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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:
key study
Study period:
2008-08-19 to 2009-01-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Version / remarks:
March 2006
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
anaerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
A sample of activated sludge was obtained from the aeration tank of Worlingworth sewage treatment works, which treats predominantly domestic waste, four days before the start of the test. On the day of collection, the sample was passed through a sieve with a mesh of 1 mm2.
A sub-sample (ca. 500 mL) was removed and centrifuged at ca. 3000 rpm for ca. 1 minute and the supernatant removed. The sample was then made up to volume with mineral salts medium (MSM, Appendix 1) and centrifuged. This procedure was repeated twice and the sample was aerated until required. Aliquots (10 mL) of a homogenised sample of the washed activated sludge were filtered through dried (approximately 105°C), pre-weighed Whatman GF/C filter papers. The filters were dried for at least one hour, allowed to cool and then reweighed. The solids level in the sludge was then calculated and an appropriate volume used to inoculate the MSM to give a final suspended solids concentration of 4 mg/L.
Duration of test (contact time):
>= 28 d
Initial conc.:
1.64 g/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Air-saturated ultrapure water was added to each of three, five-litre clear glass vessels followed by the volumes of each MSM stock solution required to prepare four litres of MSM. Each culture bottle was then inoculated and a magnetic stirring bar was added. The vessels were stoppered and each was placed on an electrically-operated magnetic stirrer. The vessels were aerated, for four days, until the day of test initiation (Day 0) with a supply of air that had been treated to remove carbon dioxide by passing it through cylinders containing fused calcium chloride and Carbosorb AS.

A formulation trial showed MMB to be sufficiently miscible in ultrapure (UHP) water to prepare a stock solution. Therefore, on Day 0, a stock solution of the test substance was prepared at a concentration of 1.64 g/L in UHP water and the pH was measured and no adjustment was necessary. Aliquots (40 mL) were then added to relevant inoculated MSM stock solutions to give a nominal concentration of 10 mgC/L. The pH of the four-day old inoculated MSM was determined and adjusted with 5N HCl to 7.4 ± 0.2. Aliquots (100 mL) from designated vessels were then transferred to Wheaton vials (160 mL capacity). Aliquots (40 mL) of the reference substance (sodium benzoate) were added as an aqueous stock solution (1.72 g/l) to the appropriate pre-aerated MSM to give a final nominal concentration of 10 mgC/L. Blank-controls contained inoculated MSM alone.

The vials were immediately sealed with aluminium coated septa and aluminium crimp seals. The vials were incubated horizontally on an enclosed reciprocating shaker. The final volume of each culture was 100 mL.
The mixtures prepared for the test are summarised below:
Vial
number Contents
1 – 23 Controls – inoculated mineral salts medium alone
1 – 23 Reference - inoculated mineral salts medium plus sodium benzoate (10 mgC/L)
1 – 23 Test - inoculated mineral salts medium plus test substance (10 mgC/L)
1 – 9 Inhibition assay - inoculated mineral salts medium plus sodium benzoate (10 mgC/L) plus test substance (10 mgC/L)

The minimum and maximum temperature of water (ca. 100 mL) contained in a culture vial under test conditions was recorded at intervals during the test and is considered to be representative of the temperature control for this test system. On Days 0, 7, 14, 21 and 28 designated cultures were injected with concentrated sodium hydroxide (nominally 7M) and shaken for a further period of approximately one hour. The content of each treated culture was allowed to stand, then carefully transferred from the culture vials to carbon analyser vials and these were immediately sealed.
Reference substance:
other: Sodium benzoate (AR grade)
Parameter:
% degradation (CO2 evolution)
Value:
78.9
Sampling time:
28 d
Details on results:
Mean production of CO2 by mixtures containing MMB was equivalent to 60% of the theoretical value on approximately Day 25 and 78.9% by the end of the test on Day 28. A biodegradation plateau was not observed. Substances are considered to be biodegradable in this test if CO2 production is equal to or greater than 60% of the theoretical value by the end of the test. Therefore, MMB was considered to be biodegradable. The pH of the test, control and reference MSM at the start of the test was 7.6. The temperature of a 100 mL volume of water held under test conditions ranged from 20.7°C to 22.6°C during the test period
Results with reference substance:
The results obtained for the biodegradation of sodium benzoate alone (97.2% of the theoretical maximum after 14 days) and for CO2 production in the controls (at most, 9.7% of the nominal organic carbon load) fulfil the validity criteria for this test.
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable, but failing 10-day window
Conclusions:
MMB is considered to be biodegradable.
Executive summary:

