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The ability of a rhamnolipid mixture produced by. Pseudomonas aeruginosa UG2 to solubilize the pesticides atrazine, trifluralin, and coumaphos was com...
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Environ. Sci. Technol. 2000, 34, 4923-4930

Effect of Rhamnolipids Produced by Pseudomonas aeruginosa UG2 on the Solubilization of Pesticides JUAN C. MATA-SANDOVAL,† J E F F R E Y K A R N S , ‡ A N D A L B A T O R R E N T S * ,† Environmental Engineering Program, Department of Civil and Environmental Engineering, University of Maryland at College Park, College Park, Maryland 20742, and Soil Microbial Systems Laboratory, Room 140 Building 001 BARC-West, 10300 Baltimore Avenue, Beltsville, Maryland 20705-2350

The ability of a rhamnolipid mixture produced by Pseudomonas aeruginosa UG2 to solubilize the pesticides atrazine, trifluralin, and coumaphos was compared with that of the surfactant Triton X-100. The values of maximum micellar solubilization capacities (Ksupra [mmol pest/mol surf]) for trifluralin and coumaphos in Triton X-100 were double those for the rhamnolipid mixture, whereas atrazine Ksupra value for the rhamnolipid biosurfactant was in the same range as that for the synthetic surfactant. Despite having the second largest Kow value of the three pesticides, coumaphos had the lowest affinity for both surfactant micellar phases. Comparison of values of aqueous-micelle solubilization rate coefficients (kOWM) obtained for trifluralin showed that the pesticide is solubilized at the same rate in both surfactant micellar phases. A much lower value of micellar-aqueous transfer rate coefficient (kOMW) for trifluralin in the rhamnolipid mixture suggests that the pesticide is bound more tightly to the biosurfactant micellar core and diffuses out to the aqueous phase at a lower speed than that observed for the synthetic surfactant. Future research can benefit from this work by studying the effect that micellar solubilization can have on the bioavailability of organic pollutants for microbial uptake.

Introduction Biodegradation is frequently proposed as a cost-effective method for the remediation of chemically contaminated sites, either by inoculation with known strains capable of degrading the contaminants or the stimulation of indigenous pollutant degrading microorganisms. However, even when appropriate microbial strains are present and environmental conditions are adequate, the extent of biodegradation may still be severely limited by the availability of pollutants to the microorganisms. It is generally accepted that only the pollutants in soil solution/groundwater (soluble) are available for microbial degradation (1). Pesticides are generally known for their low aqueous solubility and their tendency to stay sorbed in soil and therefore unavailable for microbial degradation. Surfactants at amounts above their critical micellar concentration (CMC) can greatly enhance the * Corresponding author fax: (301)405-2585; phone: (301)405-1979; e-mail: [email protected]. † University of Maryland at College Park. ‡ Soil Microbial Systems Laboratory. 10.1021/es0011111 CCC: $19.00 Published on Web 10/21/2000

