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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002-05-14 - 2002-07-23
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study following GLP. The concentration of material tested was toxic to the inoculum. The actual concentration used was not confirmed analytically, and the material is insoluble in water at the concentrations tested.
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
CYANOX® 1790 Antioxidant
IUPAC nomenclature - Tris(4-tert-butyl-3-hydroxy-2,6-dimethylbenzyl) isocyanurate
Synonym - [tris(4-t-butyl-3-hydroxy-2,6-dimethyl-benzyl)-s-triazine-2,4,6-(1H,3H,5H) trione]
Lot PA2010802
Appearance - White powder, odorless
CAS No. 40601-76-1
Molecular Formula - C42H57N3O6
Molecular Weight - 699.92 g/mole
Purity > 99%
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, industrial (adaptation not specified)
Details on inoculum:
Obtained from a domestic secondary wastewater treatment facility.
Duration of test (contact time):
>= 28 d
Details on study design:
Water used for the preparation of test media was collected from a water purification system using both ion exchange and carbon filtration treatment. The test water had a total organic carbon (TOC) content < 1 mg/l. TOC analysis was done using a DC-80 TOC analyzer system. This water was used to make a defined mineral medium containing phosphate buffer, ferric chloride (final concentration 0.25 mg/l), magnesium sulfate (final concentration 22.5 mg/l) and calcium chloride (final concentration 27.5 mg/l). The test material was not soluble enough in water to make a stock solution, so the material was weighed into weigh boats and added dry to test bottles. The weigh boats were washed with mineral medium to remove as much material from them as possible. The carbon content of the test material was analyzed with an elemental analyzer. It contained the equivalent carbon concentration of 71.9%. A stock solution of the reference material (1000 mg ai/l 99.6% pure sodium benzoate) was prepared on the day of the test which had an equivalent carbon concentration of 58.3%.

A total of six 4-liter bottles were prepared. On day minus one, 100 ml of inoculum and 2400 ml of mineral medium were added to each flask. The inoculum was the glass wool filtrate from 2 liters of secondary effluent collected on May 30, 2002 from the Loxahatchee River Environmental Control District Wastewater Treatment Plant in Jupiter, Florida. The contents of each bottle were purged with CO2-free air for 24 hours prior to test initiation. The airflow was controlled by flowmeters and regulated to 40 mg/l for each bottle. Check valves were inserted in the lines between the gas washing bottles and the test bottles.

After the bottles were purged with CO2-free air, 500 ml of mineral medium was added to two bottles that served as inoculum blanks (no test material). Sodium benzoate (103 ml of stock solution, for a concentration of 20.0 mg carbon/l) and 397 ml of mineral medium was added to another bottle. Two bottles containing test material were prepared (one containing 0.0836 g of test material and 500 ml of mineral medium, for a concentration of 20.1 mg carbon/l and another containing 0.0846 g of test material and 500 ml of mineral medium, for a concentration of 20.3 mg carbon/l). The final bottle received 0.0836 g test material, 103 ml of the sodium benzoate stock solution and 397 ml of the mineral medium (20.1 mg carbon/l test material and 20.0 mg carbon/l reference material, for a total of 40.1 mg carbon/l). Each bottle was capped with a silicone stopper. Inlet and outlet tubing was built into each stopper. All of the bottles were placed in an environmental chamber maintained at 22 +/- 2 degrees C, and aerated through the inlet tubing. The medium in each bottle was continuously stirred with a magnetic stirrer.

The evolved CO2 gas that came out of each bottle was trapped in a series of three gas-washing bottles containing 100 ml of 0.0125 M Ba(OH)2. The amount of unreacted Ba(OH)2 was quantified by titrating with a HCl solution to the phenolphthalein endpoint. During the first week of the study, CO2 was analyzed every 2-3 days. After the first week, the sampling schedule was changed to once every 3-5 days. The test was terminated on day 28. On day 28, aeration was stopped, the lids on the bottles were opened and 1 ml of concentrated HCl was added to each bottle to release any CO2 remaining in the test vessel. Final titrations were made on day 29.

