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Environmental fate & pathways

Biodegradation in water and sediment: simulation tests

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Administrative data

Endpoint:
biodegradation in water: sewage treatment simulation testing
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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1982
Report date:
1982

Materials and methods

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.

Test material

Constituent 1
Chemical structure
Reference substance name:
4-methylstyrene
EC Number:
210-762-8
EC Name:
4-methylstyrene
Cas Number:
622-97-9
Molecular formula:
C9H10
IUPAC Name:
1-methyl-4-vinylbenzene
Test material form:
liquid: viscous
Radiolabelling:
yes

Study design

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 test substance concentrationopen allclose all
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
Reference substance:
ethylene glycol

Results and discussion

% Degradation
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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

Validity criteria
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).