2,3,5,6-tetrachloropyridine

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:

16 January 1991 to 19 February 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in compliance with agreed protocols, with only minor deviations from standard test guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 A (old version) (Ready Biodegradability: Modified AFNOR Test)

Deviations:

yes

Remarks:

no reference substance was tested

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2,3,5,6-tetrachloropyridine

EC Number:

219-283-9

EC Name:

2,3,5,6-tetrachloropyridine

Cas Number:

2402-79-1

Molecular formula:

C5HCl4N

IUPAC Name:

2,3,5,6-tetrachloropyridine

Details on test material:

Lot number WP900914-748 of purity = 98.6%

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Approximately 4 L of secondary effluent was obtained from the Midland Municipal Waste Treatment Plant which receives domestic sewage. Microorganisms were concentrated from the effluent by centrifugation for 15 min at 7000 rpm and 4 °C. Cells were resuspended in an isotonic solution and kept on ice while bacterial counts were performed by eppifluorescence microscopy. The inoculum was prepared by adjusting cell concentration to 5.0 ± 3.0 x 107 cells/mL.

Duration of test (contact time):

28 d

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

A 28-day biodegradation study was performed according to the "Modified AFNOR Test" as described by OECD method 301A. Approximately 4 L of secondary effluent was obtained from the Midland Municipal Waste Treatment Plant which receives domestic sewage. Microorganisms were concentrated from the effluent by centrifugation for 15 min at 7000 rpm and 4 °C. Cells were resuspended in an isotonic solution and kept on ice while bacterial counts were performed by eppifluorescence microscopy. The inoculum was prepared by adjusting cell concentration to 5.0 ± 3.0 x 10E7 cells/mL. All reactions were prepared in sealed reaction vessels. Serum vials were amended with 150 mL of minimal inorganic salts medium and 1.5 mL of the inoculum. Reaction vials were then spiked with a stock solution of Sym-Tet in 1,4-dioxane to obtain an initial concentration of 1.0 mg/L Sym-Tet. All samples were amended with reazurin (0.0002 %) as a redox indicator. Reaction vials were then sealed with Teflon®-coated butyl rubber septa, and incubated at 25 ± 1 °C for 28 days in the dark. Sterile controls were set in a similar fashion, except 1000 mg/L formaldehyde was added in place of the inoculum.

The levels of Sym-Tet were monitored in both viable and sterile controls at time intervals of 0, 3, 7, 14, 21, and 28 days. Samples were extracted with 5 mL cyclohexane by shaking for 30 min. Sample aliquots were then diluted 200-fold, and analyzed by GC-ECD. Again, extraction efficiency was determined to be 100 %. The loss of Sym-Tet over time was used to generate degradation rate constants for the viable and sterile controls. Pseudo first-order rate constants, (k observed), were estimated from a linear regression of the natural logarithm of Sym-Tet concentration On C) versus time. Half-lives of Sym-Tet biodegradation were calculated from the following equation:
t1/2 =1n 2/ k observed Equation (1)

Parallel samples were run to determine the viability of the inoculum, as well as the inhibitory effects of Sym-Tet. These experiments were run for 5 days and are analogous to a 5-day biochemical oxygen demand (BOD) test. The concentration of Sym-Tet (1 mg/L) was similar to the levels used in the biodegradation studies described above. Three hundred mL BOD bottles were amended with secondary effluent seed (10 mL secondary effluent/L inorganic salts medium) and spiked with either glucose/glutamic acid or glucose/glutamic acid and Sym-Tet. Dissolved oxygen concentrations in the BOD bottles were measured using an Orion DO meter. Oxygen determinations for both studies were made at 0 and 5 days.

Reference substance

Reference substance:

other: glucose/glutamic acid

Results and discussion

% Degradation

Parameter:

% degradation (test mat. analysis)

Value:

ca. 0

Sampling time:

28 d

Remarks on result:

other: Study report states that "no significant biodegradation of the test chemical occurred over the 28 day test period".

Details on results:

Experiments were conducted to determine the potential for biodegradation of Sym-Tet following guidelines established in OECD method 301A. The biodegradation of Sym-Tet was examined over a 28-day period using secondary effluent from a municipal waste water treatment plant. Results from biologically active samples were compared with killed controls to assess the biodegradability of Sym-Tet. Pseudo first-order rate constants, (k observed), were estimated from a linear regression of the natural logarithm of Sym-Tet concentration (In C) versus time. Biodegradation half-lives were then estimated from the rate constants. The kobserved for the viable samples was 5.52 x 10-3 days-1 with a corresponding half life of approximately 126 days. The loss of Sym-Tet in the viable sample was most likely due to non¬biological processes, since the calculated half life for Sym-Tet in the killed controls was approximately 95 days. The results of these studies demonstrated that no significant biodegradation of the test chemical occurred over the 28 day test period.

Parallel controls were run to determine the biological activity of test inoculum and the potential inhibitory effects of Sym-Tet. The viability of the inoculum was demonstrated in a five day experiment analogous to a 5-day biochemical oxygen demand (BOD) test. BOD bottles were amended with the secondary effluent seed and spiked with either glucose/glutamic acid or glucose/glutamic acid and Sym-Tet. The concentration of Sym-Tet (1 mg/L) was similar to the levels used in the biodegradation studies described above. Dissolved oxygen (DO) concentrations in the BOD bottles were measured at 0 and 5 days. In the presence of glucose/glutamic add, DO levels decreased from 7.94 to 5.35 mg/L as the inoculum metabolized the organic substrate. In the presence of glucose/glutamic acid and 1 mg/L Sym-Tet, the inoculum consumed an equivalent amount of the theoretical oxygen demand after 5 days (decrease of DO from 7.94 to 5.45 mg/L). Thus, the viability of the inoculum was not inhibited by the presence of 1 mg/L Sym-Tet.

Applicant's summary and conclusion

Validity criteria fulfilled:

not specified

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

Experiments were performed to examine the potential for biodegradation of Sym¬Tet in municipal activated sludge. Although some loss of Sym-Tet was observed, there were no significant differences in the rate of disappearance between the killed controls and biologically active samples. In fact, both experiments showed a long half-life of 95 and 126 days, respectively. The difference in half-lives could be attributed to analytical variability. The loss of Sym-Tet over time was most likely due to volatilization and not biodegradation.

Executive summary:

Experiments were conducted to determine the potential for biodegradation of Syrn-Tet following guidelines established in OECD method 301A. The biodegradation of Sym-Tet was examined over a 28-day period using secondary effluent from a municipal waste water treatment plant. Results from biologicallyactive samples were compared with killed controls to assess the biodegradability of the substance.

Pseudo first-order rate constantswere estimated from a linear regression of the natural logarithm of Sym-Tet concentration (In C) versus time. Biodegradation half-lives were then estimated from the rate constants. The k observed for the viable samples was 5.52 x 10-³days-¹with a corresponding half life of approximately 126 days. The loss of Sym-Tet in the viable sample was most likely due to non­biological processes, since the calculated half life for Sym-Tet in the killed controls was approximately 95 days. The results of these studies demonstratedthat no significant biodegradation of the test chemical occurred over the 28 day testperiod.