The study has been conducted according to OECD 310 (CO2 in sealed vessels (Headspace Test) adopted March 2006) and under GLP. Mean production of CO2 by mixtures containing MMB was equivalent to 60% of the theoretical maximum on approximately Day 25 and 78.9% by the end of the test on Day 28. Substances are considered to be biodegradable in this test if CO2 production is equal to or greater than 60% of the theoretical value at the end of the test. Therefore, MMB was considered to be biodegradable as outlined in Commission Regulation (EC) 648/2004 as amended by Regulation 907/2006.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
2001-01-24 to 2001-03-22
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
Although the study was conducted in accordance with the GLP compliance, the biological oxygen demand in the test series varied from 21% to 103% without further discussion.
Qualifier:
according to
Guideline:
other: “Ready Biodegradability: Modified MITI Test (I) (Guideline 301C, July 17, 1992)” prescribed in the “OECD Guidelines for Testing of Chemicals”
Qualifier:
according to
Guideline:
other: “Biodegradability study of Chemical Substances by Microorganisms” stipulated in “Testing Methods relating to the New Chemical Substances” (Kanpogyo No.5, Yakuhatsu No.615, 49 Kikyoku No.392, July 13, 1974)
GLP compliance:
yes
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): Sampling time December, 2000; Sludge was sampled at the following 10 locations all over Japan:
Fushikogawa sewage disposal plant (Sapporo-shi, Hokkaido)
Kashima sewage disposal plant (Kashima-gun, Ibaraki)
Nakahama sewage disposal plant (Osaka-shi, Osaka)
Ochiai sewage disposal plant (Shinjuku-ku, Tokyo)
Kitakami River (Ishinomaki-shi, Miyagi)
Shinano River (Nishikanbara-gun, Niigata)
Yoshino River (Tokushima-shi, Tokushima)
Lake Biwa (Otsu-shi, Shiga)
Hiroshima Bay (Hiroshima-shi, Hiroshima)
Dokai Bay (Kitakyushu-shi, Fukuoka)
- Sampled activated sludge: Sewage disposal plant Returned sludge from sewage disposal plants was collected. River, lake and sea Surface water and surface soil, which was in contact with the atmosphere, was collected.
- Activated sludge preparation: To keep the homogeneity of activated sludge, 5 L of the filtrate of the mixture of activated sludge collected
from each site described above and 5 L of the filtrate of the activated sludge cultivated for approximately 3 months* were mixed to obtain 10 L, pH of the mixture was adjusted to 7.0 ± 1.0 and the mixture was aerated** in the culture tank.
* Ten L of filtrate of the mixture of activated sludge collected from each site described above was cultivated according to the procedure described in section 2.4.
** Aeration: Outdoor air was used for aeration through a pre-filter.
- Method of cultivation: Approximately 30 minutes after ceasing aeration to the culture tank, supernatant corresponding to about 1/3 of the whole volume was removed. De-chlorinated water was added to the remaining portion and total volume was adjusted to 10 L, the mixture was re-aerated (for 30 minutes or more) and then 50 g/L synthetic sewage*4 was added so that the concentration of synthetic sewage reached 0.1wt% in the added
de-chlorinated water. This procedure was repeated once every day, cultured and used as activated sludge. Cultivation temperature was set at 25 ± 2 oC.
*** Synthetic sewage used was prepared as follows: Glucose, peptone and potassium dihydrogenphosphate were dissolved in de-chlorinated water so that each component concentration reached 50 g/L. pH of the solution was adjusted to 7.0 ± 1.0 with sodium hydroxide.
-Control and use: To keep the normal state of activated sludge, excellence of sedimentation of the sludge, pH, temperature and dissolved oxygen concentration were measured during cultivation with observation of the appearance of the supernatant and formation process of activated sludge floc , and it was confirmed that they were in the range of the control standards. This result was retained as raw data. Microflora in the activated sludge was microscopically observed properly and sludge with no abnormality was used for the study. Activity of the sludge was assessed using a standard substance before the sludge was used.
- Preparation of inoculum for exposure: Test solutions were prepared in 6 test vessels. .
(a) (Water + test substance) area (n=1, vessel No.5)
Purified water of 297 mL was placed in a test vessel, 10.0 g/L test substance solution of 3 mL was added so that the test substance concentration reached 100 mg/L and then pH of the solution was measured. To prepare 10.0 g/L of test substance solution, test substance was accurately weighed using electronic analytical balance and dissolved in purified water.
(b) (Activated sludge + test substance) area (n=3, vessel No.1, 2 and 3)
The basal culture medium (volume after added activated sludge volume (2.14 mL) was deducted from 297 mL) was placed in a test vessel, 10.0 g/L test substance solution of 3 mL was added so that the test substance concentration reached 100 mg/L and then pH of the solution was measured, adjusted to 7.0 ± 0.2. To prepare 10.0 g/L of test substance solution, test substance was accurately weighed using electronic analytical balance and dissolved in purified water.
(c) (Activated sludge + aniline) area (n=1, vessel No.6)
The basal culture medium (volume after added activated sludge volume (2.14 mL) was deducted from 300 mL) was placed in a test vessel, 29.5 µL [added amount: 30 mg = 29.5 µL × 1.022 g/cm3 (density)] of aniline was sampled and added using a micro syringe so that the concentration of aniline reached 100 mg/L.
(d) (Activated sludge blank) area (n=1, vessel No.4)
The basal culture medium (volume after added activated sludge volume (2.14 mL) was deducted from 300 mL) was placed in a vessel.
- Concentration of sludge: concentration of the suspended solid reached 30 mg/L
- Pretreatment:From the TOC sample and the GC sample the Supernantant. With the Supernantant it was carried out a centrifugal seperation (1000xg, 10 min) and a sampling, 10 mL to get a 300 mL test solution


Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Details on study design:
see ´Details on inoculum´
Reference substance:
aniline
Value:
ca. 50
Sampling time:
28 d
Details on results:
Biodegradation rates after 28 days:
Percentage biodegradation by BOD 21%, 21%, 107% average 50%
Percentage biodegradation by TOC 20%, 12%, 94% average 42%
Percentage biodegradation by GC 18%, 13%, 100% average 44%

Based on the results by BOD and GC analysis, this substance was failed to meet the criteria for ready biodegradability (pass level and 10 day window).

Degradation : = 50 (±) % after 28 day(s)
Kinetic of testsubst. :
7 day(s) = 2 - 3 %
14 day(s) = 7 - 72 %
21 day(s) = 10 - 100 %
28 day(s) = 21 - 100 %
Results with reference substance:
Control substance : Aniline
7 day(s) = 40 %
14 day(s) = 78 %

Table 1: Appearances of test solution

   Test solution  Appearance  pH
    At the start of cultivation  (Water + test substance) area  Test substance was dissolved [5]5.7
 (Activated sludge + test substance) area  Test substance was dissolved [1]7.0 [2]7.0 [3]7.0
    At the completion of cultivation   (Water + test substance) area  Insoluble compound was not observed [5]6.5
  (Activated sludge + test substance) area  Insoluble compound except the sludge was not observed. Growth of the sludge was observed [1]7.1 [2]7.2 [3]7.2

Table 2: The results of test solution analysis after 28 days

            (Water + test substance)        (Activated sludge + test substance)  Theoretical amount
 Vessel-5  Vessel-1  Vessel-2  Vessel-3  
  BOD* mg   0.2  14.3  14.7  73.6  69.0
Residual amount and percentage residue of DOC*   mgC  19.2  15.3  16.8  1.1  18.3
 %  105  84  92  6  -
  Residual amount and percentage residue of test substance (GC)     mg  30.7  25.2  26.8  0  30.0
 %  102  84  89  0  -

*The values of the test solution of (activated sludge + test substance) area are shown after control blank value was deducted

Table 3: Percentage biodegradation after 28 days

 Method              Percentage biodegradation (%)
 Vessel-1  Vessel-2  Vessel-3  Average
 Result by BOD  21  21  107  50
 Result by TOC  20  12  94  42
 Result by GC  18  13  100  44
Validity criteria fulfilled:
yes
Conclusions:
The test substance was not biodegraded by microorganisms under the condition of this study
Executive summary:

