17

Protocol for Determining Bioavailability and Biodegradation Kinetics of Organic Soil Pollutants in Soil Systems to Enhance Bioremediation of Polluted Soil Sites

Henry H. Tabak, Rakesh Govind, Chunsheng Fu, and Chao Gao

1. Introduction

Treatabihty studies are critical for successful rmplementatron of both zn sztu and ex: situ bioremediatton technologies. Few contammated sites are identtcal and experience can only be applied within limits Many varrables governing the efficacy of bioremedration processes are a function of environmental condi- tions, type of contaminants, and contammated media (see Chapter 1). Protocols for conducting treatabilrty studies are required to evaluate the effectiveness of primary substrates, supplemental nutrients, electron aceptors, and their mode of dehvery.

Knowledge of biodegradation kinetics in so11 IS needed to understand the efficacy of in situ and ex situ bioremediation technologies. Laboratory studies to determine brodegradation rates can be used as screening tests to determine the rate and extent of bioremediatton that might be attained during remediation and to provide design criterra Traditionally, in situ biodegradation kinetics have been determined using soil microcosms (I) that are difficult to model mathe- matically. Laboratory studies involving soil slurry reactors have been reported by Bachmann et al. (2), Kaplan and Kaplan (3), Mihelcic and Luthy (4), and Brunner et al. (5). Currently, there IS no systematic methodology to determine biodegradation kinetics of contaminants in compacted soil systems

Biodegradation m sol1 IS a fairly complex process that mvolves diffuston of contaminants m the porous soil matrix, adsorption to the soil surface, and biodegradation in the brofilms existing on the soil particle surface and in the

From Methods In Botechnology, Vol 2 Boremedratron Protocols Edited by D Sheehan Humana Press Inc , Totowa, NJ

223

Tabak et al.

large pores as well as m the bound and free-water phase after desorption from the soil surface. In sot1 slurry reactors, biodegradation of contaminant occurs both m the liquid phase by soil microorganisms desorbed from the soil matrix and by the btoftlms immobihzed on the soil particle surface. In compacted soil systems, biodegradation occurs m the free and bound water phase primarily by the soil-immobilized microorganisms and the contrtbution of water suspended microbtota is small because of low water content.

This chapter highlights biodegradation studies on phenol, several alkyl phe- nols (p-cresol, 2,4-dtmethyl phenol, catechol, hydroquinone, and resorcmol), and selected polycychc aromatic hydrocarbons (PAHs) (naphthalene, phenan- threne, acenaphthene, and acenaphthylene). Mathematical models for sot1 slurry, wafer, column, or porous tube reactors are used to determine the conta- minant diffusivmes and btodegradatton kinetics m sot1 slurry and compacted soil systems Information on contaminant diffusivittes and biokinetics in soil can be used to evaluate the attainable end-points for various soil treatment tech- nologies, such as soil slurry biotreatment, land farming, btoventing, or uz mu btoremediatton.

7.1. Phenols and Substituted Phenols

Phenol is one of the more widely used organic compounds in existence and is the basic structural unit for a variety of synthetic orgamcs, including agricul- tural chemicals. Phenol and substituted phenols are ubiqmtous contaminants in sot1 and water. Then presence may result from the degradation of pesticides (6) and other chemicals that were applied intentionally to soils or from unmten- tional releases associated with manufacturing processes, production of energy, and waste disposal procedures (7).

Phenols were reported in wastes from combustion and conversion of fossil fuels (8). Because phenolic compounds constitute a significant fraction of water- soluble organic compounds present m liquid waste from mdustrial processes, they are important m consideration of waste disposal options and then concen- trations are subject to scrutmy in water-quality testmg. Phenolic compounds are also of concern because, unlike the polycychc compounds, they are more solu- ble and, consequently, more mobile m the soil subsurface environment. Disposal techniques to mmimize the mob&y and hazard of potentially toxic waste chem- icals must be based on a knowledge of the composition of the pollutant organics and of the mteractions between these pollutants and soil organic matter (9).

