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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1992-12-03 till 1993-06-24
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to guideline
Guideline:
other: Method C5 of 84/449/EEC (Modified Sturm Test), 1984
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
(Modified Sturm Test), 1981
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
Modified Sturm test
A sample of activated sludge was collected on the day before the test from a sewage treatment works that treated predominantly domestic waste, and was aerated in the laboratory for four hours. A sample of the mixed liquor was then homogenised in a mechanical blender and allowed to settle
for thirty minutes. An aliquot of the supernatant was passed through a Whatman's GFC filter paper to remove coarse solids and the filtrate used as
the inoculum for the test.

Bacterial inhibition test
A sample of secondary effluent was obtained on the day of the test from a trickling-filter plant at a sewage treatment works that treats predominantly domestic waste.
It was maintained under aerobic conditions in the laboratory until required, and vacuum-filtered through a Whatman GFC filter paper immediately before use. The filtrate was used as the source of inoculum for the test (1 drop/litre test medium).
Duration of test (contact time):
>= 28 - <= 73 d
Initial conc.:
10 mg/L
Based on:
test mat.
Initial conc.:
20 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Seven groups of four BOD bottles were filled with Mineral Salts Medium (MSM), inoculum and test material and/or sodium benzoate
according to the schedule shown below, care being taken to avoid the introduction of air bubbles during preparation and transfer of media to
bottles:

Group Additions to MSM (mg/L)
1 None
2 Inoculum
3 Inoculum + sodium benzoate (2)
4 Inoculum + test material (2)
5 Inoculum + test material (2) + sodium benzoate (2)
6 Inoculum + test material (10)
7 Inoculum + test material (10) + sodium benzoate (2)

Appropriate weights of BCI-MX were added, in glass ampoules, directly to the BOD bottles. Test concentrations of the reference substance were
prepared from an aqueous solution (1 g/L).

The concentrations of dissolved oxygen (DO) and thetemperatures of the contents of duplicate vessels from each group were measured, using a YSI dissolved oxygen meter fitted with a self-stirring DO/temperature bottle probe, at the start of the test and after incubation in darkness for five days
at 20·C.

The pH of each control, test and reference mixture was measured after oxygen and temperature measurement. The temperature of the incubator was measured at intervals during the test using a maximum-minimum thermometer.
Reference substance:
other: Sodium benzoate
Preliminary study:
A five-day bacterial inhibition test was performed under the conditions of the Closed Bottle Test (EEC Procedure C6, Method 301D). This showed that 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene at 2 and 10 mg/L did not inhibit degradation of the reference material sodium benzoate. In this preliminary test, 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene alone showed no evidence of biodegradation.
Test performance:
Cumulative C02 production in the control after 28 days (10.8 mg C02) was within the acceptable range for this assay system (recommended maximum 50 mg C02); cumulative C02 production after 73 days was 18.8 mg C02. These results confirm that the inoculum was viable and that the basic validity criteria were met.
Parameter:
% degradation (CO2 evolution)
Value:
31
Sampling time:
28 d
Remarks on result:
other: 10 mg/L (Test Substance)
Parameter:
% degradation (CO2 evolution)
Value:
6
Sampling time:
28 d
Remarks on result:
other: 20mg/L (Test Substance)
Parameter:
% degradation (CO2 evolution)
Value:
80
Sampling time:
73 d
Remarks on result:
other: 10 mg/l (Test Substance)
Parameter:
% degradation (CO2 evolution)
Value:
20
Sampling time:
73 d
Remarks on result:
other: 20 mg/L( test Substance)
Details on results:
1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene does not reach the required biodegradation level in 28 days to be considered readily biodegradable. Prolonged results indicate that at a concentration of 10 mg/L that the substance is ultimately biodegradable. Reference substance results indicate that the inoculum was viable and the test system was working satisfactorily.
Parameter:
COD
Value:
2 180 g O2/g test mat.
Results with reference substance:
Sodium benzoate was degraded by 68 % of its theoretical carbon dioxide production (TC02; 2.14 mg C02/m9) after nine days, 85 % after 28 days and 89 % after 73 days.

