4-methylstyrene

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: simulation testing on ultimate degradation in surface water

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 19, 1981 to November 22, 1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Principles of method if other than guideline:

A study was conducted to determine the degradation of the test substance by activated sludge over a 33 d period consisting of two phases (acclimation and biodegradation). The test system was modelled after a typical domestic sewage treatment plant.

GLP compliance:

not specified

Remarks:

GLP not applicable for this type of study at this time.

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, adapted

Details on inoculum:

- Source of inoculum: sewage treatment plant at the city of Colombia, Missouri, USA. The material used in the study was collected approximately 4 h before commencement of study
- Laboratory culture: prior to use the sludge was kept refrigerated in the laboratory
- Method of cultivation: no cultivation
- Storage conditions: prior to users such was kept refrigerator
- Storage length: maximum of 4 h
- Preparation of inoculum for exposure: solids concentration of the sludge was determined and adjusted to 2500 mg/L with local drinking water
- Initial cell/biomass concentration: microbial population was 3.8 + E7 / ml solids

Duration of test (contact time):

19 d

Initial conc.:

50 mg/L

Based on:

TOC

Remarks:

C14/C13 labeled

Initial conc.:

100 mg/L

Based on:

TOC

Remarks:

C14/C13 labeled

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

The test system employed was based on tall 3000 ml resin pots containing 2000 ccs of test solution designed to simulate a commercial sewage treatment plant. All vessels were sealed to allow trappings of released carbon dioxide and organic volatiles (samples were extracted using a siphon device to avoid introduction of air). The basic design of the system consisted of all an aeration vessel, sludge nutrient/test compound vessel containing a stirring device followed by a series of chemical traps (potassium hydroxide and ethylene glycol). The rate of aeration was controlled by a flow meter. A total of 4 individual systems were employed two of which contained the first compound, the third contained the positive control and the final vessel contained a solvent control. The study was conducted as a series all phases as follows:

1. Acclimation phase (fourteen days).
The simulated influent were prepared as follows for chamber 1.
Acetone - addition of 1.0 ml acetone to 15 ml of sludge suspension followed by dilution to two litres.
Direthylene glycol - addition of the specified quantity or diethylene glycol to 15 ml of sludge suspension followed by dilution to two litres.
Test substance - additional of the specified quantity of the test substance in 1.0 ml acetone to 15 ml of sludge suspension followed by dilution to two litres.
Non radiolabelled test substance was introduced on a daily basis into chamber No 2 (sludge nutrient/test compound chamber). Similarly systems three and four were treated with either the positive control (diethylene glycol) or the solvent (acetone). The rates of addition are shown in the table below.

Assessments conducted during this phase included measurement of suspended solids (initial value 2500 mg/L reducing to 910 - 1980 mg/L after 14 days), temperature levels 24 - 28C), any necessary pH adjustment (6.9 - 7.6) and measurement of dissolved oxygen levels in the system (0.7-4.8 ppm). During the course of this phase of the study the DOC reduction was analysed at each sampling interval and other samples of sludge were removed for microbial counting on days 0, 3, 6, 9, 11 and 15 (start of the biodegradation phase day 1).

2. Biodegradation phase (Day 15 -33).
On day 15 a 13C/14C test substance mixture was introduced into the two treatment reaction systems at 50 and 100 ppm (measured as organic carbon), introduction of additional substrate was discontinued and the gas traps were attached. Samples were taken at intervals (0.5 h, 3.0 h, 24 h, 2 days, 3 days, 4 days, 6 days, 8 days, 10 days, 13 days, 16 days and 19 days. The results are shown in the table below.

Reference substance:

ethylene glycol

Key result

Remarks on result:

not determinable because of methodological limitations

Transformation products:

not specified

Evaporation of parent compound:

yes

Volatile metabolites:

yes

Residues:

yes

Details on results:

The results show that organic carbon levels were reduced by >90 % in the presence or both the test substance and the positive standard therefore it can be concluded that the test substance did not adversely affect the ability of microorganisms to degrade organic carbon in the simulated sewage system present at levels up to100 ppm expressed as organic carbon. Similarly the results of microbial cell counts show that the test substance did not adversely affect microbial populations which, although any effects were partially masked during the acclimation phase of the study by the large amounts on substrate populations were exposed to, the authors conclude that there were no effects that resulted in for any reduction in removal of organic carbon. The ability on microflora to degrade the test substance was assessed by trapping radiolabelled carbon dioxide and radiolabelled volatiles in the biodegradation phase of the study. The amount or radiolabelled carbon dioxide varied from 10.89 - 11.81 % of the applied dose while trapped volatile organics accounted for 8.71 and 11.8 % giving total radiolabelled carbon trapped or 19.6 and 23.61 % of the applied dose. Analytical results showed that only 40 % of the initial dose rate was present in the test system within 30 minutes after dosing. In an attempt to address the volatility problem a non study system was a set up in which the efficacy of the trapping system was assessed. The results of this study showed that three minutes after spiking liquid scintillation counts showed a recovery of 67.4 % without aeration. This study showed that a large proportion of the volatiles trapped were in point of fact parent material rather than carbon dioxide.
The conclusion regarding and the biodegradation phase of the study is that the test substance when placed in an aerobic system is rapidly lost to the atmosphere therefore biodegradation is not a critical route for removal from the ecosystem.

Results with reference substance:

Diethylene glycol was used as a reference standard which is known to biodegrade in domestic sewage systems. The results obtained with this material in terms of effects on microflora and organic carbon removal were virtually identical to those obtained with the test substance. For practical purposes the test substance can therefore be considered equivalent to the reference standard and can therefore be classed as readily biodegradable.

Acclimation phase DOC analysis (ppm)

Sample description		Acclimation day number.
		12	13		14
Acetone control	Influent	351		343		372
	Effluent	14		12		9
Positive control	Influent	169		203		178
	Effluent	14		11		5
Treatment level 1	Influent	343		366		315
	Effluent	21		15		10
Treatment level 2	Influent	292		306		340
	Effluent	18		14		9

percentage removal 93.9.

 Microbial plate counts organisms/ml

Sample description	Dilution	Acclimation day number.					Biodegradation phase
		0	3	6	9	11	15	Termination
Acetone control	10+E5	292	NC	1300	335	490	335	40
Positive control	10+E5	346	NC	1625	300	320	225	53
Treatment level 1	10+E5	406	NC	1138	665	590	270	25
Treatment level 2	10+E5	360	NC	1470	590	1463	455	66

Residues in the sludge samples as parent compound equivalents

	14C-PMS 50ppm		14C-PMS 100ppm
Sample time	ppm	% of applied dose	ppm	% of applied dose
0.5 hours	19.8	36.0	41.6	37.8
3 hours	11.6	21.1	25.9	23.5
6 hours	6.59	12.0	9.36	8.5
1 day	3.7	6.7	9.86	9.0
2 days	3.73	6.8	10.23	9.3
3 days	3.97	7.2	9.26	8.4
4 days	3.54	6.4	8.6	7.8
6 days	2.34	4.3	8.07	7.3
8 days	2.21	4.0	6.69	6.1
10 days	1.77	3.2	6.07	5.5
13 days	1.63	3.0	4.93	4.5
16 days	1.91	3.5	5.06	4.6
19 Days	1.71	3.1	4.25	3.9

Based on the above the DT50 is <0.5hrs and the DT90 is 5 hrs.

Validity criteria fulfilled:

not applicable

Conclusions:

Based on the results obtained, it was postulated that the high rate of loss from the reaction vessels was due to volatilisation. A subsequent experiment introducing the test substance into a completely closed system resulted in 58.8% accountability at test start, with concentrations decreasing to 2.7% of initial values after 3 h of aeration. Since on 4.3% was recovered by trapping, the test substance was assumed to have been adsorbed by the experimental system.

Executive summary:

A study was conducted to determine the biodegradation of the test substance by activated sludge in a 33 d study consisting of two phases (acclimation and biodegradation). The sludge was obtained from a municipal wastewater treatment plant and diluted with tap water to approximately 2500 mg solids/L (ca. 3.8 E+7 organisms/mg solids). The test system consisted of 3 L standard tall aeration vessels (resin pots) and was modelled after a typical domestic sewage treatment plant. All ground glass joints were treated with sealing wax to preserve the integrity of the closed system.

In the 14 d acclimation period, non-radiolabelled test material was used at concentrations ranging from 0 to 50 ppm (vessel 1) or 0 to 100 ppm (vessel 2) following a daily ‘draw and fill’ routine. Daily operations included determination of the suspended solids in the mixed liquor, pH adjustment upon refill and measurement of dissolved oxygen levels in the mixed liquor. Aliquots of the effluent from each of the two controls and two test chambers were taken for Dissolved Organic Carbon (DOC) analysis on Days 12, 13 and 14. Aliquots of the sludge from each of the two controls and two test chambers were taken for microbial analysis on Days 0, 3, 6, 9, 11 and 15 of the acclimation period and at the end of the study.

