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Environmental fate & pathways

Biodegradation in water: screening tests

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Description of key information

Alpha methylstyrene was shown to be rapdily biodegradable. 

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Reliable results of guideline studies (OECD 301C, OECD 301D, OECD 301F and OECD 302C) investigating the ready and inherent biodegradation of alpha-methylstyrene are available.

The ready biodegradation of alpha-methylstyrene was investigated in a study conducted according to OECD Guideline 301 D (Closed Bottle Test; 1992) and EU Method C.4-E (2008) over a period of 28 days. In deviation from OECD Guideline 301D a mixed inoculum obtained from the effluent of a wastewater treatment plant and soil was used in the test. The biodegradation rate was determined by measurement of oxygen consumption. Inoculum blank, procedural/functional control with the reference substance sodium benzoate, and toxicity control using 1.13 mg/L test substance and 3.039 mg/L reference compound were performed. The functional control reached the pass level >60% after 14 d. In the toxicity control containing both test and reference item 63 % biodegradation based on oxygen consumption occurred within 21 d (78 % after 78 d) thus indicating that the test substance was not inhibitory at the concentration tested. In this close-bottle test 56 % biodegradation were obtained within 21 days, which is very close to the 60 % threshold for ready biodegradability. Regarding this test, a number of uncertainties have to be taken into account. During application of the volatile AMS to the medium in the Closed Bottle test, the spiked medium was left at least for some time in an „open system“ and so a part of the applied amount of AMS might evaporate from the medium before transferred into the test vessels or at least before the vessels were gas-tight sealed. In this case, the actual loading would have been below the nominal one and a part of the nominal concentration would have been not available for microbial biodegradation.

The latter uncertainty may also be related to the results of a study conducted according to OECD Guideline 301 F, in which alpha-methylstyrene underwent 8 % and 21 % biodegradation after 28 d at 100 and 20 mg/L, respectively, under the chosen test conditions. The part of applied test substance, which might have evaporated from the test solution, would have been no longer available to the degrading inoculum, leading to an underestimation of the degradation rate. This is supported by the higher percentage observed with the lowest nominal concentration, which suggests that the actual level of dissolved substance (and consequently actual ThOD) is closest to 20 than 100 mg/L, and probably lower than 20 mg/L.

In the study conducted according to OECD Guideline 301C (Ready Biodegradability: Modified MITI Test (I)), alpha-methylstyrene was not degraded under the test conditions employed (0% biodegradation after 14 d; initial concentration: 100 mg/l). The test method was identified by OECD as being in principal appropriate for volatile substances and an ‘improved type of study design for volatile substances’ was applied for testing AMS (no further details specified). The reason for the significant difference compared to the results of all other studies available on biodegradability can not be explained by the available references. However, it seems questionable, whether the applied MITI-I method is the best choice for testing ready biodegradation of substances exhibiting both, high volatility as well as limited water solubility.

From the experimental study results available for AMS on the endpoint ready biodegradability, no definitive proof of ready biodegradability has been obtained, when strictly applying the criteria of the OECD 301 guidelines. However, from the diversity of degradation rates it can be furthermore concluded that the substance, while being in principal not recalcitrant to biodegradability, requires a specific experimental design taking into account its significant volatility as well as limited water solubility (100-116 mg/L in pure water, i.e. lower in test medium that contains salts). This potential for biodegradability is supported by inherent test biodegradation, and ability of isolated strains to grow on AMS as sole source of carbon and energy, as shown hereafter.

The inherent biodegradation of alpha-methylstyrene was investigated in a study conducted according to OECD Guideline 302 C (Modified MITI Test (II)) over a period of 28 days and using municipal activated sludge as inoculum. The biodegradation rate was determined by measurement of oxygen consumption. Inoculum blank, procedural/functional control with the reference substance sodium benzoate, and toxicity control using 31.8 mg/L test item and 100 mg/L reference compound were performed. Alpha-methylstyrene proved to be inherently biodegradable under the test conditions employed (56% biodegradation after 28 d, as mean of duplicates). The functional control reached the pass level >60% after 14 d. In the toxicity control containing both test and reference item ca. 80% biodegradation occurred within 14 d. Based on the results and the validity criteria of the guideline (>=25% biodegradation based on ThOD) the test substance can be assumed to be not inhibitory to the inoculum. Furthermore, this study provides evidence of biodegradability up to 70 % in 28 days, as shown in one of test duplicates. Obviously, no molecular configuration is present in the substance that is resistant to the biodegradation process. On the other hand, the second replicate shows a long lag phase (minimum 10 days). It can be concluded that biodegradation of the AMS-molecule requires the presence of specific bacterial species that may be relatively infrequent, or as seen in other tests, that actual substance concentrations achieved were irregular in different replicates.

In other tests it was shown that various bacterial strains, mostly isolated from contaminated sites, were able to grow on alpha-methylstyrene and the test substance was used as sole carbon and energy source: e.g. Bacillus cereus 3, Pseudomonas aeruginosa 21, BS176 and DS13, Pseudomonas convexa S107B1, Pseudomonas putida MST. Several intermediates were isolated after incubation: 2 -phenyl-2-propen-1-ol, 3 -isopropylcatechol, 1,2 -dihydroxy-3-isopropenyl-3-cyclohexane, 2 -phenylpropenoic acid, and acetophenone.

On another hand, the ready biodegradability of alpha-methylstyrene is supported by the structure analogue substances styrene, acetophenone and cumene biodegradation results.

Styrene is structurally closely related toalpha-methylstyrene, the only difference is the presence of a methyl group in the latter. Therefore, it is expected that the same enzyme causes the initial step of biodegradation. When the methyl group is removed, the same metabolism pathway is expected for both styrene andalpha-methylstyrene.As revealed in several tests on ready biodegradability, styrene proved to be readily biodegradable under aerobic conditions. There are sufficient results from standard or near-standard tests to consider styrene to be readily biodegradable and meeting the 10-day window criterion.

Acetophenoneis structurally closely related toalpha-methylstyrene. The only difference is the presence of an oxygen atom instead of the methylene group, causing lower volatility and higher water solubility.Acetophenone was shown to be readily biodegradable (64,7 % after 14 d in OECD 301C), and this was confirmed by water simulation test giving a DT50 from 6 to 8 days.

Cumeneis structurally closely related toalpha-methylstyrenewith a double bond in the side chain being the only difference in the latter.Cumene was shown to undergo >60 % biodegradation after 10 d in a test according to US-APHA guideline.