A detailed discusston of the background literature on sorption and biodegradation of phenohc compounds has been presented earlier (10). It has been concluded that btodegradation m sot1 bioremediation may be inhibited by abtottc factors, such as sorption of compounds on soil particles and by organic matter

Bioavailability and Biodegradation Kinetics in Soil 225

1.2. Polycyclic Aromatic Hydrocarbons (PA Hs)

The physical and chemical propertres that govern the interactions of PAHs and soil, necessary for evaluatron of the transport and fate of these compounds in soil and sediment environment, have not been thoroughly characterized or quantitated. Sorption is known to be an important factor in the determmatlon of the fate of these hydrophobic molecules in water/sediment or water/sol1 sys- tems. Experience gamed from studies on biologrcal soil remediation have shown that biodegradation may be inhibited by abiotic factors. It has been sug- gested that one factor causing reduced brodegradability of compounds IS sorp- tion on soil particles and organic matter. Thus, biodegradability of soil-sorbed PAHs may have a bearing on the effectiveness of microbial degradation as well as on the assessment of toxicological risks. Since sufficient understanding of these abiotic factors is lacking, it is necessary to determine the soil characteris- tics that prevent PAH brodegradatron and to investigate the correlation of brodegradabihty and biotoxicity of sorbed PAHs. The mechamsm by which pol- lutants, such as PAHs, enter groundwater needs to be identified and studied and reliable techniques to predict their transport within aquifers need to be devel- oped. Because of the hydrophobic nature of PAHs, adsorption is very important m determining their transport and fate m subsurface sol1 systems A detailed drscussion of the previous literature on PAH partitioning, environmental fate, and biodegradation in soils has been reported earlier (10).

1.3. Determination of Biodegradation Kinetics Using Respirometry

Recently, several research studies reported the use of respirometry for evalu- ating biodegradation kmetrcs of organic pollutants m soil. Long-term resprro- metric biological oxygen demand (BOD) analysis was applied to bench-scale studies to contmuously monitor bacterial respiration during growth m mixed organic wastes from contaminated water and soil, in order to assess the potential for stimulating biodegradation of these wastes (I). This mformatron was used to make an initial determination regarding the need to explore bioremediation fur- ther as a potential remedial action technology using on-site, pilot-scale testing,

A treatability study used electrolytic respirometers and biometers to deter- mine the biodegradation potential of crude oil petroleum-based wastes (drilling mud, tarry material, and heavy hydrocarbons) as contaminants of a polluted soil site area (II). The treatabrlrty data provided biotreatment efficiencies of the petroleum wastes and were used to ascertain the bioremediation clean-up time.

The use of radroisotopes m brodegradatron studies can significantly increase the sensrtrvity of biodegradation measurements and realistic estimates of brodegradatron can be obtained with a minimum of analytical interference from an environmental matrix, at concentrations that are often outside the

226 Tabak et al.

scope of screening methods. Biodegradation products may be quantified using a combinatton of separation and radiochemical detection techniques. Recently, techniques were developed for the measurement of Monod kinetic coeffr- cients, microbial yield, and endogenous decay coefficients, through measure- ment of mmal rates m short duration batch experiments using radtolabeled substrates (12).

The application of radrolabeled substrate techniques has recently been expanded to the determination of biodegradation of organic compounds in soil. Radiolabeling techniques were used to study the persistence of pentachlorophe- no1 (PCP) (see Chapter 12) and mercuric chloride m soil, their effect on the mrcrobrota, and the reversibility of that damage in numerous soil types, at drf- ferent substrate concentrations (13). Studies were also reported on determina- tion of btommerahzatton rates of 14C-labeled organic chemicals m aerobic and anaerobic suspended soil.

Development of biodegradation models for organic pollutants m sol1 1s drffr- cult because of the existence of several comphcatmg factors, which include:

1 The presence of diffustonal barriers in sol1 macro- and mlcropores for compound, oxygen or nutrients,

2 The effect of chemical sorptton to clay and humrc constituents, 3 The presence of other biodegradable organic matter m soil; 4 Changes m mlcroblota growth resulting from protozoa parasitizing the blodegrad-

mg population, 5 The effect of compound solublllty in the aqueous phase, 6 The formation of blofllms on the sol1 surface m conJunctlon with suspended

cultures, and 7. The existence of unacclimated soil mlcrobtota

Most biodegradation kinetic models have neglected sorption of the contamr- nant on soil particles, which has been shown to be important in contaminant transport. Kinetically, sorptron is a two-phase process, with an initial fast stage (~1 h) followed by a slower long phase (days), controlled by diffusion to mter- nal adsorption sites (14).