The percent biodegradability at each time interval was calculated for each test condition using the following equation: percent biodegradability = cumulative CO2 produced (mg)/ThCO2 (mg) x 100, where ThCO2 (mg) = carbon added (mg) x 44 mg as CO2/12 mg as carbon. The amount of CO2 produced at each time interval = K(50-Vt), where K = 1.10 (the constant which converts volume of HCl titrated to mg CO2 produced), 50 = volume (ml) needed to titrate 100 ml Ba(OH02 and Vt = volume (ml) HCl titrated into the test solution. The amount of CO2 produced in the inoculum only control was subtracted from that produced by the test or reference materials to obtain the amount of CO2 produced from these materials.
Reference substance:
benzoic acid, sodium salt
Preliminary study:
None conducted
Parameter:
% degradation (CO2 evolution)
Value:
2.3
Sampling time:
5 d
Parameter:
% degradation (CO2 evolution)
Value:
5.4
Sampling time:
10 d
Parameter:
% degradation (CO2 evolution)
Value:
9.3
Sampling time:
14 d
Parameter:
% degradation (CO2 evolution)
Value:
17.2
Sampling time:
20 d
Parameter:
% degradation (CO2 evolution)
Value:
24.7
Sampling time:
28 d
Details on results:
The cumulative biodegradation rates (in %) for the two concentrations of test material at 3, 5, 7, 10, 14, 17, 20, 24, 28 and 29 days were 1.0 and 1.0, 2.1 and 2.4, 4.5 and 4.57, 4.98 and 5.83, 8.28 and 10.4, 13.3 and 15.4, 16.3 and 18.1, 17.4 and 21.8, 21.7 and 27.6 and 23.2 and 28.5, respectively. At the same time intervals, values (in %) for inoculum controls were 12.9 and 12.7, 15.1 and 15.4, 17.2 and 14.4, 22.0 and 22.7, 25.6 and 23.5, 20.7 and 21.2, 24.4 and 23.4, 25.5 and 24.5, 23.3 and 23.4 and 20.1 and 19.6, respectively.

Since a >= 60% biodegradation rate was not achieved within 28 days, the material was not deemed readily biodegradable.
Results with reference substance:
The cumulative biodegradation rate for the reference material at 3, 5, 7, 10, 14, 17, 20, 24, 28 and 29 days was 19.0, 37.0, 54.0, 62.4, 68.1, 71.6, 73.6, 74.4, 76.4 and 77.1, respectively. The cumulative biodegradation rate for the reference plus test material at 3, 5, 7, 10, 14, 17, 20, 24, 28 and 29 days was 9.5, 18.4, 26.9, 32.0, 35.4, 37.3, 38.3, 38.8, 39.6 and 40.5, respectively.

The authors stated that "the test substance was not toxic to the inoculum since biodegradation occurred in a mixed solution containing test and reference materials". However, it is clear that the test material was toxic to the inoculum, since the amount of biodegradation occurring in the flask containing both the reference and test material was less than that of the reference material alone.

Validity criteria fulfilled:
yes
Remarks:
The test was valid, since the total amount of CO2 evolution in the inoculum blank at the end of the test was within the acceptable range for a valid test, and the cumulative biodegradation of the reference material (68.1% on day 14) was > = 60% by day 14.
Interpretation of results:
not readily biodegradable
Conclusions:
Since a >= 60% biodegradation rate was not achieved within 28 days, the material was not deemed readily biodegradable
Executive summary:

The test material was not readily biodegradable within a 28-day test period when exposed to microorganisms maintained in an aerobic, aqueous mineralized environment (according to OECD TG 301B and under GLP conditions). The mean cumulative biodegradation of the test substance was 25.9% after the 28 days and did not meet the test criteria for ready biodegradability.

Description of key information

The test material was not readily biodegradable within a 28-day test period when exposed to microorganisms maintained in an aerobic, aqueous mineralized environment (according to OECD TG 301B and under GLP conditions). The mean cumulative biodegradation of the test substance was 25.9% after the 28 days and did not meet the test criteria for ready biodegradability.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable
Type of water:
freshwater

Additional information