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Environmental fate & pathways

Biodegradation in water: screening tests

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Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labeling and/or risk assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 88%

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
A fresh sample of activated sludge (mixed liquor) was collected from the aeration tank of the Wilmington, Delaware (USA) Publicly Owned Treatment Works (POTW). The inoculum was derived from the secondary effluent of the POTW and was kept aerobic prior to use. While stirring the inoculum on a stir plate at low speed, 32.0 mL aliquots were dispensed into each 4-L experimental vessel (test vessel).
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
112.2 mg/L
Based on:
test mat.
Initial conc.:
112.7 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Four liter experimental glass bottles, which were uniquely labeled and identified, were used as the test vessels. The test vessels were held at room temperature in diffused overhead lighting conditions (or in the dark when the lab was not in use) for the duration of the study. Solutions were stirred continuously using stir bars and plates.

To each of the test vessels needed to conduct the study, a total volume of 3 L was added as follows: 1) Test water: 2929 mL; 2) Mineral Media Solution: 39 mL; and 3) Inoculum: 32.0 mL. This mixture was aerated with CO2-free air for four days to purge the system of carbon dioxide. After the aeration period, test and control substances were added and three CO2 gas diffusion bottles were filled with 100 mL of 0.0125 M Ba(OH)2 solution and connected in series to the exit air line of each 4 L vessel. The CO2 produced in each experimental vessel reacts with the barium hydroxide in CO2 adsorption bottles and is precipitated as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 N HCl. On the day of CO2 measurement, one or more CO2 absorption bottles was removed for titration. The volume of Ba(OH)2 solution in the CO2 adsorption bottle was measured and transferred to a 250 mL flask. The Ba(OH)2 was then titrated with 0.05N HCl using phenolphthalein as an indicator and a stir bar and magnetic stirrer to insure good mixing. The titration was complete when the indicator became colorless. On the last day of the study, the pH of each test vessel was measured and recorded. Then 1 mL of concentrated HCl was added to each of the experimental vessels, which were then aerated overnight to remove the CO2 present in the test suspensions. On the next day, the last analysis of evolved carbon dioxide was made.

Mineral media was prepared from stock solutions of appropriate concentrations of mineral components; potassium and sodium phosphates plus ammonium chloride, calcium chloride, magnesium sulfate, and iron (III) chloride. The theoretical CO2 production can be determined either by knowing the molecular formula and purity of the test substance or by direct measurement by determining the total (organic) carbon using a total organic carbon analyzer. An amount of test substance is then added directly to the mineral medium in the appropriate test substance vessels, and toxicity control vessels. In order to check the background CO2 due to the inoculum, an inoculum control blank vessel was prepared in duplicate. The inoculum control blank was mineral medium with inoculum at the same test volumes without test or positive control substances. In order to check the abiotic degradation of the test compound, an abiotic control was prepared in duplicate. The abiotic control was mineral medium with inoculum that has been sterilized by autoclaving containing the test substance. In order to check for inhibition due to toxicity of the test substance, a toxicity control vessel was prepared. The toxicity control was mineral medium with inoculum, the test substance, and the positive control substance.

The amount of CO2 produced by the test substance during the test is measured and expressed as percent of the theoretical CO2 it should have produced (ThCO2) calculated from the carbon content of the test compound. Biodegradability is therefore expressed as percentage ThCO2. The pH of the test solutions was recorded on days 1 and 28.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
29 d
Remarks on result:
other: 0% degradation was also observed at days 1, 2, 4, 7, 11, 15, 21, and 28 for both replicates.
Details on results:
The results of the CO2 Evolution Test indicated that the test substance was not "Readily Biodegradable". It had reached 10% of the ThCO2 by about day 21 of testing but did not reach the 60% pass criteria by day 28.

Direct chemical analysis of the test substance demonstrated that it did not undergo primary biodegradation (degradation of the parent compound but no mineralization) and was not lost from the test system. Concentrations of the test substance increased about 15% in samples collected on day 28 compared to those from day 1. This increase is possibly due to evaporative water losses.


The ThCO2 produced by the test and positive control substances was determined to be 0.67 and 2.14 mg CO2/mg substance, respectively. The value for the test and control substances was determined using the molecular formula and the purity of the substances because the composition was known.

The following results were observed:
• The test substance reached 0% biodegradability by day 28. Since biodegradability was less than 60%, this chemical is not “Readily Biodegradable”.
• Sodium benzoate (the positive control substance) was greater than 60% biodegradable within 14 days confirming that the inoculum was viable; therefore, the test was valid.