So11 consists of various size pores, with about 50% of the total pore volume consistmg of pores with radn cl mm. Most soil bacteria range in size from 0.5 to 0.8 mm, and hence a srgmfrcant portion of the sol1 may be inaccessible to most bacteria.

The role of so11 aggregates and effect of then characteristics on btoremedra- tron m so11 have been analyzed m the contaminated aggregates broremediation (CAB) model (15). Sensitivity analysis of the CAB model has shown the effects of aggregate radius, partition coefficient, and initial contaminant concentratron on the time and mechanism of remedratron. Diffusion rates of substrate and

Bioavailability and Biodegradation Kinetics in Soil 227

oxygen and biodegradation kinetics were found to be controlling mechanisms for remediation in the aggregates.

The success of bioremedration processes lies in degrading the organic cont- aminants and in reducing both the toxicrty and migration potential of the haz- ardous constituents in soil. Laboratory studies were conducted using phenohc compounds to characterize overall chemical degradation and toxicity reduction in a contaminated soil. Results indicated that first-order kinetics described the loss of phenolic compounds satisfactorily and loss of contaminant in the water soluble fraction was faster than loss of the same chemicals m soil. Furthermore, toxicity of the water-soluble fraction decreased with concentration and no enhanced mobilization of the applied chemrcals was observed during the degradation process.

Scow et al. (26) reviewed biodegradation kinetics m so11 and discussed the effects of diffusion and adsorptton. They concluded that current models are based on studies of single microbial population or single enzymes and consid- ering the complexity of biodegradation processes in soil, it IS unlikely that a sm- gle model or equation would be applicable for the degradatron of all organic substrates in all types of soil environments

Based on earlier research on bioavallabillty and biodegradation kinetics with phenolic compounds and polycyclic aromatic hydrocarbons, a systematic multilevel protocol, shown m Fig. 1, was developed for evaluating bioavail- ability (sorption/desorptlon equihbrla and kinetics), contaminant and oxygen diffusion limitations, and biodegradation kmetics of organic pollutants m com- pacted soil systems. The approach involves the use of soil microcosms and three types of respnometric soil slurry, wafer, and porous tube or column bioreactors. This protocol can be applied for contaminated soil and the diffu- sivities and biodegradation kinetics obtained from the mathematical models can be used to evaluate the efficacy of treating the contaminated soil using bioremediation processes.

2. Materials

2.1. Soil Characterization

1 Soil selected for this study was uncontaminated top sol1 (17) (Faywood silty clay loam, 12-20% slopes, Famrly: fine, mixed, meuc; Subgroup: Typic Hapludalfs, Order: Alflsols) with 17.5--20% by weight sol1 morsture, 1060 (g/L) density, 0.415% total carbon content, 6.65 pH m distilled deionized water and 5 79 pH m O.OlM CaCl,.

2. The so11 was au-dned and passed through a 2-mm sieve. 3. The measured particle size distribution was as follows (urn, wt %) ~2 pm, 15.65

wt%, 2-10, 5, 10-20, 7 75; 20-75, 13.45, 75-150, 6.5, 150-300, 14 3, 300-600, 22.10; 600-1000, 16 25. The calculated average soil particle size was 0 0334 cm

228 Tabak et al.

Fig. 1. Overall protocol for determining biodegradation kinetics and attainable endpoints in soil slurry and compacted soil biotreatment systems.

4. The porosity of the soil particle, as measured by nitrogen adsorption and desorption porosimetry (micrometrics ASAP 200), was 0.03. Porosity of the compacted soil in the tube reactor, as measured by bulk density, was 0.2.

2.2. Analytical Method

The concentration of the chemical compound in the liquid sample was ana- lyzed using three methods: standard extraction (EPA method 604 and 610) with methylene chloride followed by GUMS analysis; HPLC analysis; and scintilla- tion counting of the r4C using radiolabeled compound (18). All three analytical methods were calibrated using standard solutions. The calibration data were used to convert the peak areas (GUMS or HPLC) or counts of disintegrations per minute (DPM) for radiolabeled compounds to the actual liquid concentra- tion. The GC used was a HP-5890A model equipped with a flame ionization detector. The following conditions were used to measure concentration of phe- nols: initial oven temperature 60°C initial hold time 5 min, oven temperature rate 8”C/min, final oven temperature 280°C, final hold time 5 min, injector tem- perature 225’C, detector temperature 310°C makeup gas (nitrogen) flowrate, 35 mL/min, detector gas flowrate 32 mL/min hydrogen and 435 mL/min air, carrier gas (helium) 2 mL/min, column HP-5 methyl silicon gum and 5 m x 1.53 mm x 2.54 mm film thickness, and software HP Chemstation.

