1,3-di-o-tolylguanidine

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Values of pH at the end of the test were not given, The potential inhibitory effect on the micro-organisms of the inoculum was not assessed.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

Location Collected from the following ten locations
Furukawa Processing Center (Sapporo, Hokkaido)
Nakahama Processing Center (Osaka)
Kitakamigawa (Ishinomaki, Miyagi Prefecture)
Yoshinogawa (Tokushima)
Hiroshima Bay (Hiroshima)
Fukashiba Processing Center (Kashima Gun, Ibaragi Prefecture)
Ochiai Processing Center (Shinjuku, Tokyo)
Shinogawa (Tottori)
Lake Biwa (Otsu, Shiga Prefecture)
Doukai Bay (Kita Kyushu City, Fukuoka Prefecture)
Time Period December 2003

Sludge Collection
Sewage Processing Centers Returned sludge
Lakes, rivers and bays Surface water and top soil at the point of contact with air

Preparation of the Activated Sludge
To maintain consistent activated sludge, 5L of filtered solution of a mixed sludge from each of the above locations where collected was mixed with 5L of filtered solution of activated sludge*1 incubated for approximately three months to make a total of 10 L, the pH was adjusted to a level of 7.0±1.0 and this was aerated*2 in an incubation tank.
*1 10L of filtered solution from the mixture of sludge from each location collected as indicated above was incubated according to 2.4 below to produce the activated sludge.
*2 Aeration was conducted using outside air through a pre-filter.

Incubation
After approximately 30 minutes of aeration in the incubation tank, supernatant of about 1/3 of the total amount was removed. Dechlorinated water was added to get a total of 10L, which was aerated again (at least 30 minutes), and then 50g/L of synthetic sewage*3 was added so the concentration of synthetic sewage in the added dechlorinated water was 0.1wt%. This operation was repeated once a day to produce the incubated activated sludge. The incubation temperature was 25±2°C.

*3 Glucose, peptone, gallium dihydrogen phosphate were dissolved in purified water to make 50g/L of each, and the pH was adjusted to 7.0±1.0 using sodium hydroxide.

Administration and Use
To maintain the normal state of the activated sludge, the external appearance of the supernatant and the generated state of the activated sludge were monitored, and the excellent settling properties, pH, temperature and dissolved oxygen concentration were measured and verified to be within the range of administrative standards (refer to “Test Methods Relating to New Chemical Substances”). These results were saved as raw data. The activated sludge biota was observed using a standard optical microscope, and it was cleared for testing upon verification of the absence of abnormalities.

Inspecting the Level of Activation of the Activated Sludge and Date Use Started
The level of activation was checked using standard product before beginning to use the activated sludge.
Date Use Started 1/13/2004

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

other: During the incubation period, changes in BOD of the test solution were continuously measured and automatically recorded with the data processing device

Parameter followed for biodegradation estimation:

test mat. analysis

Details on study design:

Conducting the Biodegradation Testing

Test Preparations

(1) The suspended substance concentration was measured to determine the amount of activated sludge addition.
Measurement Method Conducted according to “Factory Sewage Test Method, Suspended Substances” (JIS K 0102-1998 – 14.1).
Measurement Dates 2/16/2004
Measurement Results The suspended substance concentration of the activated sludge was 4400mg/L.

(2) Preparation of the Incubation Base
Purified water (manufactured by Takasugi, Japan Pharmacopeia) was added to 3mL each of solution A, solution B, solution C and solution D stipulated in “Factory Sewage Test Method, Biochemical Oxygen Demand” to make 1L, and the pH was adjusted to 7.0.

(3) Control Substance
To verify that the sludge had sufficient activated for the actual testing, aniline (manufactured by Showa Chemicals, reagent grade, Lot # SO-32360) was used as a control substance. Evaluation and production of the chemical substance was performed according to the test method stipulated in the laws relating to standards as well as in conformance with OECD test guidelines.

Preparation of the Test Solution
6 test containers were prepared, and the test solution was prepared according to the following method.

(I) Addition of the Test Substance and Aniline
(a) (water + test substance) group (1, test container #6)
300mL of purified water was placed in the test container and 30mg of the test substance was precisely measured using an electronic analysis scale and added so the test substance concentration was 100mg/L.