Percentage biodegradation by BOD at the termination of cultivation in a test vessel out of 3 of (activated sludge + test substance) area was 107% and high, but in other 2 vessels it was 21% and low in both vessels (see Table-1 and Fig.1). Percentage biodegradation by TOC and GC analysis for each solution was similar (see Table-2,3 and fig.3). Because residual percentages of DOC and the test substance were almost identical in vessel-1 and 2 of (activated sludge + test substance) area, it was considered that any other organic substances than test substance was not present. Based on these results it was detected that the test substance was biodegraded by microorganisms only in a test vessel, and most of the test substance was not biodegraded by microorganisms and remained as the test substance in 2 vessels.

Results:

(1) Percentage biodegradation by BOD 21%, 21%, 107% average 50%

(2) Percentage biodegradation by TOC 20%, 12%, 94% average 42%

(3) Percentage biodegradation by GC 18%, 13%, 100% average 44%

Conclusion:

Test substance was not biodegraded by microorganisms under the condition of this study.

Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))
GLP compliance:
yes
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): returned sludge from sewage disposal plants was collected; surface water and surface soil, which was in contact with the atmosphere, was collected

Sludge was sampled at the following 10 locations all over Japan. Fushikogawa sewage disposal plant (Sapporo-shi, Hokkaido); Fukashiba sewage disposal plant (Kashima-gun, Ibaraki); Nakahama sewage disposal plant (Osaka-shi, Osaka); Ochiai sewage disposal plant (Shinjuku-ku, Tokyo); Kitakami River (Ishinomaki-shi, Miyagi); Shinano River (Nishikanbara-gun, Niigata); Yoshino River (Tokushima-shi, Tokushima); Lake Biwa (Otsu-shi, Shiga); Hiroshima Bay (Hiroshima-shi, Hiroshima); Dokai Bay (Kitakyushu-shi, Fukuoka)

Duration of test (contact time):
28 d
Initial conc.:
30 mg/L
Based on:
test mat.
Details on study design:
Sampling of activated sludge
Sewage disposal plant: Returned sludge from sewage disposal plants was collected
River, lake and sea: Surface water and surface soil, which was in contact with the atmosphere, was collected
Sampling Sites: Sludge was sampled at 4 sewage disposal, 3 river and 2 bay sites all over Japan

Activated sludge preparation
To keep the homogeneity of activated sludge, 5 L of the filtrate of the mixture of activated sludge collected from each site described above and 5 L of the filtrate of the activated sludge cultivated for approximately 3 months (see cultivation) were mixed to obtain 10 L, pH of the mixture was adjusted to 7.0 ± 1.0 and the mixture was aerated (aeration: outdoor air was used for areation through a pre-filter) in the culture tank.

Cultivation
About 30 minutes after ceasing the aeration of the solution obtained above approximately 1/3 of the whole volume of the supernatant is removed. An equal volume of 0.1 per cent synthetic sewage (glucose, peptone and potassium dihydrogenphosphate dissolved in dechlorinated water so that each component reached 50 g/L and pH adjusted to 7.0 with sodium sodium hydroxide) is added to the remaining portion of the supernatant, and the mixture is aerated again. This procedure is repeated once every day. The culturing is carried out at 25 +/-2°C.

Control
To keep the normal state of activated slugde, sedimentation of the sludge, pH, temperature and dissolved oxygen concentration were measured during cultivation with observation of the appearance of the supernatant and formation process of activated sludge flock, and it was confirmed that they were in the range of the control standards (see methods OECD TG 302 C). This result was retained as raw data. Microflora in the activated sludge was microscopically observed properly and sludge with no abnormality was used for study.

Implementation of biodegradation study

Addition of test compound and preparation for test
(1) A test vessel containing water to which is added 30 ppm (w/v) of the test compound
(2) A test vessel containing the basal culture medium, to which is added 30 ppm (w/v) of test compound; the pH of this solution is adjusted to 7 before the inoculation of active sludge
(3) A test vessel containing basal culture medium to which is added 100 ppm (w/v) of aniline as control substance
(4) A test vessel for the control blank test, containing only the basal culture medium.

