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Environmental fate & pathways

Biodegradation in water: screening tests

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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Remarks:
Conducted according to guideline in effect at time of study conduct
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 22.82% solids in water

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, adapted
Details on inoculum:
Secondary activated sludge from the Elkton, Maryland (U.S.A.) Publically Owned Treatment Works (POTW) was used as microbial inoculum. The effluent was kept in glass containers under aerobic conditions in the period between sampling and application on return to the laboratory.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
12 other: mg C/L
Based on:
other: measured total carbon of the test substance, confirmed by dissolved organic carbon measurement at start of test
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Test conditions:
The test vessels (4-L glass bottles) 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. They also were continuously aerated with CO2-free air.

Test conduct:
To each of the test vessels needed to conduct the study, a total volume of 3 L of test water, four mineral media stock solutions, and inoculum were added. The four individual stock solutions of appropriate concentrations of mineral components; potassium and sodium phosphates plus ammonium chloride, calcium chloride, magnesium sulphate, and iron (III) chloride were added separately. While stirring the inoculum on a stir plate at low speed, 22.5 mL aliquots were removed and dispensed into each test vessel to achieve 30 mg solids/L. Test and reference substances were added using the final 76 mL of test water for quantitative transfer of the test and reference substances to bring the final volume to 3 litres. Seventy six mL of test water was also added to the inoculum control blanks. 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.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Test performance:
The viability of the inoculum and validity of the test were supported by the results of the reference substance, sodium benzoate, and the toxicity control. The toxicity control, which included both the test substance and the reference substance in the same vessel, attained greater than 25% biodegradation within 14 days. The reference substance attained a biodegradation level of greater than 60% within 14 days. The reference substance attained 99% DOC elimination within the 28 day test period.
% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
30
Sampling time:
28 d
Remarks on result:
other: average of 2 replicates
Details on results:
The ThCO2 produced by the test and reference substances was determined to be 0.212 and 2.11 mg CO2/mg substance, respectively. The test substance reached a final biodegradability of 30%. The test substance attained greater than 10% biodegradation by day 2. Greater than 60% biodegradation was not achieved, however, within 10 days of reaching 10% biodegradation. Additionally, biodegradation was less than 60% at the end of the test on day 28. The test substance did not meet the criteria for Ready Biodegradation. Sodium benzoate (the reference substance) was greater than 60% biodegradation within 14 days confirming that the inoculum was viable. In the Toxicity Control, which included the test and reference substances in the same vessel, biodegradation was greater than 25% biodegradation within 14 days. Therefore, the test substance was not inhibitory to microorganisms in the inoculum. There was a degradation phase from about day 2 to 5. Greater than 60% was not reached by day 28, however. The degradation phase is from 10% biodegradation until biodegradation reaches a plateau. The pH of the Test, Reference, Tox, and Inoculum Control Blank test solutions on day 28 varied by less than 0.3 pH units from those measured on day 0. The total CO2 evolution in the Inoculum Control Blanks at the end of the test averaged 93 mg CO2, which is equivalent to 31 mg CO2 / L. The difference of extremes of replicate values of the test substance at the end of the test was less than 20%.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
The test substance was not Ready Biodegradable under conditions of the test. The test substance did not reach the 60% pass criteria by day 28.
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 Elkton, Maryland (U.S.A) Publicly-Owned Treatment Works (POTW).

The biodegradation process was monitored on days 2, 5, 8, 12,16, 21 and 28 when 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 from the Inoculum Control Blank) is expressed as a percentage of the total CO2 that the test substance could have theoretically produced (ThCO2) based on the measured total carbon (TC) content of the test substance.

Findings:

• The test substance reached a maximum biodegradability of 30%.

• Greater than 60% biodegradability was not reached within 10 days of exceeding 10% biodegradation.

• The toxicity control, which included both the test substance and the reference substance in the same vessel, attained greater than 25% biodegradation within 14 days.

• The reference substance attained a biodegradation level of greater than 60% within 14 days.

Conclusions:

• The test substance did not meet the criteria for “Ready Biodegradation” under conditions of this test.

• The test substance was not inhibitory to microorganisms in the inoculum.

• The test was valid.