3,4-Dichloro-N-(5-chloro-4-{[2-({4-[(2-hexyldecyl)oxy]phenyl}sulfonyl)butanoyl]amino}-2-hydroxyphenyl)benzamide

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:

from 2005 february 28th to 2005 march 1st

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed according to the OECD Guideline 301B.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

GLP compliance:

yes

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

SRID or Lot No. : DEV75332
Physical State and Appearance : Tan powder

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, adapted

Details on inoculum:

On arrival at the laboratory, the activated sludge was aerated before use. A sample of the mixed liquor
was homogenized for two minutes with a mechanical blender. It was allowed to settle for
approximately 60 minutes. The supernatant was pipetted to provide sufficient volume of inoculum for
each carboy. Viability of the supernatant was confirmed by using an EasicultB TTC dip slide to
estimate microbe numbers.

Duration of test (contact time):

ca. 28 d

Initial test substance concentration

Initial conc.:

ca. 20 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

The C02 scrubbing apparatus was set up to remove C02 at a constant rate from the air supplied to the
carboy solutions. This was done by diverting the air through a drying column (containing Drierite@), a
C02 absorption column (containing Ascarite II@), and flow meters before being bubbled through the
test carboys. The air passes through the system at a rate of approximately 50-100 mL/min (one to two
bubbles per second), although this was not measured. All test carboys were covered with aluminum
foil for the duration of the study to protect the solutions from the light.
Five inoculated carboys were required for the testing of the test substance: two for the Inoculum
Blank, one for the Positive Control, and two for the test substance. BSM was prepared first (see
Table 1). The carboys were filled with 2350 mL of BSM. Next, 150 mL of the prepared inoculum
supernatant was added to each carboy. This mixture was aerated with C02-free air for approximately
24 hours to purge the system of carbon dioxide.
After the aeration period, the test substance was directly added to two of the carboys with 500 mL,
purged BSM to begin the test period. Purged BSM (500 mL) was added to each of the carboys used as
Inoculum Blank controls (containing no test substance). A small plastic weigh boat was added to
Inoculum Blank #1, as concurrent testing of another test substance used this for a carrier vehicle. The
Positive Control Stock Solution (20 mg DOC/L) was added to the carboy used for the Positive
Control. The final volume in each carboy was 3000 mL. Each carboy was agitated with a magnetic
stir bar. Three C02 absorber bottles were connected in series to the exit airline of each carboy. Each
absorber bottle contained 100 mL of 0.0125M Ba(OH)2.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

% Degradation

% Degradation 1

Parameter:

% degradation (CO2 evolution)

Value:

ca. 1

Sampling time:

6 d

Remarks on result:

other: test 2

% Degradation 2

Parameter:

% degradation (CO2 evolution)

Value:

ca. 2

Sampling time:

10 d

Remarks on result:

other: test 2

% Degradation 3

Parameter:

% degradation (CO2 evolution)

Value:

ca. 3

Sampling time:

14 d

Remarks on result:

other: test 2

% Degradation 4

Parameter:

% degradation (CO2 evolution)

Value:

ca. 3

Sampling time:

27 d

Remarks on result:

other: test 2

% Degradation 5

Parameter:

% degradation (CO2 evolution)

Value:

ca. 4

Sampling time:

28 d

Remarks on result:

other: test 2

Details on results:

At test end, cumulative biodegradation of the test substance in Test vessel #1 reached -l%, and 4% in Test
vessel #2. Less carbon dioxide was evolved from Test vessel #1 than from the Blank, resulting in a
negative value after subtraction of the Blank (this procedure corrects for background C02 evolution). The
Positive Control, sodium benzoate, yielded 74% of the theoretical carbon dioxide possible over the course
of the test. The graph showing percent biodegradation for both Test vessels and the Positive Control is
provided in Figure 1.
The pH of the BSM at test start was 7.508. No unusual variation in pH was noted from Day 0 to Day 27
in any of the test carboys. The pH ranged from 7.206 to 7.394 on Day 27. The Inoculum Blanks released
an average of 95.7 mg C02 (31.9 mg C02/L) over the test period. The barium hydroxide stock solution
needed 48.3 +- 0.1 mL of titrant per titration compared to 47.5 +- 0.2 mL for the airline control. This
indicates that the airline did not contain carbon dioxide after scrubbing. The air temperature during the
test period ranged from 22 to 23°C.
A 28-day test for ready biodegradability using unacclimated microorganisms as the inoculum showed -1 %
and 4% degradation of the test substance at 20 mg DOC/L (theoretical), based on carbon dioxide
evolution. These values may not be significantly different from zero given the accuracy of measurements
of the test. In order for the test substance to be classified as readily biodegradable, it must first reach 10%
degradation. Then there is a 10-day time window in which 60% degradation must occur. The test
substance did not reach 10% during the course of the study. Due to the stringency of this test, low C02
evolution does not necessarily mean that the test substance is not degradable under environmental
conditions, or after waste water treatment.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

These results indicate that the test substance is not readily biodegradable under the conditions of this
study.