Concentrations of the test substance increased about 15% in samples collected on day 28 compared to those from day 1. Test substance concentrations measured in the control blanks were less than the Limit of Quantitation (LOQ = μg mL-1).

In the toxicity test, which includes the test substance and the positive control substance in the same vessel, the substances yielded greater than 25% biodegradation within 14 days. Therefore, the test substance was not inhibitory to microorganisms in the inoculum.

The lag phase lasted from day 0 to about day 21 when an average of 10% biodegradation was attained. The degradation phase of the test substance occurred from about day 21 until end of the study on day 57, it reached a maximum of 25% biodegradation.

The pH of the test and positive control substances and the inoculum blank on days 1 and 28 varied by 0.3 pH or less from that on day 1.

The total CO2 evolution in the control blanks at the end of the test averaged 16 mg CO2 / L, since a total of 48 mg CO2 evolved from test vessels that contained 3 L inoculum control.

The difference of extremes of replicate values of the removal of the test chemical at the end of the test was less than 20%.

BOD5 / COD results

Results with reference substance:
Sodium benzoate (the positive control substance) was greater than 60% biodegradable within 14 days, confirming that the inoculum was viable; therefore, the test was valid.

Any other information on results incl. tables

The ThCO2 produced by the test and positive control substances was determined to be 0.67 and 2.14 mg CO2 mg substance, respectively. The value for the test and control substances was determined using the molecular formula and the purity of the substances because the composition was known. 

 

Concentrations of the test substance increased about 15% in samples collected on day 28 compared to those from day 1. Test substance concentrations measured in the control blanks were less than the Limit of Quantitation (LOQ). In the toxicity test, which included the test substance and the positive control substance in the same vessel, the substances yielded greater than 25% biodegradation within 14 days. Therefore, the test substance was not inhibitory to microorganisms in the inoculum. There was no lag phase for the test substance. The lag phase is the period from inoculation until the degradation percentage has increased to about 10%. The 10-day window is the 10-day period immediately following the attainment of 10% biodegradation.

 

The pH of the test solutions of the test and positive control substances and the inoculum blank on days 1 and 28 varied by 0.5 pH or les from that on day 1. The total CO2 evolution in the control blanks at the end o the test averaged 16 mg CO2/L, since a total of 48 mg CO2 evolved from test vessels that contained 3 L inoculum control. The difference of extremes of replicate values of the removal of the test chemical at the end of the test was less than 20%.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test substance was not “Ready Biodegradable“, did not undergo primary degradation nor was lost from the test system and was not inhibitory to microorganisms in the inoculum.
This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
Executive summary:

The test substance, was tested for ready biodegradability using the 28-day CO2 Evolution test for “Ready Biodegradation” according to OECD Guideline 301B in the version dated July 17, 1992. This test is also known as the Modified Sturm Test. The biological system used was secondary activated sludge from the,() Publicly-Owned Treatment Works (POTW). On days 1, 2, 4, 7, 11, 15, 21, and 28 carbon dioxide (CO2) trapped in barium hydroxide was measured by titration of the residual hydroxide. Additionally, traps were titrated on day 29 to quantify any remaining CO2 after acidifying the test systems on day 28. The amount of CO2 produced from the test substance (corrected for that derived from the blank inoculum) was expressed as a percentage of the total CO2 that the test material could have theoretically produced based on its carbon composition (Th CO2). Test substances giving a result of greater than 60 percent yield of CO2  (within 28 days) should be regarded as readily biodegradable. This level must be reached within 10 days of biodegradation exceeding 10 percent within the 28-day period of the test.

 

The test substance reached a maximum biodegradability of 0% by Day 28. Greater than 60% biodegradability was not reached within 10 days of exceeding 10% biodegradation. Direct chemical analysis of the test substance showed an increase in concentration of about 15% in samples collected on day 28 compared to those from day 1. The reason for this increase is unknown. In the toxicity test, which includes both the test substance and the positive control chemical in the same vessel, the substances yielded greater than 25% biodegradation within 14 days. The reference chemical attained a biodegradation level of greater than 60% by day 10.

 

Based on the results of this study, it was concluded that the test substance was not “Ready Biodegradable”. The test substance did not undergo primary degradation nor was lost from the test system. The test substance was not inhibitory to microorganisms in the inoculum. The test was valid.