Pergamon

~

Pergamon

War. Res. Vol. 28, No. 8, pp. 1827-1833, 1994

Elsevier Science Ltd. Printed in Great Britain

0043-1354(93)E0039-U

BEHAVIOR OF N I T R O G E N - S U B S T I T U T E D N A P H T H A L E N E S IN F L O O D E D S O I L - - P A R T II. EFFECT OF BIOAVAILABILITY O N B I O D E G R A T I O N KINETICS BILAL AL-BASHIR [, JALAL HAWARI 2., RI~JEAN SAMSON2 a n d ROLAND LEDUC 3

IDepartment of Civil Engineering and Applied Mechanics, McGill University, Montreal, Quebec, Canada H3A 2K6, 2Environmental Engineering Group, Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec, Canada H4P 2R2 and 3Department of Civil Engineering, University of Sherbrooke, Sherbrooke, Quebec, Canada JIK 2RI (First received February 1993; accepted in revised form November 1993)

Abstract--Mineralization of l-aminonaphthalene, 2-aminonaphthalene and I-amino-2-methyl-naphthalene under aerobic conditions in flooded soil was found to proceed with a biphasic pattern, an initial fast phase followed by a slower second one. Also the sorption isotherms of these substrates were found to be hyperbolic and were best described by the Langmuir model. When initial mineralization rates were expressed in terms of initial aqueous-phase concentrations, they gave rise to simple hyperbolic kinetics that obeyed Michaelis-Menten model for enzyme-catalysed reactions. These initial mineralization rates were found to be directly proportional to the substrate aqueous concentration reaching their maxima at about 100 #g g-t (aminonaphthalene/soil slurry). Whereas the second phase mineralization rates were found to be first order with respect to the adsorbed fraction of the substrate and showed no sign of saturation, thus indicating that biodegradation is controlled by the rate of desorption. Key words--aminonaphthalene, mineralization, sorption, bioavailability, Michaelis-Menten, first-order kinetics.

INTRODUCTION

Biodegradation kinetics of pollutants in the soil environment provides a basis for a better understanding of their fate, persistence and potential threat to living organisms. Several studies have shown that bioavailability plays a crucial role in determining the biodegradation of various organic contaminants and several models have been developed to understand biodegradation kinetics. Scow et al. (1986) have reported that the mineralization of the aromatic amine, aniline, in soil is biphasic and is attributed to several factors, i.e., the presence of two states of the substrate bioavailability, the involvement of different populations of organisms, or the accumulation of intermediate metabolites. Consequently, a two-compartment model has been developed (Scow et al. 1986) to describe the biphasic phenomena encountered in the mineralization of aniline. Others (Steen et al. 1980) have modified a secondorder model, applicable to biodegradation of pollutants in natural waters, by incorporating a partition coefficient in it. The model assumes that adsorption affects biodegradation solely by decreasing concentration of the pollutants in the aqueous *Author to whom all correspondence should be addressed.

phase, i.e. only the aqueous fraction is bioavailable and the adsorbed fraction is totally unavailable. Also, Hamaker (1972) proposed a biodegradation model based on simple hyperbolic kinetics and on the assumption that soil contains both active and inactive biological sites. Accordingly, the rate of degradation is proportional to the amount of contaminant adsorbed on the active sites and adsorption on active and inactive sites is proportional to the concentration of the soluble substrate. Mihelcic and Luthy (1991) have reported that mineralization of naphthalene in a soil-water suspension under denitrifying conditions is dependent on solute partitioning between soil and water. They described the overall change of soluble naphthalene in the aqueous phase by developing a model which combines Michaelis-Menten kinetics with those of sorption/desorption in soil. The model assumes that the soluble and the sorbed substrate were in equilibrium throughout the course of the experiment and suggests that complete mineralization of the adsorbed fraction is attainable. The latter two assumptions deal with a specific case in which desorption is instantaneous and shows no hysteresis, i.e. desorption is first order with respect to aqueous concentration, and does not take into consideration the limiting case when desorption turns zero order with respect to aqueous concentration.

