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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
December 2011 - February 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Remarks:
Test performed according GLP issued by State Environmental Protection Administration of the People's Republic of China Test performed according the guidelines of SEPA with reference to OECD 302C (INHERENT BIODEGRADATION TEST). Test meeting validity criteria. Inoculum used is according guidelines but the chosen inoculum is not optimal. Inoculum is enrichment of glucose and peptone degrading microorganisms, microbial diversity is lost when inoculum is first preconditioned on glucese and peptone.
Qualifier:
according to
Guideline:
OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))
Deviations:
no
GLP compliance:
no
Remarks:
Test performed according GLP issued by State Environmental Protection Administration of the People's Republic of China
Oxygen conditions:
aerobic
Inoculum or test system:
mixture of sewage, soil and natural water
Details on inoculum:
Preparation of the Inoculums
Activated sludge, surface soil and surface water weresampled from ten sites distributed in four districts throughout city,such asChengdong, Chengbei, Baguazhou andJiangning. of the sludge, soil and water were collected and mixed thoroughly together. After removing floating matter, the mixture was allowed to stand and then the supernatant isfiltrated through0.22μmMillipore filter. After that the filtrate was adjustedthe supernatant to pH 7.0 with sodium hydroxide or phosphoric acid .Finallyan appropriate volume of the filtered supernatant wastransferred to a fill-and-draw activated sludge vessel and aeratedfor about 23.5 h.Lot No.: I20111215.
Thirty minutes after stopping the aeration, about one third of the whole volume of supernatant was discarded.Then an equal volume ofthesolution (pH 7.0) containing 0.1% each of glucose, peptone and potassium orthophosphate,was added into the settled material and aerated again.This procedure was repeated once per day during 5 d.
Before usethe mixture was allowed to stand, and the supernatant was removed. A small quantity ofsludge was taken to becentrifuged (10000 rpm×10 min) and then weighed. Then the sludge was dried in the oven and weighed again in order to calculate the content of dry sludge was 10%. At lastofcentrifugedsludge was dilutedwith basal culture medium (BSM) to get activated sludge suspension with a concentration of 1000mg/L (dry basis).


PROCEDURE PREPARATION OF INOCULUM IS ACCORDING GUIDELIN: A MICROBIAL DIVERSE INOCULUM IS USED AT THE START, DUE TO THE FIVE DAYS ENRICHMENT ON GLUCOSE AND PEPTONE THE DIVERSITY IS LOST! AN ENRICHMENT OF MICROORGANISMS GROWING ON EASY BIODEGRADABLE ORGANICS (GLUCOSE AND PEPTONE )IS USED TO INOCULATE THE TEST. FOR THIS REASON THIS INOCULUM IS NOT SUITABLE TO TEST INHERENT BIODEGRADABILITY!!!
Duration of test (contact time):
28 d
Initial conc.:
30 mg/L
Based on:
test mat.
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Parameter:
% degradation (test mat. analysis)
Value:
< 1
Sampling time:
28 d
Validity criteria fulfilled:
yes
Remarks:
percentage of degradation of reference substance is >40% after 7 days and 65% after 14 days. Recovery of analysis is ok
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
During the test, the temperature was kept at (25±1)°C. The test was valid because the level of biodegradation of the reference substance sodium benzoate exceeded 40% after 7 days, and 65% after 14 days.
The results showed that biodegradation of meta-Diisopropanolbenzene were 0% after 28 days.
Residual amounts of the test substance meta-Diisopropanolbenzene in “test” vessels after completion of the biodegradation test and in “abiotic” vessel before and after completion of the biodegradation test were determined by UPLC. Based on the residue analysis, biodegradation of the test chemical was <1% in the “abiotic” vessel during the testing period.
Therefore, the test substance has no inherent biodegradability under this test condition.
Test performed according GLP issued by State Environmental Protection Administration of the People's Republic of China
Test performed according the guidelines of SEPA with reference to OECD 302C. Test meeting validity criteria, no certificate of analysis but sufficient info on test substance present. Inoculum used is according guidelines but the chosen inoculum is not optimal. Inoculum is enrichment of glucose and peptone degrading microorganisms, microbial diversity is lost when inoculum is first preconditioned on glucese and peptone. This might be the reason for not finding biodegradation. Lack of biodegradation in this test (with this inoculum) does not mean that the test substance does not biodegrade under environmental conditions.
Executive summary:

The inherent biodegradation test of meta-Diisopropanolbenzene was performed according to “The guidelines for the testing of chemicals” (HJ/T 153 -2004), “The guidelines for the testing of chemicals” (SEPA.: Environmental Sciences Press. 2004), and Procedureof the ‘Guidelines for Testing of Chemicals’ of the OECD: “Inherent Biodegradability: Modified MITI Test (II)” (1981)etc.The inherent biodegradability of meta-Diisopropanolbenzene was determined in a 28 -day Biochemical Oxygen Demand (BOD) test and the analysis of residual chemical of meta-Diisopropanolbenzene in BOD bottles in an aerobic, aqueous medium. During the test, the temperature was kept at (25±1)°C. The test was valid because the level of biodegradation of the reference substance sodium benzoate exceeded 40% after 7 days, and 65% after 14 days. Based on the residue analysis, biodegradation of meta-Diisopropanolbenzene was 0% in the “abiotic” vessel during the testing period. The BOD results showed that biodegradation of meta-Diisopropanolbenzene was <1% after 28 days. Therefore, meta-Diisopropanolbenzene has no inherent biodegradability under this test condition.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
December 2011 - April 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Remarks:
Test performed according GLP issued by State Environmental Protection Administration of the People's Republic of China Test performed according the guidelines of SEPA with reference to OECD 301D. Test meeting validity criteria. Inoculum used is according guidelines but the chosen inoculum is not optimal.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
number of bottles differ
Principles of method if other than guideline:
Four groups of "inoculum control", "test", "reference control" and "toxicity control" were assigned in this test with 2 replicates each. Oxygen measurements performed on day 5, 7, 11, 14, 18, 21, 25 and 28.
According OECD 301D oxyen measurement should be performed in duplicate and bottles are discarded after oxygen measurement.
GLP compliance:
no
Remarks:
according GLP issued by State Environmental Protection Administration of the People's Republic of China
Oxygen conditions:
aerobic
Inoculum or test system:
other: secondary effluent of sewage treatment plant treating predominantly domestic sewage
Details on inoculum:
Samples of secondary effluent were obtained as the test inoculum from a sewage plant treating predominantly domestic sewage.
Test inoculum was prepared by filtering the sample of sewage effluent through an course filter and keeping the filtrate under aerobic conditions until use.
1 ml of this filtered inoculum was added / Liter medium
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: The nutrient medium of the closed bottle test contained per litre of deionized water: 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.4 mg Na2HPO4.2H2O, 0.5 mg NH4Cl, 22.5 mg MgSO4.7H2O, 27.5 mg CaCl2, 0.25 mg FeCl3.6H2O.
- Test temperature: 20+/-2°C
- Aeration of mineral medium: yes



TEST SYSTEM
- Culturing apparatus: incubator
- Number of culture flasks/concentration:
2 bottles containing only inoculum = inoculum blank
2 bottles containing test substance (2 mg/L) and inoculum =test
2 bottles containing test substance (2 mg/L) and sodium benzoate (2 mg/L) and inoculm = toxicity control
2 bottles containing sodium benzoate (2 mg/L) and inoculum = reference control
Concentration inoculum is 1 ml/L
- Method used to create aerobic conditions: aeration with pressured air
- Measuring equipment: oxygen meter.


SAMPLING
- Sampling frequency: day 5, 7, 11, 14, 18, 21, 25 and 28
- Sampling method: the bottles from day5 were used also for measuring the other days

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes
- Reference control: yes



CALCULATIONS:
THOD test substance = 2.56 mg/mg
THOD sodiumbenzoate = 2.38 mg/mg

Oxygen consumption (mg/l) (BOD) = mean oxygen concentration (mg/l) inoculum blank - mean oxygen concentration (mg/l) test (or reference)
Biodegradation (%) = BOD/THOD *100



Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Details on results:
Temperature during test = 20 +/-2 C
Biodegradation of refernce substance is 67% within 14 days
Oxygen consumption in inoculated blanks is 1.2 mg O2/L after 28 days
Oxygen concentration in bottles > 0.5 mg O2/L during the test
Differences in duplicates at end test <20%
Results with reference substance:
The reference substance reached 67% biodegradation within 14 days.
Validity criteria fulfilled:
yes
Remarks:
see Details on results
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
meta-Diisopropanolbenzene was biodegraded 0% at day28 in the closed bottle test and therefore should not be classified as readily biodegradable. This lack of biodegradation may be due to the stringency of the test (choice of the inoculumn could also be the reason). Lack of biodegradation in a ready biodegradability test does not mean that the test substance does not biodegrade under environmental conditions.
Executive summary:

