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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted under GLP

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2,4,4-tetramethylcyclobutane-1,3-diol, mixed isomers
EC Number:
EC Name:
2,2,4,4-tetramethylcyclobutane-1,3-diol, mixed isomers
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report): PM 1265 (internal company product designation) and Cyclobutanediol, 2,2,4,4-Tetramethyl
- Substance type: production sample
- Physical state: solid
- Lot/batch No.: TS060505
- Storage condition of test material: Ambient conditions

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Activated sludge was collected from Denton Wastewater Treatment Plant, Denton, Maryland on July 17, 2006. The Denton facility treats predominantly residential wastes. The sludge was sieved using a 2-mm screen and then aerated for approximately four hours. After the aeration period, an aliquot of the sludge was homogenized in a blender at medium speed for approximately two minutes and then allowed to settle for approximately 30 minutes. The supernatant was used as the inoculum the same day that it was prepared. A total suspended solids measurement and standard plate count were performed on the inoculum. Plates were incubated at 20±3ºC for approximately 48 hours. The results of the standard plate count and TSS measurement performed on the inoculum were 5.3 x 105 CFU/mL and 260 mg/L, respectively.
Duration of test (contact time):
49 d
Initial test substance concentration
Initial conc.:
10 mg/L
Based on:
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
The test medium was a modified biochemical oxygen demand (BOD) test dilution water and was prepared using high quality water. The test chambers were amber 4-liter bottles. The air entering the chambers was passed through Drierite to remove ambient moisture and then through Ascarite to produce CO2 free air. The air exiting the test chambers was passed through a series of three gas washing bottles, each containing approximately 100 mL of 0.5 N KOH to trap the CO2 that had evolved within the chamber. An additional set of gas washing bottles that were not connected to a chamber were maintained concurrently with the traps connected to the chambers. These bottles contained approximately 100 mL of 0.5 N KOH and the amount of CO2 detected in the KOH solution was subtracted from the CO2 in the blank control traps to determine the amount of CO2 produced by the
inoculum in the blank control. The test was conducted at 20±3ºC. Magnetic stir bars and stir plates were used to mix the contents of the test chambers. All chambers were aerated with CO2-free air for approximately 24 hours at a rate of 50 to 100 mL per minute to purge the systems of CO2. After the aeration period, the flow of CO2-free air was stopped, and three CO2 traps, each containing approximately 100 mL of 0.5 N KOH, were connected to the exit air lines of each chamber. Sufficient volumes of reference substance stock solution to achieve a nominal concentration of 10 mg C/L were added to the appropriate chambers. Sufficient volumes of test substance stock solution to achieve a nominal concentration of approxiamtely10 mg C/L were added to the appropriate chambers. The final volume within all chambers was brought up to 3000 mL by the addition of NANOpure water and the airflow was restarted on the system. The biodegradation test was started by bubbling CO2-free air through each of the test chambers
at a rate of 50 to 100 mL per minute. The CO2 produced from the degradation of organic carbon sources within the test chamber was trapped as K2CO3 in the KOH solution and the amount of inorganic carbon in the trapping solution was measured at various intervals during the study, using a
Shimadzu Model TOC-5000 carbon analyzer.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradation
% degradation (CO2 evolution)
St. dev.:
Sampling time:
49 d
Details on results:
There was a lag period of approximately 22 days before degradation of the test substance began. Approximately 22 days later (day 44) greater than 60% degradation occured. At test temination on day 49 the cumulative degradation reached 72.6% and based upon the slope of cumulative degradation degradation had not plateaued (see figure).

BOD5 / COD results

Results with reference substance:
The viability of the inoculum and validity of the test were supported by the results of the reference substance, sodium benzoate, from which an average of 105.1% of theoretical CO2 was evolved. An average percent biodegradation of greater than 60% was achieved by Day 14, thereby fulfilling the criteria for a valid test of reaching the pass level by Day 14.

Applicant's summary and conclusion

Validity criteria fulfilled:
Executive summary:

A ready biodegradation study conducted according to the procedures specified in the OECD Guideline for Testing of Chemicals, Method 301B was conducted on PM-1265. Degradation of the test substance was not observed until approximately day 22 of the study (lag phase). Approximately 22 days later (day 44) greater than 60% degradation occured. At test temination on day 49 the cumulative degradation reached 72.6% and based upon the slope of cumulative degradation degradation had not plateaued. The reference substance, sodium benzoate, exhibited a normal degradation curve and achieved greater than 60% degradation by day 14. Based upon the amount of CO2 evolved and the degree of degradation observed, it is clear that the substance will undergo ultimate degradation. However, under the conditions of this test there was a considerable lag period which prevents the substance from being considered readily bioegradable.