1827

I828

BILAL AL-BASHIR el al.

These previous studies indicate that there is a need to further investigate the extent to which bioavailabilit5, affects biodegradation kinetics. F u r t h e r m o r e , studies on the behavior of amino-substituted P A H s are seriously lacking in the literature. In the preceding paper (Al-Bashir et al. 1994), the mineralization of three a m i n o n a p h t h a l e n e s , i.e., 1-aminonaphthalene, 2 - a m i n o n a p h t h a l e n e and I-amino-2-methyl-naphthalene, was found to be biphasic and it was suggested that bioavailability considerations were responsible for such behavior. It is the purpose of this study to investigate the kinetics of mineralization of these c o m p o u n d s and to examine the mechanisms and the extent to which bioavailability affects their kinetics.

MATERIALS AND M E T H O D S

The aminonaphthalenes, l-amino-[l-J4C]-naphthalene and 2-amino-[8-14C]-naphthalene were prepared from a mixture of their corresponding radiolabelled and unmarked naphthols (Sigma Chemical Co., St. Louis, Mo, U.S.A.) using the Bucherer reaction (Vogel, 1978). The compounds had a specific activity of 32,874 and 36,283 dpmmg -l, respectively, and a final purity of 99 _+ %. [8-14C]-l-amino-2methytnaphthalene was prepared by reducing [8-~4C]-l-nitro-2-methylnaphthatene using Urushibara catalysts (Hata, 1971) which, in turn, was prepared by direct nitration of [8>4C]-2-methylnaphthalene. For more details on the synthesis of these compounds see A1-Bashir et al. (1994). The soil sample was obtained from Jarry Park, Montreal, Quebec and was characterized as clayey loam with soil organic matter~ organic carbon, pH and cation exchange capacity as 3.96%, 1.60%, 7.27 and 1 9 . 9 4 m e / l O O g soil, respectively. Varying initial concentrations of each compound were investigated. For 1-aminonaphthalene, these were, 5, 10, 20, 30, 40, 50, 100 and 150 Hg l-aminonaphthalene g ~ of soil slurry. The initial concentrations for 2-aminonaphthalene were 5, 15, 30, 50, 100 and 150~gg ~ and for l-amino-2methylnaphthalene were 10, 20, 35, 50, 100 and 200,ug g- ~. The amino-compounds were added as a methanol stock solution and the total volume of methanol added was constant at 200t~I per vial. Batch equilibrated adsorption experiments were conducted for autoclaved slurry samples at pH 6.5 following the procedure described previously (AI Bashir et al., 1990). Biodegradation was carried out in 100-ml glass vials containing 30 g soil slurry (30% w/w soil/water) and housing a KOH trap. The aqueous phase was a mineral growth medium prepared by the method of Thomas et al. (1986). Biodegradation was carried out under pure oxygen and the pH was kept between 6.5 and 7.0 using HC1 (1 N). Pure oxygen was also replenished every sampling session. For each concentration, six replicates were prepared of which three were analysed regularly for ~4CO2 in the alkali trap without acidification, two were sacrificed by acidification to make corrections for any CO, in the soluble form and the sixth one acted as control by receiving 500 pg/g HgCI> The evolved ~4CO2 was captured in KOH traps comprising of 10-ml glass tubes that shared the same head space with the slurry. Radioactivity in the alkali was measured using a scintillation counter (Packard Tri-Carb #4530, Downers, ILL, U.S.A.). On few occasions, duplicate samples of 2-ml slurry were taken from the biologically-active vials. These were centrifuged at 15,600 × g for 10 min (Centrifuge 5414, Eppendorff, Hamburg). The supernatant was then analysed for radioactivity.