Bioavailability and Biodegradation Kinetics in Soil 229

2.3. Preparation of Stock Solutions of Contaminant

For phenols, make 1.0 L or more of main phenol stock solution with a con- centration of 2.5 g/L of phenol in double-distilled water. Using the main stock solution, make 50-200 mL of experimental stock solution with a phenol con- centration of 500 mg/L or 1 g/L, as needed. In the case of PAHs, weigh each chemical equal to 80% of standard solubility in hexane. Dissolve the weighed chemical completely in hexane. Obtain standard solutions containing 60,40,20, and 10% standard solubility levels by diluting with hexane. Use an HPLC col- umn (Supelco LC-PAH 15 x 4.6 cm) to obtain peak areas for each standard solution. Make a plot of peak areas vs the concentration to obtain the standard calibration curve for each chemical. For chemicals with extremely low solubil- ity (~1 ppm), use radiolabeled compounds with scintillation counting. Make standard solutions in the same manner and calibrate with counts of disintegra- tions per minute (DPM). Make stock solutions of each PAH chemical by first weighing chemical equal to at least twice standard solubility level in water. Mix the chemical thoroughly with ultrapure water using teflon coated magnetic stir bar for 48 h. Allow the solution to settle for 48 h. Decant the saturated solution and filter using a 5-pm millipore filter to remove all suspended particles. Analyze the liquid concentration using either HPLC or scintillation counting. Obtain other stock solutions by diluting with ultrapure water to obtain solutions with 80, 60,40, 20, and 10% standard solubility levels.

2.4. Preparation of Contaminated Soil

In the case of phenols, add the phenol stock solution directly to soil while preparing the soil slurry, soil wafer, porous tube, or soil column reactors. In the case of PAHs, which exhibited low water solubilities, dissolve the PAH com- pound in acetone and use the acetone solution to contaminate the soil. Specifically, dissolve 700 mg of naphthalene in 0.5 L acetone. Mix the acetone well to ensure complete dissolution of added naphthalene. Spike 1 kg of the uncontaminated sieved soil with the 0.5 L acetone solution containing 700 mg of naphthalene. Mix the soil thoroughly while adding the contaminated solution, Spray the soil and then spread on an inert surface as a thin layer and leave open in the fume-hood for 24 h to allow the acetone to evaporate. Periodically turn the soil to expose fresh surface during the 24 h time period. Take four samples from the soil before and after the contaminant solution is added and determine the soil concentration of naphthalene using the standard EPA extraction procedure.

2.5. Preparation of Stock Solution of Nutrients The nutrient solution used in the respirometer was an OECD synthetic medium

containing the following constituents in double-distilled water (19): KH2P04 (85 mg/L), K,HP04 (217.5 mg/L), Na2HP04 a 2Hz0 (334 mg/L), NH&l (25 mg/L),

230 Tabak et a/.

MgS04 . 7H20 (22.5 mg/L), CaC& (27.5 mg/L) and FeC13 + 6H20 (0.25 mg/L), MnS04 . Hz0 (0.0399 mg/L), H3B03 (0.0572 mg/L), ZnSO, . 7H20 (0.0428 mg/L), (NHJ~Mo~O~~ (0.0347 mg/L), FeC& . EDTA (0.1 mg/L), and yeast extract (0.15 mg/L). The soil served as a source of inoculum.

3. Methods

3.1. Measurement of Soil-Bacterial Adsorption Isotherm

1. Incubate soil microbiota with radiolabeled phenol in a respiromettic reactor until an oxygen uptake plateau is obtained, indicating that all phenol had biodegraded into 14C biomass and 14C02, which is absorbed in the KOH solution.

