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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
Study period:
October-December 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
GLP compliance:
yes
Remarks:
GLP compliance statement is included in attached full study report

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Batch #: 191016
Expiration Date: October 15, 2020
0.9% (9306 mg/L) mineral complexed organic matter (based on total organic carbon content of 0.51%)
1124 mg/L Fe
3333 mg/L Na
2187 mg/L total inorganic carbon
pH=7.28
Density: 1.011 g/mL at 25 degrees C

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
Inoculum
Identification: Activated sludge
Lot No: Q190089.01
Source: Brazos Valley River Sewer Sludge
Collection Date: 25 October 2019

Inoculum Preparation
The inoculum was aerated from the time of receipt until use (~6 days). Coarse solids were allowed to settle out. Suspended solids were determined by filtering a measured portion of the supernatant onto a pre-weighed glass fiber filter and drying the filter before weighing again. The measured difference was 4.36 g/L.
Duration of test (contact time):
29 d
Initial test substance concentration
Initial conc.:
> 10 - < 20 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Test Vessel Preparation and Dosing
On the day before dosing, aliquots of the inoculum (20.6 mL) were brought to a final volume of 2500 mL with mineral medium. This solution was added to each blank, reference, and test flask (8 flasks total). Each flask was placed on a stir plate and mixed, and aeration was initiated using the gas system described earlier.

On Day 0, a stock solution was prepared for each test group by dissolving the appropriate amount of test or reference material into the appropriate volume of mineral media. Five hundred milliliters of the reference stock solution were added to each of two flasks designated as Reference (A and B). Next, 500 mL of test substance stock solution were added to each of two flasks designated as Test Substance (A and B). Each test substance replicate has a test concentration of 15 mg TOC/L. Finally, 500 mL of the toxicity reference stock solution were added to each of two flasks designated Toxicity Reference (A and B). Five hundred milliliters of mineral media were added to each of two flasks designated Blank (A and B). The flasks were maintained in the dark at a monitored temperature between 23-25C for the duration of the test. Each flask was stirred continually using magnetic stirrers. CO2-free air was pumped through the flasks and from the flasks to the CO2 absorbers continuously. Three CO2 absorbing bottles containing 100 mL of 0.0125 M Ba(OH)2 were connected in series to each flask. The pH in each flask was checked and adjusted to 7.6 using diluted HCl.

Sampling
On Days 3, 4, 5, 6, 7, 8, 9, 12, 15, 18, 21, 24, 26, 27 and 28, the collecting bottles nearest to the test flasks from each blank, reference, and test flask were removed and replaced. Bottles were labeled to indicate what batch of Ba(OH)2 they contained, which flask they were connected to, and the numerical order in which they were added. The Ba(OH)2 was titrated against 0.05 N HCl to measure the amount of CO2 evolved by difference.

On Day 28, at the end of the study, the pH of all test and reference flasks was recorded. Then all test flasks were acidified using 6 N HCl to a pH of 3.1 to 3.3 s.u. All flasks were re-stoppered and aerated for 24 more hours. After being aerated overnight, all remaining Ba(OH)2 bottles were removed for titration on Day 29.

Test Validity
For this test to be considered valid, the reference substance must yield >60% of theoretical maximum CO2 within a 10-day window. The 10-day window starts when the reference substance yields 10% biodegradation and ends ten days later.

Evaluation of Results
The test substance will be considered readily biodegradable if it meets the 10-day window validity requirements by Day 28 of the study. The test substance will not be considered biodegradable if it does not meet this requirement.
Reference substance
Reference substance:
aniline
Remarks:
Substance: Distilled aniline Manufacturer: Sigma-Aldrich Lot: SHBG3091V Expiration: 26 July 2021

Results and discussion

Test performance:
Test Validity
For this test to be considered valid, the reference substance must yield >60% of theoretical maximum CO2 within a 10-day window. For the reference substance, distilled aniline, the 10-day window started on Day 4 with a mean biodegradation of 20.80%, and ended on Day 8 with a mean biodegradation of 69.53%; therefore, the test was considered valid.

Evaluation of Results
A test substance is considered readily biodegradable if it meets the 10-day window validity requirements by Day 28 of the study. The 10-day window started on Day 9 with a mean biodegradation of 10.33%. After 28 days, FBS Transit yielded a mean biodegradation of 25.00%, and achieved a maximum mean degradation of 25.07% on Day 29 when the study was terminated. Therefore, the test substance, FBS Transit, was not considered readily biodegradable. Individual and average titrations for each blank at each sampling time are presented in Table 1 of the attached report. The net amount of CO2 evolved from each reference and test system, as determined by titration of Ba(OH)2, is presented in Tables 2-4 of the attached report. The progression of biodegradation in test and reference systems as a graph is presented in Appendix C of the attached report.
% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
ca. 25
St. dev.:
5.9
Sampling time:
29 d
Remarks on result:
other: FBS Transit was not considered readily biodegradable
Remarks:
FBS Transit did not yield >60% of the theoretical maximum CO2 by Day 28 of the study. It achieved a maximum mean degradation of 25% on day 29 when the study was terminated.
Details on results:
See Tables 1-4 in attached final report.

BOD5 / COD results

Results with reference substance:
For this test to be considered valid, the reference substance must yield >60% of theoretical maximum CO2 within a 10-day window. For the reference substance, distilled aniline, the 10-day window started on Day 4 with a mean biodegradation of 20.80%, and ended on Day 8 with a mean biodegradation of 69.53%; therefore, the test was considered valid.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Because FBS Transit is isolated from natural organic matter in an aquatic environment, it has already undergone microbial degradation in situ. The organic matter isolated using our proprietary manufacturing process is likely the refractory portion of organic matter that remains after all labile organic matter is utilized. For these reasons, this organic matter is also partially resistant to biodegradation by the inoculum utilized here and as such, persists for a longer period of time.