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Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2010-02-24 to 2010-07-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to the OECD Guideline and it is GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

-Test system: Activated sludge from the sewage plant at Hildesheim is well suited as it receives predominatly municipal sewage and hardly any industrial chemical waste.
-Source: Municipal sewage treatment plant, D-31137 Hildesheim.

Duration of test (contact time):

28 d

Initial conc.:

20 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

-Test vessels: 5000mL, brwn glass
-Volume of the test medium: 3000mL
-Test temperature: 20.0-23.5ºC
-Aeration: 30-1000mL/min

Reference substance:

benzoic acid, sodium salt

Preliminary study:

No data.

Parameter:

% degradation (CO2 evolution)

Value:

98

Sampling time:

28 d

Remarks on result:

other: Functional control 20mg/L

Parameter:

% degradation (CO2 evolution)

Value:

100

Sampling time:

28 d

Remarks on result:

other: Test item 20mg/L

Parameter:

% degradation (CO2 evolution)

Value:

88

Sampling time:

28 d

Remarks on result:

other: Test item 20mg/L

Parameter:

% degradation (CO2 evolution)

Value:

97

Sampling time:

28 d

Remarks on result:

other: Toxicity control 20mg/L test item+20mg/L reference item

	Study Day (6)	Study Day (14)	Study Day (21)	Study Day (28)
Test Item, 1st Replicate 20 mg/L	31	69	91	100
Test Item, 2nd Replicate 20 mg/L	24	56	71	88
Fuctional control 20 mg/L	49	76	85	98
toxicity Control: 20mg/L test item + 20mg/L Reference item	36	67	83	97

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The first item replicate reached the 10% level (beginning of biodegradation) after 3 days and the 60% pass level after 12 days. the second test item replicate reached the 10% pass level within the 10 -d-window and came to 94% after 28 days.
The test item is classifed as readily biodegradable in the 10-d-window and after 28 days.

Executive summary:

The ready biodegradability of the test item was determined with a non adapted activated sludge over a test period of 28 days over a test period of 28 days in the Modified Sturm test. the study was conducted according to the OECD 301B. The test item was tested at a concentration of 20 mg/L in duplicates, corresponding to a carbon content (TOC) of 12.8 mgC/L in the test vessels. The biodegradation of the test item was followed by titrimetric analysis of the quantity of CO2 produced by the respiration of bacteria. The degradation was stopped on day 28 by acidification of the test solutions. The last titration was made on day 29, after residual CO2 had been purged from the test solutionst solutions over a period of 24h. The percentage CO2 production was calculated in relation to the theorical CO2 production (ThCO2) of the test item. The biodegradation rate was calculated for each titration time.

The first item replicate reached the 10% level (beginning of biodegradation) after 3 days and the 60% pass level after 12 days. the second test item replicate reached the 10% pass level within the 10 -d-window and came to 94% after 28 days.

The test item is considered as readily biodegradable.

This study is considered as relevant. All the validity criteria were fulfilled and no deviations were observed from the OECD 301B guideline.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 12/09/2007 to 07/03/2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to the Method and it is GLP

Qualifier:

according to guideline

Guideline:

other: ISO 11734 Evaluation of the "ultimate" anaerobic biodegradability of organic compounds in digested sludge

Version / remarks:

Version 15-Dec-1995

Deviations:

no

Principles of method if other than guideline:

Not relevant.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

None.

Oxygen conditions:

anaerobic

Inoculum or test system:

other: washed digested sludge from a municipal sewage treatment plant

Details on inoculum:

Methanogenic sludge from the biological sewage treatment plant at Taunusstein-Bleidenstadt dealing predominantly with domestic sewage was used for this test. The sludge was obtained directly from the digester passing through a sieve (Z 2mm) into a plastic container (10 L volume) previously filled with CO2. The sludge was used five days after it was obtained. In the meantime, the container with the sludge was stored at approximately 36°C. Prior to use in the test, the sludge was washed in order to reduce inorganic carbon content. For this purpose, the sludge was centrifuged at approximately 3000 rpm for five minutes in sealed tubes being flushed with nitrogen. The supernatant after centrifugation was discarded, and the pellet was resuspended in anaerobic test medium. The resulting washed sludge exhibited a dry matter of 27.2 g/L. 50 mL of that sludge was used in =450 mL of final test solution volume resulting in 2.7 g dry matter per liter of final volume in the test solutions.