The study was conducted according to the guidelines of SEPAHJ/T 153-2004, “the guidelines for the testing of chemicals”; and with the reference to OECD Procedure 301D etc. Under valid conditions, the ready biodegradability of meta-Diisopropanolbenzene was determined in a 28-day dissolve oxygen depletion using secondary effluent from a domestic waste water treatment plant. During the test, the temperature was kept at (20±2)°C. The test is valid because the level of biodegradation of the reference substance reached 67.2% within 14 days. Oxygen consumption in inoculum blanks was 1.19mg O2/litre after 28 -day test. The test substance did not inhibit the degradation of the reference substance and was therefore not toxic to the inoculum. Thus the study met the acceptability criteria prescribed by the protocol and was considered valid. Finally the results showed that under valid conditions, the biodegradation rate of meta-Diisopropanolbenzene was 0% biodegradation after 28 -days; while the reference substance (sodium benzoate) reached 67.2% after 14 days, which indicated that meta-Diisopropanolbenzene is not readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2012-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with minor deviation
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
The difference in extremes of the replicate test bioreactors was 22.2% which exceeds the 20% limit specified in the Test Guideline (Reference 1) for a valid test. The evolution of CO2 from the test bioreactors was low resulting in low individual cumulative biodegradation values of 1.8% (Test 1) and 2.2% (Test 2) at Day 28; these low values are judged to be the reason why the test failed to satisfy this validity criterion. Furthermore 0.3% of the cumulative biodegradation recorded in Test 2 was accounted for in the final trap on Day 29 suggesting that some influx of atmospheric CO2 had occurred in this trap prior to analysis. In the absence of this anomalous result the difference in extremes between the test bioreactors is 5.6%.
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
A sample of activated sewage sludge (ca 2 x 5 L) was obtained from Haddington Municipal Sewage Works (a local sewage processing plant situated in Haddington, East Lothian, UK, which handles predominantly domestic sewage) on 02 November 2012. On arrival at the laboratory, the sludge was allowed to settle before a portion of the overlying supernatant was siphoned off from one 5 L sample to concentrate the solid sewage inoculant portion to the required level. The resultant sludge was then aerated and shaken by hand to homogenise thoroughly. Sub-samples (5 mL) of the homogenised sludge were dried in an oven at approximately 105°C and the suspended solids content determined to be 1.85 g/L, which was outwith the required range of 3-5 g/L.

To increase the solids content, the sludge remaining in the second 5 L bottle (some supernatent had been lost in transport) was combined with the homogenised sludge sample and the combined sludge shaken by hand then allowed to settle for ca 1 hour. Following settling a portion of the overlying supernatant was removed and the remaining sludge sample shaken by hand. The solids content was re-determined; a value of 2.27 g/L obtained. The sludge was allowed to further settle for ca 1 hour after which a portion of the supernatant was removed and the solids content of the hand homogenised sludge re-determined; a value of 3.60 g/L was obtained, this was within the protocol required range of 3-5 g/L.

The resultant sludge was aerated until required for use. Prior to use it was allowed to settle for ca 1 hour after which the supernatant was clear; portions of the supernatant were added to the bioreactors for use as the microbial inoculum.
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
other: Total Organic Carbon
Initial conc.:
26.05 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Remarks:
The levels of carbon dioxide (CO2) evolved were measured at intervals throughout the test and compared with the maximum theoretical CO2 evolution to determine the extent of biodegradation.
Details on study design:
The test was conducted using 6 bioreactors (2 litre capacity), 2 each for control (Control 1 and Control 2) and test item (Test Item 1 and Test Item 2) and one each for the reference item (Reference) and toxicity control (Toxicity Control).

A totally sealed system was constructed and supplied, on a positive pressure basis, with CO2-free air. CO2 was removed by passing through self-indicating soda lime granules (Durasorb). The CO2-free air was passed through a glass manifold and subsequently through a secondary soda lime tube prior to entering each bioreactor. A non-return valve was then placed in-line to prevent back-flow between the bioreactor and the first trap. The flow rate in the system was adjusted so the flow rate in each bioreactor was similar and in the protocol range of 30 100 mL/min; the recorded flow rates during the main test were in the range 30-45 mL/min.