Inoculation of activated sludge
The activated sludge prepared under the conditions described under cultivation was added to each test solution (2) and (4) so that the concentration of the suspended solid reached 100 mg/L, and added to solution (3) so that the concentration of the suspended solid reached 30 mg/L.

Conditions
Concentration of test chemicals: 30 ppm (w/v)
Concentration of activated sludge: 100 ppm (w/v)
Test temperature: 25 +/-2°C
Period: 28 days.

Preparation of basal culture medium
Solution A, B, C and D of 3 mL, which are prescribed in ‘Measurement method of factory drain,
Bio-chemical oxygen consumption’ (JIS K 0102-1998, section 21), were mixed up to 1 L with
purified water (Takasugi Seiyaku Co., Ltd., Japanese Pharmacopeia), and then pH of this solution
was adjusted to 7.0.

Performance of test
The BOD curve is obtained continously and automatically for 28 days at 25°C (Closed system oxygen consumption measuring apparatus: Ohkura Electric Co., Ltd; 300 ml culture bottle; carbonic acid gas absorbent: Soda lime; magnetic stirrer). During the cultivation period, state of test solution was observed daily by inspection.
After 28 days of testing, pH, residual chemicals and intermediates in the testing vessel are analysed.
The test chemicals in the testing vessel without activated sludge are also analyzed in order to determine whether there is any change in the test chemical during the testing period, or any loss of the original test chemical by evaporation or by adsorption by the walls of the test vessels.
Reference substance:
aniline
Parameter:
other: BOD
Value:
110
Sampling time:
28 d
Parameter:
other: TOC
Value:
97
Sampling time:
28 d
Parameter:
% degradation (test mat. analysis)
Value:
100
Sampling time:
28 d

Appearances of test solution

   Test solution  Appearance  pH
    At the start of cultivation  (water+test substance (1)) area  Test substance was dissolved 5.95.9
 (activated sludge+test substance (2)) area  Test substance was dissolved 7.17.07.0
    At the completion of cultivation  (water+test substance (1)) area  Insoluble compound was not observed  10.49.2
 (activated sludge+test substance (2)) area  Insoluble compound except the sludge was not observed.Growth of the sludge was observed  7.78.37.8

The results of test solution analysis after 28 days were as follows:

               (water+test substance)        (activated sludge+test substance)  theoretical amount
 1  2  Vessel 1  Vessel 2  Vessel 3  
 BOD*7  mg  -  -  22.4  22.0  23.8  20.7
    residual amountand % residue of DOC  mgC  5.3  5.7  0.2  0  0.3  5.5
 %  96  104  3  0  6  -
    residual amount and% of test substance (GC)  mg  8.5  8.8  0  0  0  9.0
 %  95  98  0  0  0  -

Percentage biodegradation after 28 days was as follows:

 Method            Percentage biodegradation %  
 Vessel 1  Vessel 2  Vessel 3  Average
 Result by BOD  108  106  115  110
 Result by TOD  97  100  94  97
 Result by GC  100  100  100  100
Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable
Conclusions:
The test substance was biodegraded by microorganisms under the condition of this study.

Description of key information

Based on the results of two reliable studies, it is concluded that MMB is not readily biodegradable. However, the substance is inherently, ultimately biodegradable, with 78% biodegradation observed by day 28 in in the key study according to OECD TG 310. 

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable but failing 10-day window

Additional information

Three studies on MMB were undertaken to determine the substance's biodegradability, including OECD Guideline 301 C (Ready Biodegradabilty: Modified MITI Test (I)); OECD Guideline 302C (Inherent Biodegradabilty: Modified MITI Test (II)); OECD Guideline 310 (Ready Biodegradabilty- CO2 in Sealed Vessels (Headspace Test) (March 2006).

MMB is inherently, ultimately biodegradable, based on the results of two reliable tests (OECD 310 and OECD 302C). High degradation were observed in these tests, including 78,9% (CO2 evolution) in an OECD 310 test, however this was not achieved within sufficient window to meet the criteria for ready biodegradability and 110% (BOD), 97% (TOC) in an OECD 302C test.

One study was disregarded since one of three replicates showed a BOD of 107% whereas the other two replicates yielded a BOD of 21% respectively.