Biodegradation results over the test period

 Day

  Reference % Total organic carbon   10 mg/L  % Total organic carbon      20 mg/L  % Total organic carbon

 1

1
 2  27  1
 3  42  2
 6  58  4  1
 9  68  8  2
 14  78  14
 20  82  19
 27  84  28  6
 28  85  31  6
 37 86  39  9
 42 86  44  9
 48 86  49  11
 51 86  52   11
 56 86  58  13
 59 87  61  14
 64 87 67  16
 70 88  74  18
 72  89  78  19
 73 89  80  20
Validity criteria fulfilled:
yes
Interpretation of results:
other: Biodegradable in an enhanced ready biodegradation screening test (61% biodegradation after 59 days).
Conclusions:
Cumulative CO2 production by mixtures containing 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene at 10 and 20 mg/L respectively was equivalent to 31 % and 6 % of its Total organic carbon (2.44 mg CO2/mg) after 28 days and 80 % and 20 % after 73 days. 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene is considered to be "Not Readily Biodegradable". The reference substance sodium benzoate showed 68 % after 9 days and 85 % degradation after 28 days.
Executive summary:

The ready biodegradability of 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene has been assessed in the Modified Sturm Test (EEC Procedure C5, OECD Procedure 301D).

In the modified Sturm Test, 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene was added to inoculated mineral salts medium, to give nominal test concentrations of 10 and 20 mg/L. Control vessels contained inoculated mineral salts medium alone or inoculated mineral salts medium plus sodium benzoate (20 mg/L). Test and control vessels were aerated for 73 days with air that had been treated to remove carbon dioxide (C02). The CO2 produced by each culture was trapped in a series of Drechsel bottles containing barium hydroxide which were connected to the outlet from each test vessel. The residual barium hydroxide was determined at intervals by titration.

Cumulative CO2 production by mixtures containing 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene at 10 and 20 mg/L respectively was equivalent to 31 % and 6 % of its Total organic carbon (2.44 mg CO2/mg) after 28 days and 80 % and 20 % after 73 days

Sodium benzoate was degraded by 68 % of its theoretical carbon dioxide production (Total organic carbon; 2.14 mg C02/mg) after 9 days, 85 % after 28 days and 89 % after 73 days.

Description of key information

Jenkins, 1993:
Cumulative CO2 production by mixtures containing 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene

at 10 and 20 mg/L respectively was equivalent to 31 % and 6 % of its Total organic carbon (2.44 mg CO2/mg) after 28 days and 80 % and 20 % after 73 days. 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene is considered to be "Not Readily Biodegradable". The reference substance sodium benzoate showed 68 % after 9 days and 85 % degradation after 28 days.
Yoshida, 1994:
Within 28 days, a degradation rate of 5 % was determined. 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene is considered to be "Not Readily Biodegradable". The reference compound aniline showed 73 % degradation after 14 days.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable, not fulfilling specific criteria

Additional information

Jenkins (1993) reported:

Cumulative CO2 production by mixtures containing 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene at 10 and 20 mg/L respectively was equivalent to 31 % and 6 % of its Total organic carbon (2.44 mg CO2/mg) after 28 days, 61% after 59 days and 80 % and 20 % after 73 days.

The biodegradation rate was monitored prolonged in intervals of 1 to 5 days up to 73 days. No lag phase was seen but the production of carbon dioxide was nearly linear with time. The higher rates were seen in the test with 10 mg/L. Prolonged results indicate that at a concentration of 10 mg/L that the substance is ultimately biodegradable:

After 28 days, the biodegradation rate was 31%.

After 40 days, the biodegradation rate was in the range of 39 to 44%.

After 60 days, the biodegradation rate was in the range of 61 to 67%.

After 73 days, the biodegradation rate was 80%.

A report (Flach, 2016) using the closed-bottle test with about 20 mg/L loading yielded similar results.

Therefore, 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene is considered to be "Not Readily Biodegradable". However, significant biodegradation occurs under enhanced biodegradation screening conditions and the pass level of 60% of theoretic CO2 evolution is reached with day 60.

According to REACH Guidance document R11 (2014) p. 37, a substance is not P or vP when the substance is proven to be biodegradable in an enhanced screening test.