring DOC removeal achieved 83% degredation after 28 days.
Executive summary:

The behaviour of pentaerythritol was similar in this exercise to that in the previous ring-test. The median delay time (td) was 13 days, with five laboratories reporting values >18 days. The mean %ThOD in previous tests was 74% compared with 64% for this exercise. Just over half (13/25) of the participants attained >60%ThOD after (td+ 10 days), rising to 16/25 after 28 days. A further six participants reported %ThOD values >44% and four of these achieved DOC removals 80%, so that 'passes' could be said to be 20/25. Only one of the five 'failures' reported a tdvalue of >18 days.

Generally a high degree of degradability was indicated and this can be viewed as supportive evidence of ready biodegradability.

Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Although study performed according to internationally accepted guidelines, it was not conducted to GLP nor was there evidence of Quality Assurance. At least one major parameter described in the test guidelines was not included in the test (e.g. reference substance). This ommision limits the ability to assess the validity of the inocolum.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3200 (Zahn-Wellens / EMPA Test)
Qualifier:
according to guideline
Guideline:
OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, industrial, adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Waste Water Outlet from Perstorp on-site industrial WWTP
-1g suspended solids/litre
Duration of test (contact time):
28 d
Initial conc.:
365 mg/L
Based on:
DOC
Initial conc.:
ca. 0.694 g/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST SYSTEM
- Culturing apparatus: glass vessel
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: aerated with compressed air dispersed at the bottom of the glass vessel

- Measuring equipment: not specified
- Test performed in closed vessels due to significant volatility of test substance: not specified
- Test performed in open system: not specified


SAMPLING
- Sampling frequency: 0, 12, 24, 36 and 48 hours and at day 3, 5, 7, 14, 21, and 28.
- Sampling method: not specified
- Sterility check if applicable: not specified
- Sample storage before analysis: not specified
Reference substance:
not specified
Parameter:
% degradation (DOC removal)
Value:
99
Sampling time:
28 d
Validity criteria fulfilled:
not specified
Interpretation of results:
inherently biodegradable
Conclusions:
The test substance (Penta) was 99% degraded after 28 days and therefore can be considered inherently biodegradable.
Executive summary:

Several substances, including Penta A, were tested for inherent biodegradability with adapated industrial activated sludge, according to OECD TG 302B. At the start of the test, the DOC level was 365 mg/L. After 28 days the measured DOC was 53.7 mg/L. Based on the DOC contributed by the inoculum, the resultant degradation of the test substance is considered to be 99% after 28 days. Therefore, the test substance can be considered inherently biodegradable.

Description of key information

Numerous studies have been done to test the biodegradability of pentaerythritol, each under different conditions, each assessing biodegradability by measuring a different or slightly different endpoint. Upon review of the available studies, it is clear that pentaerythritol is readily biodegradable.  

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

A number of resources are available to evaluate the ready biodegradability of pentaerythritol. Several tests employing different test methodologies are presented.  The two factors that influenced the rate of degradation were inoculum concentration and pre-adaptation of inoculum. 

In the majority of tests pentaerythritol was observed to degrade by at least 60% over 28 days under all conditions. The biodegradation usually occurred after a brief lag time and achieved 60% within 10 days (Malta and Blom, 1990; Birch and Fletcher, 1991). When tested against activated sludge that was pre-adapted to industrial chemicals (Zhan-Wellens), the rate of biodegradation increased and no lag time was observed. After 28 days, nearly 100% biodegradation was observed (ANOX, 1994). 

Based on the results of Birch and Fletcher, the test substance is readily biodegradable when the inoculum concentration is at least 0.05% vol/vol of secondary effluent or ~ 3 x 105viable cells/litre. This concentration is well within the recommended concentration provided in the relevant guideline and is far below that usually encountered in a standard STP.

Based on several studies, pentaerythritol is considered to be readily biodegradable. As such removal in the STP is expected to be efficient with the major fraction of any emissions being removed at this point. Any emissions to soil, surface water and sediment would be expected to degrade rapidly by microbial action.

Given the ready and rapid biodegradability of pentaerythritol it is not expected to pesist in any environmental compartment.