RESULTS

The effect of initial N-substituted n a p h t h a l e n e concentration on the mineralization of l - a m i n o n a p h thalene, 2 - a m i n o n a p h t h a l e n e a n d I-amino-2-methyln a p h t h a l e n e is shown in Fig. 1. C o n c e n t r a t i o n s reported in the legend of Fig. 1 represent total initial concentration of /~g of c o m p o u n d per g of soil slurry. A t relatively low concentrations, no lag period was observed for any of the studied compounds. However, a lag period became more p r o n o u n c e d for b o t h 1- and 2 - a m i n o n a p h t h a l e n e at 1 5 0 # g g ~ and for l - a m i n o - 2 - m e t h y l - n a p h t h a l e n e at 2 0 0 ~ g g (Fig. 1). This is manifested in the lapse of 1-2 weeks between the spiking of the soil samples with the c o n t a m i n a n t s and the onset of an active biodegradation process. After the lag phase, mineralization of each of the studied a m i n o n a p h t h a l e n e c o m p o u n d s showed a two-phase process; an initial rapid phase followed by a second one with a diminished rate (Fig. 1). The diminished mineralization rates, observed for the second phase, are attributed to the partial unavailability of the c o n t a m i n a n t s to microbial uptake (Al-Bashir et al., 1994). Figure 1, also, shows that a m o u n t s mineralized ( y g contamin a n t g ~ slurry) increased with increasing initial concentration in slurry (/lg of c o n t a m i n a n t g ~ soil slurry) but the latter decreased when expressed as a

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200ua/a

ZOO

Z50

Fig. 1. Mineralization of (a) I-aminonaphthalene, (b) 2aminonaphthalene and (c) I-amino-2-methyl-naphthalene at various initial concentrations in flooded soil.

Nitrogen-substituted naphthalenes in flooded soil--part I1 O.Z

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error bar represents the standard deviation of three measurements of the aqueous phase concentration. Table 1 reports estimates of K L and M for the three aminonaphthalenes. These estimates were obtained using the Lineweaver-Burk method for the linear transformation of the Langmuir model (Kinniburgh, 1986). Accordingly, a plot of l/q versus 1/c yields a straight line such that KL=intercept/slope and M = l/intercept. Table 1 reports the r-values for the linear fits and the 90% confidence intervals for the estimated parameters. The confidence intervals of the two functions: (1) the inverse of slope and (2) the intercept over the slope, were obtained following the procedure outlined by Ang and Tang (1975). When initial-phase mineralization rates are expressed as a function of contaminant aqueousphase concentrations, they yield hyperbolic curves (Fig. 4) that fit Michaelis-Menten kinetics:

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1829

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0.15

V = ( R m a x C ) / ( K m + C)

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40 80 lZO 1SO ZOO Initial concentration in slurry (/~/g)

Fig. 2. Initial mineralization rates as a function of total concentration in slurry of (a) l-aminonaphthalene, (b) 2-aminonaphthalene and (c) l-amino-2-methylnaphthalene.

percent of the total amounts added. Furthermore, the initial mineralization rates for the three aminonaphthalenes (/Jg contaminant g - ' slurry d a y - ' ) increased with increasing initial total concentration in the slurry and reached their maxima at about 100/zgg -~ as shown in Fig. 2. In this figure and subsequent ones, an error bar indicates the standard deviation of three measurements. These errors are independent from each other as opposed to the dependent errors incorporated in the cumulative observations of Fig. 1 (Callas and Gehr, 1989).

(2)

where, v, is mineralization rate (g g ml-J day-t), c, is solute concentration (#g m l - ' ) in the liquid phase, Rm= is the maximum attainable rate (/zg ml-' d a y - ' ) and K,, is the half-saturation constant (/zgml-'). Michaelis-Menton model assumes a reversible formation of a catalyst-substrate complex followed by a first order decomposition of the complex to a product. At relatively low substrate concentrations mineralization is first order with respect to the substrate concentration, i.e., v = (Rm,~/Km)c. While at very high concentrations, all of the catalyst is presumably complexed, and the mineralization rate thus reduces to v = R .... i.e., zero order kinetics with respect to substrate concentration (Fig. 4). The linear transformation of the Michaelis-Menten equation using Lineweaver and Burk method gives (Piszkiewicz, 1977): 1/v = (Km/RmaxC) -