(b) (sludge + test substance) group (3, test containers #2, 3, 4)
The incubation base [the amount of activated sludge added (2.05mL) subtracted from 300mL] was added to the test containers and 30mg of the test substance was precisely measured using an electronic analysis scale and added so the test substance concentration was 100mg/L.

(c) (sludge + aniline) group (1, test container #5)
The incubation base [the amount of activated sludge added (2.05mL) subtracted from 300mL] was added to the test container and 29.5uL [addition amount 30mg = 29.5µL x 1.022 g/cm3 (density)] of aniline was measured using a microsyringe and added so the test substance concentration was 100mg/L.

(d) sludge blank group (1, test container #1)
The incubation base [the amount of activated sludge added (2.05mL) subtracted from 300mL] was placed in the test container.

(II) Innoculating the Activated Sludge
Activated sludge were inoculated in test solutions (b), (c) and (d) in a suspended substance concentration of 30mg/L.

Test Solution Incubation Apparatus and Environmental Conditions

(1) Test Solution Incubation Apparatus
Closed oxygen demand measurement apparatus
Constant temperature vat and measurement unit (Mfg by Asahi Techneion Co., Ltd.)
Data processing device (Mfg by Asahi Techneion Co., Ltd.)

Test Containers: 300mL incubation chambers (improved incubation chambers)
Carbon Dioxide Gas Absorbent: Soda lime, No. 1 (Mfg by Wako Pure Chemical Industries for carbon dioxide absorption)

(2) Environmental Conditions
Incubation temperature of test solution 25±1°C
Time of test solution incubation 28 days
Agitation method Rotating agitation with a magnetic stirrer

Observations, Measurements, etc.
(1) Observations
During the incubation period, the status of the test solutions was visually observed on a daily basis. Additionally, the operational status of the apparatus was periodically checked.

(2) Measurement of Biochemical Oxygen Demand (BOD)
During the incubation period, changes in BOD of the test solution were continuously measured and automatically recorded with the data processing device. Additionally, the temperature inside the tank was measured and recorded on a daily basis.

Reference substance:

aniline

Preliminary study:

No preliminary study

Key result

Parameter:

other: BOD

Value:

0 - 1

Sampling time:

28 d

Parameter:

% degradation (test mat. analysis)

Value:

0

Sampling time:

28 d

Details on results:

See graph

Results with reference substance:

The biodegradation determined by the BOD for the aniline after 7 days and 14 days was 72% and 77% respectively, and this test was deemed valid.

Validity criteria fulfilled:

yes

Remarks:

see conclusions

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

Validity criteria:
The oxygen uptake of the inoculum blank did not exceed 60 mg 02/L in 28 days. The difference of extremes of replicate values of the removal of the test substance at the end of the test, was less than 20% and the percentage degradation of the reference substance calculated from the oxygen consumption exceeded 40% after 7 days and 65% after 14 days. Even if no pH values were given, the test was considered as valid.

Executive summary:

The ready biodegradability of 1,3-di-o-tolylguanidine was evaluated in a study performed in accordance with OECD testing guideline 301 C and GLP requirements. The maximum level of biodegradation was 1% in 28 days. Therefore, according to these results, 1,3-di-o-tolylguanidine is not considered as readily biodegradable.

The sterile control did not show abiotic degradation. The potential inhibitory effect of 1,3-di-o-tolylguanidine on the micro-organisms of the inoculum was not assessed.

Validity criteria were fulfilled.

Description of key information

1,3-di-o-tolylguanidine is not considered readily biodegradable.
Study performed in accordance with:
- OECD testing guideline 301 C and 
- GLP requirements. 

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

The ready biodegradability of 1,3-di-o-tolylguanidine was evaluated in a study performed in accordance with OECD testing guideline 301 C and GLP requirements.

The maximum level of biodegradation was 1% in 28 days. Therefore, according to these results, 1,3-di-o-tolylguanidine is not considered as readily biodegradable.

The sterile control did not show abiotic degradation.The potential inhibitory effect of 1,3-di-o-tolylguanidine on the micro-organisms of the inoculum was not assessed.

Validity criteria were fulfilled.