2. Allow the soil suspension to settle for 30 min. 3. Sample 1 mL of supernatant and measure 14C activity by liquid scintillation

counting (Packard scintillation counter). 4. Determine the equilibrium amount of 14C biomass absorbed to the soil by subtract-

ing the 14C present in the biomass in suspension and the 14C present as carbon diox- ide absorbed in the KOH solution from the total 14C added initially. The ratio of the biomass adsorbed to the soil and the biomass present in the suspension gives the biomass/soil adsorption isotherm parameter.

3.2. Soil Microcosm Reactors

The methodology for the establishment of the bench-scale microcosm reac- tors, including the reactor configuration and supportive equipment description, undisturbed soil cube sampling for inclusion in the microcosms, procedure for contamination of soil bed with homologous series of organic compounds, description of the application of nutrients, and the operation of the microcosm reactors (CO2 generation analysis and chemical analysis of soil samples and reactor leachates for the parent compound and metabolites) has been fully described elsewhere (20). A schematic of the soil microcosm reactor is shown in Fig. 2. A microcosm reactor is an airtight rectangular container (50 x 30 x 30 cm.) constructed of glass and supported by stainless steel panels. The nutrients and appropriate contaminants are sprayed from the top using liquid atomizing sprays. The bottom of the reactor is equipped with ports to allow the drainage of leachates. A controlled flowrate of carbon dioxide-free air is passed through the reactor and the exit gas is bubbled through potassium hydroxide solution to quantify the average evolution rate of carbon dioxide in the reactor.

Each microcosm reactor represents a controlled site, which eventually selects out the acclimated indigenous microbial population in the soil for the contaminating organics. Samples of soil are then taken from the microcosm reactors and used as source of acclimated microbial inoculum for measuring oxygen uptake respirometrically; carbon dioxide generation kinetics in shaker flask reactors; and for studies with other soil reactor systems. The microcosm

Hoavailability and Biodegradation Kinetics m Soil 231

Adlustable Hb Ftoiameter

Valve

Filter

Vacuum Pump b / \

u Hydroc Atr ( _

arbon Free IMinder r-l Temporary Leachate Holding Tank

Fig. 2. SchematIc of the so11 microcosm reactor.

reactor units are also being used directly to evaluate the biodegradability of the pollutant organics and to measure their average biodegradation rate in this intact, undisturbed soil bed.

Some macrocosm reactors were contaminated with a mixture of phenolic compounds dissolved m double-distilled water so that the total chemical oxygen demand per kilogram of soil in the microcosm reactor was 300 mg. Equal con- centrations of phenol, resorcinol, catechol, 2,4-drmethyl phenol, cresol, and hydroquinone were used in the mixture. Other microcosm reactors were conta- minated with 25 ppm each of several PAHs dissolved m a mixture of double- distilled water and 0.5% solution of a surfactant, Triton X-100 Control macrocosm reactors were uncontaminated soil systems that were sprayed with an equal volume of double-distilled water and reactors with soil contammated with 0.5% solutton of surfactant, Triton X-100.

3.3. Adsorption Studies (21,22)

1 Air dry the soil naturally and then sieve it to pass a 2 00-mm sieve 2 Use batch well-stirred bottles m a constant temperature room for this study (21,22)

232 Tabak et al.

3 Place 10 g of sot1 sample m each bottle and mix with 100 mL of double-distilled water contammg various concentrations of the compound and mercuric chloride to mmimtze biodegradation. Express the soil:solution ratio as the oven-dry equivalent mass of adsorbent m grams per volume of solution

4 Sample the liquid after 2,4, 6, 8, 10, 12, 14, 16, 18, 20, and 36 h. 5 Centrifuge the bottle contents and take the liquid sample using a syringe connected

to a 0.45~urn porous silver membrane filter holder The filter prevents sot1 particles from entering the sample

6 Analyze the liquid samples using HPLC, GCMS analysis, and hqurd scmtillation analysts for radiolabeled compounds (see Note 1)

7 Obtain the amount of compound adsorbed m the sot1 by subtracting the amount of compound present m the hqmd phase from the total initial amount (see Note 2)