 2000 American Chemical Society

apparent aqueous solubility of hydrophobic organic compounds (HOC) and can be successfully used in conjunction with remediation technologies such as soil washing/flushing and pump-and-treat. However, synthetic surfactants sometimes prevent microbial action on pollutants solubilized in their micellar phase (2). Since they are often toxic to microorganisms and they have considerable affinity for soil surfaces, synthetic surfactants tend to accumulate and pollute subsurface systems (3, 4). Biosurfactants are amphipathic molecules produced by a wide variety of bacteria, yeast, and filamentous fungi (5). Rhamnolipids are extra-cellular glycolipid biosurfactants produced by several strains of Pseudomonas species, growing on diverse carbon substrates such as long-chain hydrocarbons, carbohydrates, glycerol, and vegetable oils (5-7). Vegetable oils are among the substrates that promote the highest rates of rhamnolipid production when used as the sole carbon source by some P. aeruginosa strains (5-8). Recent studies have proved that rhamnolipids can be as good or even better than their synthetic counterparts in enhancing aqueous solubility of alkanes, polycyclic aromatic hydrocarbons (PAH), aromatics, and polychlorinated biphenyls (PCB) (9-15). Most biosurfactants have lower CMC values and higher micellar aggregation number than synthetic ones. These properties suggest that biosurfactants may be more efficient than synthetic surfactants in bioremediation efforts, since lower amounts of biosurfactant can provide equal levels of solubilization of organic pollutants. Being microbially produced, biosurfactants are expected to be more compatible with microorganisms as compared to synthetic ones and enhance both mobilization and biodegradation of pollutants in subsurface environments. However, biosurfactants can also be toxic to microorganisms and act by disrupting microbial membranes (16). Rhamnolipids may even act as virulence factors in strains of P. aeruginosa associated with opportunistic infections (17) Most of the research regarding the use of surfactants in soil remediation is limited to PAHs and PCBs, and so far no information is available on their application for remediation of sites contaminated with pesticides. Although general agricultural usage probably accounts for the majority of pesticides contamination potential, point sources from wastes generated after rinsing agricultural equipment from farms and pesticide formulating retailers have created sites sufficiently contaminated to qualify as Superfund sites (18). The purpose of this research was to evaluate and compare the effectiveness of a rhamnolipid mixture and Triton X-100 in enhancing aqueous and micellar phase solubility of trifluralin, coumaphos, and atrazine. These are pesticides whose low solubility and high hydrophobicity can potentially reduce their bioavailability in subsurface environments. Being respectively the first and the fourth most used pesticides in the U.S., atrazine and trifluralin are of particular concern for contamination of soil and groundwater (19). Rotation of corn and soybean crops is a common practice that increases the possibility of finding sites contaminated with both pesticides. For instance, Winterlin et al. (20) describe a highly contaminated agricultural aviation facility with a wide range of different compounds that include from DDT and toxaphane to atrazine, trifluralin, parathion, and linuron in the upper levels. Coumaphos degrading consortia have been used by Karns et al. (21), to degrade coumaphos in cattle dips from vats to low ppm levels. The solubilization enhancement of coumaphos as a way to increase its bioavailability and allow degradation of the pesticide to lower levels has been also a concern that have lead the authors to investigate its behavior VOL. 34, NO. 23, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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in the presence of surfactants. Thus, the three pesticides examined in this study include a broad range of chemical structures that, at least partly, represents the diversity of compounds likely to be found in sites with mixed agricultural wastes Although the present research is centered in the effects of surfactants to solubilize solid pesticides into the aqueous and micellar phases, the obtained aqueous and micellar maximum solubilization capacities (Ksub and Ksupra) can be incorporated into more complex models able to describe the distribution of organic pollutants in multiphase systems where soil is present (22).