Duration of test (contact time):

84 d

Initial conc.:

>= 20 - <= 100 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

inorg. C analysis

Remarks:

IC measured at the end of the test

Parameter followed for biodegradation estimation:

other: headspace pressure

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

A stock solution of the test item was prepared in anaerobic ultrapure water (Sera|, Purelab plus). For this purpose, 1.3910 g of the test item was weighed directly into 100 mL of anaerobic ultrapure water containing a few crystals of dithionite. The pH of this stock solution was 6.89 and thus was not adjusted. 1 mL of this solution was used per =450 mL of test solution which was prepared as described below, and the final concentration was 7.7966 mg C/=450 mL (=16.93 mg C/L). In the same way, a stock solution of the control item sodium benzoate was prepared at 7.7498 g/100 mL of anaerobic test medium containing a few crystals of dithionite. The pH of this stock solution was 6.95. pH was not adjusted. 1 mL of this solution was used per =450 mL of test solution, and the final concentration was 45.2 mg C/=-450 mL (=98.71 mg C/L). According to the ISO 11734 it was necessary, that the volumes of the liquid VL (medium + inoculum) and that of the headspace V“ are the same in all vessels. For this purpose, the volumes of the flasks were measured, and after completion of the test solutions, individually, respective amounts of anoxic test medium (without any test or reference item) had to be added in order to adjust the volume of the liquid and that of the headspace. Therefore, the individual concentrations of the test item and the control item are slightly different, but total amounts of test or control items are the same in all vessels. By the addition of resazurin it could be demonstrated, that the test solutions were all strictly anaerobic.
At to the test solutions were prepared. The vessels with the test solutions were incubated at constant temperature of 32-35 °C in a water bath in the dark. Approx. 1h after the test solutions had been transferred into the water bath, pressure in the flasks was equilibrated by passing a tubule connected to the gas-tight syringe through the rubber stopper of the test vessel. There was no red resazurin in the test solutions assuring that the test solutions were anaerobic enough. Periodically gas production in the test solutions was measured by aid of a pressure transducer connected to a tubule which was passed through the rubber stoppers. Measured pressures (in millibars) were recorded, and excess pressure was vented after each measurement. The resulting new (neutralized) pressure was recorded too, which became the initial pressure value for the calculation of the increasing pressure of the next measurement. Then, incubation was continued. The incubation at 32-35 °C was followed until day 84. Then inorganic carbon in the liquid phase was measured by aid of a carbon analyzer in order to quantify the percentage of test item transferred into CO2, CO3- and HCO3-. The anaerobic conditions could be verified by the fact that the redox indicator resazurin remained colourless all over the incubation period.
Inorganic Carbon (TIC) and DOC (Dissolved Organic Carbon) were measured by aid of a carbon analyzer.

Reference substance:

benzoic acid, sodium salt

Preliminary study:

None.

Parameter:

other: the measured carbon

Value:

87

Sampling time:

84 d

Details on results:

For calculation of the biodegradation, the following formulas had to be considered:

Carbon in the Headspace :
1 mol of methane and 1 mol of CO2 each contain 12 g of carbon. The mass of carbon in a given volume of evolved gas was calculated according:
m = 12 *103*n
where
m is the mass of carbon, in milligrams, in a given volume of gas evolved
12 is the relative atomic mass of carbon
n is the number of moles of gas
n is calculated from the gas laws using n = pV/RT
where
n is the number of moles of gas;
p is the pressure, in pascals, of the gas
V is the volume, in m3, of the gas
R is the molar gas constant [8.314 J/(moI*K)]
T is the incubation temperature, in kelvins
The net mass of carbon (subtraction of the corresponding blank values) produced as gas in the headspace from the test compound was calculated using
mh = [12000 * 0.1 (Ap * Vh)] / RT
where
mh is the mass, in milligrams, of net carbon produced as gas in the headspace
Ap is the mean of differences between initial and final pressures, in millibars, in the test vessels minus those in the blank vessels
Vh is the volume, in liters, of headspace in the vessel
0.1 is the conversion factor for both newtons per square meter to millibars and m3 to liters
For a normal incubation temperature of 35 °C (308K) the following equation was used:
mh = 0.468 (Ap * Vh)
The course of biodegradation was followed by plotting the cumulated pressure increase Ap, in millibars, against time. Final degradation values were generated by addition of the amount of carbon in the liquid to that in the headspace.

Carbon in the liquid:
The mass of carbon in the liquid of the test vessels was calculated following
mi = Pic, net* Vi
where
mi is the mass, in milligrams, of carbon, in the liquid
Pic, net is the mean concentration of inorganic carbon, in milligrams per liter, in the test vessels minus that in the control vessels at the end of the test
Vi is the volume, in liters, of liquid in the vessel

Total gasified Carbon:
The total mass of gasified carbon in the vessel was calculated using
mt = mh + mi
where
mt = is the total mass, in milligrams, of gasified carbon
DOC-Elimination
The % DOC-elimination was calculated according to
At = [(C0 – Ct) * 100] / C0
where:
At = % degradation at time t
Co = theoretical concentration of the test or control item in the test solution at time to
Ct= DOC-concentration within the test solutions with the test or control item at time t

Results with reference substance:

None.