3.4. Desorpfion Studies (21,22) 1 Mix 100 mL of double-distilled water with 20 g of soil and specific mittal con-

centration of chemical, as m the case of adsorption. 2. After adsorption equihbnum is attamed (see Note 2), add 100 mL of double-distilled

water containing 2 g/L of mercuric chloride to inhibit btodegradation (see Note 3) 3 Withdraw 20-mL samples usmg a syringe at 4,8, 16,24,48,72,96 and 120 h, each

sample being withdrawn from a separate bottle and 1s filtered through a 0 45-pm filter attached to the syringe

4. Analyze the concentration of compound m the hquld samples using either methyl- ene chloride extraction followed by GC/MS or HPLC analysis 14C Scmtillatton counting analysis may be used for radtolabeled compounds (see Note 1)

3.5. Radiochemical Techniques for Determining Biodegradation of Organics in Soil (21,22)

1 Set-up experimental and control reactors m a respirometer 2 Add 1 mL of 14C fully tagged radiolabeled compound (concentratton equals

1 @i/mL) to each of the experimental and control reactors One ~CI equals 2 22 x lo6 DPM

3 Add 5 mL of 2N KOH solutton m each resptrometer reactor holder to absorb the 14C02 released from the solution

4. Withdraw the 5-mL KOH solutton after approprtate acclimation time, depending on the compound being studied Add 5 mL of fresh 2N KOH solutton to contmue the 14C02 adsorptton.

5 Mix the 5-mL KOH sample with a cocktail solution (Ultima Gold) m a 1.10 ratio (1 mL of KOH with 9 mL of cocktail solutton) Analyze the sample using hqmd scmttllatton counting (Packard TRI-CARB 2500 TR Liquid Scmttllatton Analyzer)

3.6. Respirometric Bioreactor Studies

The concentration of selected soil in the reactor flask varred from 2 to 10% by weight, using dry weight of sot1 as the basis. The total volume of the slurry m the flask was 250 mL. Three types of bioreactors were used to determine the btokinetlc

Bioavailability and Biodegradation Kinetics in Soil 233

parameters of the suspended and lmmobllized microbiota and the transport parameters of contaminant and oxygen in the soil matrix. These were (21,22):

1. Slurry bioreactor, where soil at 5% slurry concentration is vigorously mixed with the contaminant, dissolved In water with nutrients,

2. Wafer reactor, where a thm wafer of sol1 is spiked with contaminant and nutrients dissolved in water, to obtain a 50% total soil moisture content; and

3. Porous tube or column reactor, where sieved soil with contaminant 1s packed m a porous glass tube or column with moisture content identical to the wafer reactor

Schematics of the soil slurry, wafer, porous tube, and column bloreactors are shown in Figs. 3 and 4. In the soil slurry reactor, there are no limitations of oxy- gen, which freely diffuses into the well-stirred slurry and nutnents that are ml- tially dissolved in the aqueous phase. Hence, the biodegradation rate in sol1 slurry reactors depends on the intrinsic biokinetic rate, microorganism concen- tration in the sol1 matrix, and inherent diffusivity of the contaminant. In the sol1 wafer reactor, oxygen diffuses freely through the thm soil matrix, and hence biodegradation rate 1s controlled by the water content m addition to the other intrinsic parameters, as in the case of the soil slurry reactor In the soil column or porous tube reactor, biodegradation rate is controlled by the water content and oxygen dlffusivity and other intrinsic biokinetlc parameters. The sol1 col- umn or porous tube reactor provides a better estimate of biodegradation rates for in situ bloremediation than the sod wafer and soil slurry reactors.

3.6.1. Soil Slurry Reactors

1. Mix 25 g of spiked soil with 250 mL of double-distilled water, OECD nutrients (8), and 7 mL of moculum (see Note 4) from the shaker flask using a Teflon-coated stir bar. Set up at least duplicate flasks to ensure reproducibility.

2 Set up duplicate control reactors contammg 25 g of uncontaminated sol1 with 250 mL of double-distilled water, OECD nutrients, and 7 mL of moculum (see Note 4) from the shaker flasks.

3. Connect the flasks to a resplrometer that continuously measures cumulative oxygen uptake and carbon dioxide evolution (see Note 5 and ref. 23)

In the case of phenols, 20 g of uncontaminated soil was mixed with 250 mL of double-distilled water and 2.5-10.0 mL of experimental stock solution to produce the desired phenol concentrations m each flask.