1/Rma

x

(3)

When l/v for each substrate, i.e., l-aminonaphthalene, 2-aminonaphthalene, and I-amino-2-methylnaphthalene, is plotted against its respective l/c for initial mineralization rates, a linear relationship is

Initial-phase mineralization As Fig. 3 indicates the adsorption of the studied compounds gave rise to hyperbolic isotherms that were best described by the Langmuir model:

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where q is the sorbate concentration (gg g-~ soil) in the solid phase and c is solute concentration (/~gml-') in the liquid phase, KL is the Langmuir affinity parameters (ml/~g-~ ) and M is the adsorption maximal (#gml-J). To find q in equation (1), the mass of contamination in the solid phase is calculated by subtracting the mass in aqueous phase from the total mass added initially and this in turn is divided by the weight of soil in the soil slurry. In Fig. 3, an WR

2S/S--K

200 oo

~ n o - 2 - m e t h y l naphthalene

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ZO 30 40 SO SO Concentration in liquid (/Jg/rnl)

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Fig. 3. Partitioning of (a) l-aminonaphthalene, (b) 2aminonaphthalene and (c) l-amino-2-methyinaphthalene between the solid and the aqueous-phases of the soil-water suspension.

1830

BILAL AL-BASHIR et al. Table 1. Estimation of adsorption and mineralization kinetic parameters of the studied amino-PAHs Michaelis-Menten kinetics parameterst

Langmuir p a r a m e t e r s *

KL

M

r

K~ (/~g/ml)

Rm~. (#g/ml.day)

r

k,(day) I x 10 4

r

0.16_+0.06 0,16 + 0.03 0.33 + 0.15

765 + II0 355 + 49 1053_+441

0.998 0.997 0,997

4.0+2.1 1.90+ 0.19 1.10+ 0.11

0.42_+0.21 0.13 + 0.01 0.14 + 0.06

0.973 0,996 0,992

6,1 +_0.2 5.7 + 0.6 2.9 + 0.2

0,998 0.988 0,996

Compound I-aminonaphthalene 2-aminonaphtbalene I-amino-2-methyl-naphthalene

First-order kinetics parameters:~

*KL is Langmuir partition coefficient (ml/#g), M is adsorption maximum (ug/g), r, is the correlation coefficient. "['Rma~ is maximum mineralization rate attainable with increasing concentration [from equation (2)], K~ is the half-saturation constant [from equation (2)]. .~k. is the uptake coefficient from equation (3). The ( _+ ) error establishes the 90% confidence interval

obtained with correlation coefficients, r, reaching 0.973, 0.996 and 0.992, respectively. Table 1 summarizes the parameters Rmax and Km for all studied aminonaphthalenes and their corresponding 90% confidence intervals. Not all of the compounds originally present in the aqueous phase were mineralized during the initial phase, especially those at concentrations leading to maximum mineralization rates. Presumably, part of the soluble fraction of the aminonaphthalene underwent further irreversible adsorption (i.e., chemisorption) onto the soil, e.g., humic materials. To confirm this observation, the concentration of the contaminant in the aqueous phase was determined and was found to be negligible. Bollag et al. (1983), Parris (1980) and Hsu and Bartha (1974) have all reported that aromatic amines and their enzymatic metabolic products

cross-link to humic substances in the soil through chemical bonding. S e c o n d - p h a s e mineralization

The second-phase mineralization rates remained fairly constant during the experiment (Fig. 1), increasing linearly with the contaminant concentration in the soil part of the slurry and showing no sign of saturation (Fig. 5). The relationship between the mineralization rate and concentration in the solid phase can be expressed mathematically as:

where ku is the substrate uptake coefficient from the solid-phase ( d a y - ' ) and q is the substrate concentration in the solid-phase (#gg-~). Equation (4) represents first order kinetics in which the rate is 0.3

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