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Biodegradation in water: screening tests

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biodegradation in water: screening tests
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to well documented methods.

Data source

Reference Type:

Materials and methods

Test guideline
according to guideline
other: Sanseverino, et al 1994
Principles of method if other than guideline:
Test followed the methods developed in Sanseverino, et al (1994)

Sanseverino J, Werner C, Fleming J, Applegate B, King JM, Sayler GS. 1994. Molecular diagnostics of polycyclic aromatic hydrocarbon biodegradation in manuactured gas plant soils.Biodegradation. 1993-1994;4(4):303-21.

Center for Environmental Biotechnology, University of Tennessee, Knoxville 37932.
GLP compliance:
not specified

Test material

Constituent 1
Reference substance name:
Details on test material:
14C-Labeled compounds were obtained from NEN LifeScience Products (Boston, MA) and had a purity >97%.

testosterone (specific activity of 2.05-2.10 GBq/mmol) were labeled on the C-4 carbon of the steroid backbone.

Study design

Oxygen conditions:
Inoculum or test system:
other: Biosolids in Wastewater Treatment Systems in Tennessee U.S.A.
Details on inoculum:
Biosolids samples (0.5-1 L) were obtained from the aerationcbasin of four different WTPs on at least two separate dates for each plant between March, 1998 and April, 2000. All of these treatment plants processed primarily municipal sewage, employing aerobic biological treatment. Information on the efficiency of plant operations during the sampling period was obtained from water discharge permits maintained at EPA ( through Surf your Watershed and Envirofacts Warehouse.

Effluent water temperatures were available for one plant for approximately 3 years. In this plant, effluent temperatures averaged 16.7 °C (( 4.2) and ranged from a low of 8.2 °C in January to 24 °C in July. The industrial WTP has an influent soluble total organic carbon (STOC) load of about 50 000 kg per day of strictly aqueous chemical waste (i.e., no sanitary wastewater). This load consists mainly of short-chained organic acids and alcohols.
Duration of test (contact time):
72 h
Initial test substance concentration
Initial conc.:
99 µg/L
Based on:
other: radio-labelled test material
Parameter followed for biodegradation estimationopen allclose all
Parameter followed for biodegradation estimation:
CO2 evolution
Parameter followed for biodegradation estimation:
radiochem. meas.
Details on study design:
Biosolids obtained from WTPs were employed in mineralization experiments within 4 h of collection. Accordingly, 5-mL aliquots were placed into sterile 40-mL Eagle Picher vials (Fisher Scientific, Pittsburgh, PA). Other 8-mL vials containing 0.5 N NaOH were added to each 40-mL vial to serve as 14C-CO2 traps. Individual vials were set up for 17â-estradiol, testosterone, and glucose mineralization assays. Each assay had at least three time points (ranging from 0- to 72-h incubation), and each time point included three experimental and two killed-control vials. Killed control samples were generated by acidification with 2 N H2SO4 before the addition of the radiolabeled compounds, and experimental samples were killed at the appropriate incubation time points, with 2 N H2SO4. A total of 2.5 μL (1 106- 9 106 dpm) of radiolabeled compounds was added to the mineralization vials; the same quantity of radiolabeled compounds was also added to three separate scintillation
vials, containing 1 mL of H2O, to determine the initial radioactive counts. Mineralization vials were incubated on a gyratory shaker at ca. 200 rpm at appropriate temperatures.

The starting concentration of 14C -testosterone was 364 nM (99 μg/L).

The glucose (specific activity 12.1-13.0 mCi/mmol; 0.4477-0.48 GBq/mmol) was uniformly labeled on all carbons and was used as a control for biosolids viability and metabolic activity.

Results and discussion

% Degradationopen allclose all
% degradation (radiochem. meas.)
as 14C-CO2
Sampling time:
24 h
Remarks on result:
other: WWTP3-municipal
% degradation (radiochem. meas.)
as 14C-CO2
ca. 55
Sampling time:
24 h
Remarks on result:
other: industrial biosolids (value not reported in text, only in graph, hence the range.
% degradation (radiochem. meas.)
St. dev.:
Sampling time:
72 h
Remarks on result:
other: mineralised to CO2

Applicant's summary and conclusion

Interpretation of results:
readily biodegradable
Under test conditions using municipal water treatment plant innoculum, up to 68% of the test substances (C-14 Testosterone) was mineralised to CO2 within 72 hours of test initiation. There was no significant difference in mineralization of testerone in the municipal and industrial waste biosolids. Although the test was not run according ot OECD test guidelines for ready biodegradability, the test substance meets the criteria to be classified as readily biodegradable.
Executive summary:

Laboratory mineralization assays using 14C labeled estrogens and testosterone were performed with biosolids from four municipal treatment plants and one industrial system.  In these samples, 64.1% ( ±5.5) of the 14C-Testosterone was mineralized to14CO2 after 24 hrs. In waste water treatment plant number 3, mineralization of testosterone was 68% percent 14C-CO2 after 24 hrs. Mineralization rates after 72 hrs were essentially the same. Total removal of14C-testosterone from the aqueous phase was 95%. First-order rate constants k were obtained for the mineralization and removal from the aqueous phase of natural and a synthetic steroid hormone in biosolids from one WTP. In these biosolids14C-testosterone was rapidly mineralized to 0.0152 ( 0.0021 min-1, respectively), Changes in temperature of approximately 15 °C had a statistically significant effect on the rate of mineralization and removal of14C-17 estradiol from the aqueous phase but not for14C-testosterone or14C-17R-ethinylestradiol. These results suggest that biosolids in municipal plants in this region have the capability to remove natural steroid hormones in their influents over a range of temperatures.