On Day 15 of acclimation, 14C/13C-labelled test substance was introduced with the daily influent at 50 and 100 ppm (as organic carbon). Daily ‘draw and fill’ was discontinued at that time. Trapping solutions for the collection of 14C-CO2 and 14C-labelled volatile organics in effluent air, and representative aliquots of the mixtures in each test vessel were taken at 0.5, 3, 6 and 24 h, then on Days 2, 4, 6, 8, 10, 13, 16 and 19. Analysis was conducted using Liquid Scintillation counting.

The outcomes of this 33 d two-phase study showed that the inclusion of the test substance at two rates (50 and 100 ppm, calculated as carbon equivalents) did not adversely affect the wastewater treatment process or affect the sludge microflora. The test substance was found to be eliminated principally as volatile organic carbon and/or C02. After only 0.5 h, test substance concentrations in the sludge were measured to be less than 40% of the dose rate. Less than 5% of the dosed substance remained after 19 d. Elimination as volatile compounds peaked by the second day, then decreased over the remainder of the test period.

Based on the results obtained, it was postulated that the high rate of loss from the reaction vessels was due to volatilisation. A subsequent experiment introducing the test substance into a completely closed system resulted in 58.8% accountability at test start, with concentrations decreasing to 2.7% of initial values after 3 h of aeration. Since only 4.3% was recovered by trapping, the test substance was assumed to have been adsorbed by the experimental system (Cranor, 1982).

Description of key information

The results of the simulation testing with adapted micro-organisms clearly demonstrates that p-methylstyrene is readily accepted as a food source that does not adversely affect microbial populations or the removal of organic carbon.

Additional information

A study was conducted to determine the biodegradation of the test substance by activated sludge in a 33 d study consisting of two phases (acclimation and biodegradation). The sludge was obtained from a municipal wastewater treatment plant and diluted with tap water to approximately 2500 mg solids/L (ca. 3.8 E+7 organisms/mg solids). The test system consisted of 3 L standard tall aeration vessels (resin pots) and was modelled after a typical domestic sewage treatment plant. All ground glass joints were treated with sealing wax to preserve the integrity of the closed system. In the 14 d acclimation period, non-radiolabelled test material was used at concentrations ranging from 0 to 50 ppm (vessel 1) or 0 to 100 ppm (vessel 2) following a daily ‘draw and fill’ routine. Daily operations included determination of the suspended solids in the mixed liquor, pH adjustment upon refill and measurement of dissolved oxygen levels in the mixed liquor. Aliquots of the effluent from each of the two controls and two test chambers were taken for Dissolved Organic Carbon (DOC) analysis on Days 12, 13 and 14. Aliquots of the sludge from each of the two controls and two test chambers were taken for microbial analysis on Days 0, 3, 6, 9, 11 and 15 of the acclimation period and at the end of the study. On Day 15 of acclimation, 14C/13C-labelled test substance was introduced with the daily influent at 50 and 100 ppm (as organic carbon). Daily ‘draw and fill’ was discontinued at that time. Trapping solutions for the collection of 14C-CO2 and 14C-labelled volatile organics in effluent air, and representative aliquots of the mixtures in each test vessel were taken at 0.5, 3, 6 and 24 h, then on Days 2, 4, 6, 8, 10, 13, 16 and 19. Analysis was conducted using Liquid Scintillation counting. The outcomes of this 33 d two-phase study showed that the inclusion of the test substance at two rates (50 and 100 ppm, calculated as carbon equivalents) did not adversely affect the wastewater treatment process or affect the sludge microflora. The test substance was found to be eliminated principally as volatile organic carbon and/or C02. After only 0.5 h, test substance concentrations in the sludge were measured to be less than 40% of the dose rate. Less than 5% of the dosed substance remained after 19 d. Elimination as volatile compounds peaked by the second day, then decreased over the remainder of the test period. Based on the results obtained, it was postulated that the high rate of loss from the reaction vessels was due to volatilisation. A subsequent experiment introducing the test substance into a completely closed system resulted in 58.8% accountability at test start, with concentrations decreasing to 2.7% of initial values after 3 h of aeration. Since only 4.3% was recovered by trapping, the test substance was assumed to have been adsorbed by the experimental system (Cranor, 1982).

 

Therefore, the results of the simulation testing with adapted micro-organisms clearly demonstrates that p-methylstyrene is readily accepted as a food source that does not adversely affect microbial populations or the removal of organic carbon.