Addition of m/p DIOL to each test bioreactor and to the toxicity control bioreactor was effected via 100 mL of a solution in mineral medium. The application solution was prepared by dissolving m/p DIOL (260.40 mg) in CO2-free mineral medium (500 mL) such that 100 mL of solution contained 52.08 mg of m/p DIOL (see "any other information on materials and method incl. tables").

A 1 g/L stock solution of the reference item was prepared by dissolving sodium benzoate (0.99975 g) in 1000 mL of deionised water. Aliquots of this stock (69 mL) were added to each appropriate bioreactor to give an addition rate of 20 mg dissolved organic carbon (DOC)/L.
To prepare the bioreactors, 1600 mL of mineral medium and 20 mL of microbial inoculum were added to each bioreactor and then purged with CO2-free air overnight. The appropriate volumes of test item solution and/or reference item stock were then quantitatively added to the appropriate bioreactors. Finally, and following pH measurement and adjustment if necessary, the bioreactors were made up to a final volume of 2000 mL with CO2-free mineral medium. Details of bioreactor preparation are given in the table (see "any other information on materials and method incl. tables").

Each bioreactor was connected to 3 traps, each containing 100 mL of 0.0125M Ba(OH)2. At trap collection the trap closest to the bioreactor was taken for titration and the 2 remaining traps were moved one place closer to the bioreactor. A new trap containing 100 mL of 0.0125M Ba(OH)2 was then placed third in line from the bioreactor.

Trap changes and/or titrations were conducted on Days 2, 3, 5, 7, 10, 12, 15, 20, 25, 28 and 29; the Day 29 trap analysis gave additional results for Day 28 following an acidification procedure to terminate the biodegradation process and release dissolved CO2.

Each sampled trap was titrated with 0.05M HCl; a few drops of phenolphthalein were added as the indicator, utilising a colour change from pink to colourless as the end point. The percent biodegradation was calculated from the titre.

The pH was determined in each bioreactor (Argus Sentron pH meter) on Days 0, 28 (pre-acidification) and 29.

The test was conducted in a laboratory where the temperature was monitored over the duration of the test using a digital maximum/minimum calibrated thermometer. The temperature was maintained at 20-22°C during the test period. The bioreactors were covered with aluminium foil to exclude light.
Reference substance:
benzoic acid, sodium salt
Remarks:
stated purity of ≥99.5%, obtained from Fisher (Batch No. 0761409), expiry date of July 2013.
Parameter:
% degradation (CO2 evolution)
Value:
2
Sampling time:
28 d
Details on results:
The mean cumulative biodegradation of the test item at Day 28 was 2.0%.

The TOC content of the toxicity control bioreactor was equivalent to the combined TOC of the individual test and reference bioreactors. The cumulative biodegradation observed for the toxicity control expressed in terms of the total TOC added was 48.2% which correlates well with the mean (48.9%) cumulative biodegradation for the separate test (2.0% (mean)) and reference (93.5%) item data. This close correlation indicates that the test item was not inhibitory to the microbial inoculum under test conditions. This conclusion is supported by the fact that the extent of biodegradation of the reference item is similar in the reference bioreactor (93.5%) and toxicity control (93.8%); calculated by subtracting the mean biodegradation of the test item (2.0%) from the biodegradation expressed in terms of reference item addition only (95.8%)).
Results with reference substance:
The reference item was readily biodegradable under the conditions of the test as >60% biodegradation was achieved by Day 5

Trap Sampling and Titre Volumes (0.05 M HCl)

 

Day No.

Background Titre (mL)

Bioreactor

Control 1
(mL)

Control 2
(mL)

Test Item 1
(mL)

Test Item 2
(mL)

Reference Item
(mL)

Toxicity Control

(mL)

2

52.0

49.7

49.6

49.6

50.8

8.2

9.2

3

52.0

49.9

50.0

49.9

50.0

20.5

22.2

5

52.0/51.7*

49.8

49.7

49.1

49.3

26.0

25.8

7

52.5

50.5

50.6

50.1

50.2

38.5

36.7

10

51.8

49.9

50.1

49.7

49.5

41.9

41.2

12

51.4

49.6

49.4

49.6

49.4

46.0

45.2

15

52.4

50.5

49.8

50.6

50.4

47.9

47.0

20

52.0

50.3

49.9

50.3

50.1

47.9

48.0

25

51.6

51.0

50.9

50.3

50.1

49.4

48.8

28

51.4

51.2

51.1

50.8

51.4

50.7

50.3

29**

51.7

50.9

51.3

51.3

51.0

50.8

50.4

29**

51.8

50.9

51.1

51.2

50.8

51.1

50.8

29**

51.9

51.6

51.8

51.7

51.3

51.0

51.2

 

* Different background titre applied to Trap 3 analysed on Day 5; this was due to Trap 3 being filled with a different batch of 0.0125M barium hydroxide
** The biodegradation process was terminated following Day 28 analysis and trap change; all traps remaining were taken for analysis on Day 29


Cumulative Biodegradation (%)

 

Day No.