Carbon Content of the Test Item

1 mL of a stock solution with 33.7 mg test item was prepared in 100mL ultrapure water. This solution exhibited a carbon content of 188.89 mg C/L (mean of two parallels). Thus, the carbon content of the test item was determined as being 560.5 mg TOC/g.

Calculation Amount Carbon in the Headspace with the Test Item

mh 1) Net-Carbon Content in the Headspace in mg Cabsolute

Date	Time (d)	TS1	TS2	TS3	TS4	TS5	TS6	TS7	TS8	TS Mean	TS Sum Mean 2)	%Degradation
01.10.2007	7	2.85	2.83	2.54	2.50	0.91	0.39	0.67	1.18	1.73	1.73	22
08.10.2007	14	0.52	0.46	0.46	0.00	1.12	0.07	1.14	0.76	0.57	2.30	30
15.10.2007	21	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	2.30	30
22.10.2007	28	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	2.30	30
29.10.2007	35	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	2.30 0	30
05.11.2007	42	1.50	0.45	0.46	0.46	0.29	0.41	0.89	1.60	0.76	3.06	39
12.11.2007	49	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	3.06	39
19.11.2007	56	2.12	1.86	1.78	1.64	1.86	0.17	0.00	0.00	1.18	4.24	54
26.11.2007	63	0.00	0.00	0.22	0.00	0.22	0.22	0.22	0.00	0.11	4.35	56
03.12.2007	70	1.42	0.87	0.00	1.76	1.55	1.80	1.90	1.16	1.31	5.66	73
10.12.2007	77	0.00	0.00	0.00	0.77	0.17	0.51	0.50	0.27	0.28	5.93	76
17.12.2007	84	0.94	0.94	0.95	0.59	0.96	0.96	0.98	0.40	0.84	6.77	87

Validity criteria fulfilled:

yes

Interpretation of results:

other: anaerobically biodegradable

Conclusions:

The substance is degraded under anaerobic conditions.

Executive summary:

The substance was investigated for biodegradation under anaerobic conditions according to guidance ISO 11734. The test was performed under standard conditions until day 84, the incubation temperature being 32 -35ºC. Under these conditions of the test item was degraded 87% within 84 days expressed as the sum of the measured carbon in the headspace and the carbon in the liquid. The results of this test give evidence that the test item is degraded under anaerobic conditions. At the end of the test after the prolonged incubation phase of 84 days, a plateau was still not yet reached indicating that the reported degradation value is not the final one. Between week two and week 5 there was a pause of degradation indicating that the bacteria had to adapt metabolites or parts of the molecule of the test item.

The test is valid, as all conditions for validity were met: the control item was degraded >60% within the time frame given by the guideline. No contamination of the test solutions with oxygen could be observed at any time of the course of the study.

This study is considered as relevant. No deviations from the guidance were observed and all conditions for validity were met.

Description of key information

The ready biodegradability of the test item was determined with a non adapted activated sludge over a test period of 28 days in the modified Sturm. The study was conducted according to the OECD 301B under GLP conditions.

The substance is considered as readily biodegradable and was almost completely mineralised at the end of the study.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

Two studies (a key study and a supporting study) are available to assess the readily biodegradation of the test substance.

In the first study wich was evaluated as a key study, the test item is classified as readily biodegradable in the 10-d-window and after 28 days.

The ready biodegradability of the test item was determined with a non adapted activated sludge over a test period of 28 days over a test period of 28 days in the Modified Sturm test. the study was conducted according to the OECD 301B. The test item was tested at a concentration of 20 mg/L in duplicates, corresponding to a carbon content (TOC) of 12.8 mgC/L in the test vessels. The biodegradation of the test item was followed by titrimetric analysis of the quantity of CO2 produced by the respiration of bacteria. The degradation was stopped on day 28 by acidification of the test solutions. The last titration was made on day 29, after residual CO2 had been purged from the test solutionst solutions over a period of 24h. The percentage CO2 production was calculated in relation to the theorical CO2 production (ThCO2) of the test item. The biodegradation rate was calculated for each titration time.

The first item replicate reached the 10% level (beginning of biodegradation) after 3 days and the 60% pass level after 12 days. the second test item replicate reached the 10% pass level within the 10 -d-window and came to 94% after 28 days.

In the second study evaluated as a supporting study, the susbtance was investigated for biodegradation under anaerobic conditions according to ISO 11734. The test was performed under standard conditions until day 84, the incubation temperature being 32 -35ºC. Under these conditions of the test item was degraded 87% within 84 days expressed as the sum of the measured carbon in the headspace and the carbon in the liquid. The results of this test give evidence that the test item is degraded under anaerobic conditions. At the end of the test after the prolonged incubation phase of 84 days, a plateau was still not yet reached indicating that the reported degradation value is not the final one.