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Environmental fate & pathways

Biodegradation in water and sediment: simulation tests

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biodegradation in water: sewage treatment simulation testing
Type of information:
experimental study
Adequacy of study:
supporting 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
other: OEDC 314B
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Dibutyl terephthalate
EC Number:
EC Name:
Dibutyl terephthalate
Cas Number:
Molecular formula:
1,4-dibutyl benzene-1,4-dicarboxylate
Details on test material:
Identification: 1,4-Dibutyl [1-14C] terephthalic acid [1,4-14C]
Description: brown residue
Batch: 120403
Purity: >99%
Storage conditions: approximately 4 °C in the dark
The sample was synthesied by American Radiolabeled Chemicals. Inc., St. Louis, MO from terephtbalic add [1,4-14C] and butatanol [1-14CJ. The product was purified by preparative HPLC.


Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms was obtained on 15 May 2012 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage. The activated sewage sludge sample was sieved through a 2 mm sieve prior to use. The sieved sample was then maintained on continuous aeration in the laboratory at a temperature of
approximately 20 °C until used. The suspended solids concentration was equal to 3548 mg/L prior to use. The pH was 7.60. The abiotic sludge was prepared by adding 20 mL of mercuric chloride solution (1000 mg/L) to 2 liters of the sieved sludge. This was then autoclaved for 90 minutes at approximately 121 °C and a pressure of 15 psi. The pH was adjusted to 7.61 with 1M NaOH.
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
0.004 mg/L
Based on:
test mat.
Initial conc.:
59 910 other: Bq radioactivity
Based on:
test mat.
Parameter followed for biodegradation estimation:
radiochem. meas.
Details on study design:
An amount of the test item dose solution (180 uL ) was dispersed directly into 2 liters of the activated sludge to give a final nominal concentration of 0.0045 mg/L, the reported water solubility of the test item. Two test systems were prepared. One contained 2 liters of activated sludge (biotic system) and one contained 2 liters of sterilized sludge (abiotic system). Each test system consisted of the test vessel, two sodium hydroxide vessels to trap evolved carbon dioxide and one ethylene glycol vessel to trap any volatile components. The test vessels were sealed and C02-free air bubbled through the solution and stirred continuously by magnetic stirrer. The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 2M sodium hydroxide. Samples (2 x 1 mL) were taken from the first sodium hydroxide trap at 5, 15, 30, 45, 60 and 90 minutes after dosing with additional samples taken after 2, 3 and 5 hours. Further samples were taken on Days 1, 2, 3, 6, 7, 8, 12, 14, 16, 21 and 28. The second sodium hydroxide traps and ethylene glycol traps were all sampled on Days 7, 14 and 28. The mixed liquor was analyzed on Day 0 and Day 28.

Results and discussion

% Degradation
% Degr.:
ca. 98
radiochem. meas.
Sampling time:
28 d
Half-life of parent compound / 50% disappearance time (DT50)
other: wastewater
ca. 60 h
(pseudo-)first order (= half-life)
20 °C
Mineralization rate (in CO2):
0.011 h-1
Details on results:
At Time 0 the actual radioactivity dosed to the Biotic system was 59910 Bq and the actual radioactivity dosed to the Abiotic system was 64530 Bq determined by LSC. Analysis of the Biotic mixed liquor at time 0 showed that a mean of 73.3% AR was in the aqueous phase and a mean of 9.8% AR was extracted from the solid phase. A mean of 2.1% AR was in the combusted solids. Analysis of the Biotic system at Day 28 showed that a mean of 4.8% AR was in the mixed liquor and a mean of 102% AR was evolved C02. Of the activity found in the mixed liquor, this was comprised mainly of dissolved C02 with a small amount associated with the solid residue (2% AR). The total mean recovery for the Biotic system was 85.1% AR at Time 0 and 107% AR at Day 28. Analysis of the Abiotic mixed liquor at time 0 showed that a mean of 7.5% AR was in the aqueous phase and a mean of 96.8% AR was extracted from the solid phase. A mean of 0.3% AR was in the combusted solids. Analysis of the Abiotic mixed liquor at time 28 showed that a mean of 99.0% AR was in the mixed liquor. The aqueous phase contained 92.5% AR. A small amount of radioactivity was detected in the solid extract samples but the levels were below the LOQ of the instrument and so not calculated. The total mean recovery for the Abiotic system was 105% AR at Time 0 and 99.0% AR at Day 28.

Given the rapidly degradable nature of the test item, it was considered likely that the small amount of 14C that was associated with the solid residue was the result of incorporation of the 14C into the microbial biomass and not due to undegraded test item. Of the 4.8% radioactivity remaining in the Biotic system mixed liquor at the end of the test, this was split approximately evenly between the aqueous phase and the solid residue. The radioactivity in the aqueous phase was shown to be due to dissolved C02 and, as discussed above, the remainder in the solid residue was considered to be 14C incorporated into the microbial biomass.

Applicant's summary and conclusion

Executive summary:

A study was performed to assess the biodegradation of the test item in activated sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2008) No. 314B, "Biodegradation in Activated Sludge". The test item, at a nominal concentration of 0.0045 mg/L, was exposed to activated sewage sludge micro-organisms in sealed culture vessels in the dark at approximately 20C for 28 days. A second system containing abiotic sewage sludge was also prepared. The test item was dissolved in an auxiliary solvent prior to being added to the test medium. Using this method the test item was evenly distributed throughout the test medium. The degradation of the test item was assessed by the determination of carbon dioxide produced. Lack of degradation in the abiotic system confirmed that degradation was due to the microorganisms in the sludge. After 28 days, 102% of the applied radioactivity (AR) in the biotic system had degraded to 14C02 with 2% AR remaining bound to the solids. This equates to 98% of the total activity detected degrading to 14CO2 and 2% remaining associated with the solids. In the abiotic system 99% AR remained in the mixed liquor after 28 days. Due to the rapid degradation of the test item, it would be expected that the test item would be degraded in a waste water treatment plant with a small amount incorporated into the microbial biomass as 14C rather than as test item.