Test Item: m/p DIOL

Reference Item:
Sodium Benzoate

Toxicity Control
Test and Reference Items

Replicate 1

Replicate 2

A

B

2

0.0

0.0

30.9

30.1

15.1

3

0.0

0.0

52.8

50.7

25.5

5

0.6

0.5

70.6

68.6

34.5

7

0.9

0.7

79.5

78.9

39.7

10

1.1

1.0

85.5

85.5

43.0

12

1.1

1.1

88.1

88.7

44.6

15

1.1

1.1

89.8

91.0

45.8

20

1.1

1.1

91.4

92.6

46.6

25

1.5

1.7

92.5

94.2

47.4

28*

1.8**

2.2**

93.5

95.8

48.2

 

* A trap change was conducted on Day 28, after which the biodegradation process was terminated and final trap analysis conducted on Day 29. The tabulated result represents the sum of all analyses

 

** Mean cumulative biodegradation for Test Bioreactors = 2.0%

 

The cumulative biodegradation determined in the Toxicity Control bioreactor is expressed in terms of the total organic carbon content resulting from the reference item only (A) and for the total organic carbon resulting from the reference and test items (B).

Validity criteria fulfilled:
no
Remarks:
see "executive summary"
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The Reaction Mass of α, α, α’, α’-Tetramethyl-m-xylene-α, α’-diol and α, α, α’, α’ -Tetramethyl-p-xylene- α, α’-diol (m/p DIOL), was not readily biodegradable under the conditions of the test. A mean total of 2.0% degradation was achieved by Day 28.
Executive summary:

The ready biodegradability of the Reaction Mass of α,α,α’,α’-Tetramethyl-m-xylene-α,α’-diol and α,α,α’,α’ -Tetramethyl-p-xylene-α,α-diol: (m/p DIOL) was assessed over a 28 day period by the CO2Evolution (Modified Sturm) Test. The study was conducted in accordance with procedures outlined in OECD Guideline 301B (1992).

 

The results of the test were as follows (28 day percent biodegradation values):

 

% Biodegradation

m/p DIOL
(Mean)

Sodium Benzoate

 Toxicity Control*

A

B

2.0

93.5

95.8

48.2

 

*The cumulative biodegradation determined in the Toxicity Control bioreactor is expressed in terms of the total organic carbon content resulting from the reference item only (A) and for the total organic carbon resulting from the reference and test item (B).

 

The test item (The Reaction Mass of a,a,a’,a’-Tetramethyl-m-xylene-a,a’-diol and a,a,a’,a’ -Tetramethyl-p-xylene-a,a’-diol: (m/p DIOL) was not readily biodegradable under the conditions of the test as it failed to achieve a transition from 10% to 60% degradation in a 10 day window during the 28 day test. The test item was not considered to be inhibitory to the microbial inoculum since the biodegradation observed in the toxicity control bioreactor was similar to that expected from the individual test and reference bioreactors.

 

The test failed to meet the validity criterion with respect to the differences in extremes of percent biodegradation in the test bioreactors. However, this was judged to be due to very low biodegradation values observed (1.8 and 2.2% for Test Bioreactors 1 and 2 respectively) and the possible influx of atmospheric CO2 into the final trap on Day 29. The test is therefore considered valid.

Description of key information

α,α,α',α'-tetramethyl-m-xylene-α,α'-diol (m-diol) is considered not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

α,α,α',α'-tetramethyl-m-xylene-α,α'-diol (m-Diol) is considered not readily biodegradable based on ready and inherent biodegradability studies, as well as a

valid study according to OECD 301B and GLP with the Reaction Mass of α, α, α’, α’-Tetramethyl-m-xylene-α, α’-diol and α, α, α’, α’ -Tetramethyl-p-xylene- α, α’-diol (m/p Diol).