3.6.2. Soil Wafer Reactors

1. Mix 25 g of spiked sol1 with 250 mL of double-distilled water, OECD nutrients, and 7 mL of moculum (see Note 4) from the shaker flask using a Teflon-coated stir bar

2 Allow the water from the flask to evaporate until the desired soil moisture content was obtained.

Tabak et al.

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Fig. 3. Schematic of the sol1 slurry (A) and sod wafer (B), broreactors used m thts Chapter. Number 4 represents a magnetrc stnring bar m (A) and a so11 wafer m (B), all other numbers correspond to the followmg in both parts: 1, respirometrtc flask, 2, con- ducttvtty probe, 3, magnettc stnring bar, 5, oxygen generates flask; 6, conductrvrty meter, 7, computer for on-line data recording, 8, multtplexer.

3 Place the sot1 m the reactor flask as a thin layer (see Note 6). 4. Contaminate the soil with the stock solutton of the compound and mix the soil with

a glass rod to ensure uniform compositton 5 Allow the excess water to evaporate at room temperature to attain the desired

moisture content 6. Set up control reactors wtthout the contaminant (exclude steps 4 and 5) 7 Connect the flasks to the respnometer (23)

Bioavailabilify and Biodegradation Kinetics in Soil 235

Fig. 4. Schemattc of the porous tube (A) and soil column (B) bloreactors used m this Chapter. Number 4 represents a tube m (A) and a soil column m (B) See the caption to Fig 3 for a description of the other elements m the figures.

In the case of phenols, 20 g of uncontaminated soil mixed with 20-30 mL of double-distilled water was placed m the reaction flask and it was well mixed to give uniform biomass concentration m the so11 matrix. Water from the reaction flask was evaporated at room temperature until the sot1 had attained the desired water content. The soil wafer was contaminated wtth 2.5-10.0 mL of experimental stock solutton, depending on the desired con- centration, and the soil wafer was mixed with the syringe needle while the stock solution was injected.

236 Tabak et al.

3.6.3. Sod Column Reactors

1. Mix 100 g of spoked so11 with OECD nutrients, and 28 mL of moculum (see Note 4) from the shaker flask usmg a Teflon-coated stir bar

2 Allow the water from the flask to evaporate unttl the desired sot1 moisture content IS obtained.

3. Pack the soil tn a porous glass tube or in the flask as a column (see Note 7) 4 Contammate the sot1 with the stock solution of the compound and mix the soil with

a glass rod to ensure umform compostnon 5 Allow the excess water to evaporate at room temperature to attain the desired

moisture content 6 Set up control reactors without the contaminant (exclude steps 4 and 5) 7 Connect the flasks to the resplrometer (23)

In the case of phenols, porous glass tubes made of vycor glass, with an aver- age pore diameter of 40 nm, were used. The pore size was chosen because it was found to be best at holding all of the soil and water wtthm the porous tube while allowing free flow of oxygen from the surroundmg an. In this way, the glass tube dtd not affect the results of the oxygen uptake expenments and served only to support the soil during contaminatton and btodegradatton. Experiments with these tubes allowed the degradation of chemtcals by sot1 mtcroorgamsms m intact contaminated soils to be even more closely simulated.

Abtotic adsorption and desorption kinetics of the contaminant into the sot1 matrix and oxygen uptake data obtained for the sot1 slurry, sot1 wafer, and col- umn reactors are used m conjunctton with detailed mathematical models to derive the intrinsic biokinettc and transport parameters. The protocol described m this chapter was applied to phenol, alkyl phenols, and PAHs and the results obtamed have been presented in another paper (24). A modified electrolytic respirometer, discussed m a recent paper (23), is used continuously to monitor the oxygen uptake and carbon dioxide evolutton. In addttton, a radiolabeled contaminant was used to confirm the oxygen uptake from the contaminant rather than resultmg from normal sot1 respiration.

4. Notes

1. HPLC has the distmct advantage of requmng no extraction with a solvent, as m the case of CC/MS analysts However, the HPLC method did not have enough sensitiv- ity for liquid phase concentrations below 1 ppm Solvent extraction was ttme-con- summg, used excessive amounts of solvent, and often dtd not yield 100% recovery of the compound 14C scintillation countmg required a radlolabeled compound but was fast and easy to use However, a hmited number of 14C compounds are available and they are expensive. Furthermore, scmttllatton countmg methods result m the generation of radioacttve waste that requires proper handling and disposal. For hq- utd phase concentrattons below I ppm, 14C scmttllation countmg is the preferred

Bioavailability and Biodegradation Kinetics in SOI/ 237

method For higher concentrattons, and for compounds with low octanol-water par- tition coeffictent, HPLC 1s better than extraction with organic solvent and GC/MS analysts. In this study, results from all three analytical methods agreed closely.

2. Equihbnum 1s defined as the state at which the liquid concentration reaches a sta- tionary value, which is usually attamed m 24 h The eqmhbrmm data are used to obtain Freundlich isotherm parameters.

3. Imtially, the liquid phase was being decanted after centnfugation, and then double- disttlled water was being added to allow the compound to desorb from the soil. However, it was found that, even after centnfugatton, the water phase contamed colloidal particles that may have a significant amount of compound adsorbed. Attempts to use smaller pore stze filters required a large pressure gradient to force the hquld through the filter.

4. Set up several shaker flasks, each containing 100 g of sot1 from the acchmated macrocosm reactor, and mtx with 1 L of double-distilled water containing 4 mL of secondary activated sludge and OECD nutrients (19) Inoculum was withdrawn by taking a fixed volume of the slurry from the shaker flask.

5. The sot1 slurry experiments provide Insight on contaminant degradation in both the soil and aqueous phases Bacteria and contaminant diffuse from the soil phase into the aqueous phase attammg equihbrmm, whereas contammant degradation occurs m both the soil and water phases. The cumulattve oxygen uptake and carbon dtox- ide evolution are controlled by contaminant diffusion and transport from within the sod particle to the bulk solutton.

6 Unlike the soil slurry system, the water present m the wafer reactor is significantly smaller and stationary, which increases the contammant concentration and decreases the mass transfer within the liquid phase. Oxygen uptake and carbon dioxide evolution are greatly affected by these differences.

7. The sot1 column reactor 1s slmtlar to the porous tube reactor smce it allows the effect of oxygen profile on biodegradation rate m compacted sot1 systems to be studied. The reason for using soil columns for the PAHs study was mainly because porous tube systems can be used when small amounts of soil, typically ~30 g, have to be analyzed When compounds exhtbitmg low water solubthty, such as PAHs, have to be studied, spiking with water saturated with the contaminant results in low contam- inant concentrations in the soil. Furthermore, low contaminant concentrations may have to be used to prevent inhibition effects. When the contaminant concentration m the soil is low, more soil has to be used m the reactor to achieve significantly higher cumulative oxygen uptake than the uncontammated sot1 control reactors Hence, the so11 column reactor was developed, m which significantly greater amounts of soil (exceedmg 30 g) can be tested as compared to the porous tube reactor.

5. Conclusions A three-step experimental protocol for determining the Important kinetics

parameters for the in situ biodegradation of toxic chemicals m ~011s was devel- oped using phenol and naphthalene as the test compounds. The protocol was developed so that the experimental schemes that were used grew m complexity

Tabak et al.

toward the actual zn sztu case, but remained simple enough to allow them to be adequately modeled The data gained for each of the schemes agreed with expectations. In the case of phenol, both the rate and extent of biodegradation decreased with the increase in the complexity of the soil systems m the experi- mental schemes. The amount of phenol degraded in the wafer reactor was less than in the slurry reactor primarily because of lower water content and mass transfer rates, since phenol, being a water-soluble chemical, did not partition significantly into the soil phase. In the case of the porous tube reactor, the amount of phenol degraded was even lower, primarily because of oxygen diffu- sion limitations In the case of naphthalene, all three reactors produced com- parable cumulative oxygen uptakes. This was mainly because naphthalene partitioned mainly m soil organic matter, being a strongly hydrophobic com- pound, and biodegradation of naphthalene occurred mamly m the soil phase. The aqueous concentration of napthalene was so small that the contribution of biodegradation in the aqueous phase was negligible. Modeling procedures applied to the three experimental schemes proved useful for determining the brokmetrc parameters for the degradation of phenol. Model predictions were found to agree very well with experimental data. Further, the model parameters were useful in simulating the treatment endpoints for the various types of sot1 reactors. The application of this protocol to other chemicals is feasible with only minor alterations in the methodology.

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