Experimental Section Materials. Rhamnolipid mixture produced by P. aeruginosa UG2 was obtained and purified according to the method described below. Its composition has been previously identified as a mixture of Rh2C10C10 dirhamnolipid (60% w/w), RhC10C10 monorhamnolipid (21%), with the remaining 19% composed of a mixture of Rh2C10C12 dirhamnolipid and Rh2C10C12-H2 “dehydro-dirhamnolipid” (23). Triton X-100, a heterogeneous nonionic octylphenol ethoxylate surfactant, was obtained from Pierce Chemical Co., Rockford, IL (100 g/L solution). Trifluralin (98.7%) was obtained from Eli Lilly & Co. Indianapolis, IN, atrazine (94% w/w) from Ciba Geigy Corp., Greensboro, NC, and coumaphos (97.1%) from Bayvet Division of Cutter Laboratories Inc., Shawnee, KS. All the chemicals were used as supplied. Microorganism and Cultivation Conditions. P. aeruginosa UG2 was provided by the Department of Environmental Biology at the University of Guelph, Ontario, Canada. Microorganisms were maintained on L-agar (Gibco) plates throughout the experiments. For growth of P. aeruginosa UG2 in liquid culture, the minimal media of Hylemon and Phibbs (24) as modified by Tomasek and Karns (25) was used. A further addition of (NH4)2SO4 and trace metals in concentrations equal to 5 times those recommended by the authors was performed at the 10th day of cultivation (8). A total concentration of 12.75 g/L (15 mL/L) of corn oil (Mazola 100% corn oil CPC International Inc.) was added to the media as carbon source. Preliminary experiments (not shown) proved that this substrate concentration was optimal for obtaining high product yield combined with a minimum of impurities (mainly nonmetabolized fatty acids). A constant pH of 7.0 was kept as recommended by Mata-Sandoval et al. (8). All the cultivation was performed in 2.8 L Fernbach flasks with a total broth volume of 1 L. Starter cultures (100 mL in 300 mL Tunaire flasks) were inoculated from L-agar plates. After 24 h, 50 mL was used for the inoculation of Fernbach flasks. Once inoculated, all flasks were incubated at 37 °C on a gyratory shaker at 220 rpm (New Brunswick Scientific incubator shaker series 25) for 16 days. Rhamnolipid Extraction and Purification. Biosurfactant extraction and purification was performed exactly as described by Mata-Sandoval et al. (8, 23). Briefly, the biosurfactant was extracted from culture supernatant using a mixture of CHCl3/CH3OH 2:1. The rhamnolipid product was concentrated from the pooled organic phase using a rotary evaporator and redissolved in methanol, filtered, and concentrated again by evaporation of the methanol. Solubilization of Pesticides at Equilibrium. Aqueous solutions of surfactants were prepared at different concentrations ranging from 0 to 3200 µM (0-2000 mg/L) of total rhamnolipids or Triton X-100. The solutions were buffered with NaHCO3 (15 mM) at pH 8, and NaCl was added to 10 mM to keep a constant ionic strength. Sodium azide (Sigma Chemical Co., St. Louis, MO) was added to the solutions at 0.01% w/w to suppress microbial activity. A 5 mL volume of each solution was placed in screw cap tubes, and 5 mg of 4924

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pesticide (1.5 µmol trifluralin, 2.3 µmol atrazine, or 1.4 µmol coumaphos) was added as a solid to ensure saturation in all the solutions once equilibrium was reached. The tubes were shaken for 72 h at a constant temperature of 25 °C. The samples were centrifuged (3000g) for 10 min and filtered (0.45 µm Millipore Milex HV) before analysis by high performance liquid chromatography (HPLC). The initial amount of 5 mg of any pesticide for the experiments was chosen after preliminary experiments which proved that this amount exceeded, at least 10 times, the maximum amount of pesticide able to be solubilized in 5 mL of the most concentrated TX-100 solution of 3200 µM (2000 mg/L). Previous experiments proved also that 72 h of shaking procedure was enough, since concentration close to saturation (98%) for all pesticides was reached within 48 h. The slopes obtained after linear regression of the values of each pesticide’s maximum solubility at different surfactant concentrations above and below its CMC values correspond to the aqueous and micellar maximum solubilization capacities Ksub and Ksupra (mmol pest/mol surf) for each pesticide in each surfactant. Such values are presented in Table 2. Trifluralin Solubilization Kinetic Experiments. While the solubilization experiments at equilibrium were done for the three pesticides trifluralin, atrazine, and coumaphos, the kinetic experiments were performed exclusively for trifluralin. Three important parameters were kept constant in order to obtain reliable values of the kinetic rate constants kOWM and kOMW: (1) the total surface of the solid pesticide in contact with the aqueous phase, (2) the agitation speed, and (3) the temperature of the system. For this purpose, trifluralin was ground and passed through a 0.7 mm stainless steel sieve to obtain a regular particle size and similar total solid surface per mass unit of pesticide. Aqueous solutions containing 64, 400, and 1600 µM (40, 250, and 1000 mg/L) of total rhamnolipids were prepared as well as Triton X-100 solutions of 160, 400, and 1600 µM (100, 250, and 1000 mg/L). All solutions were buffered and conditioned identically to those used in the equilibrium experiments. Volumes of 90 mL of each solution were placed in flasks on a gyratory shaker (New Brunswick Scientific incubator shaker series 25) at 240 ppm. The experiment started by introducing 0.65 g (1.94 mmol) of sieved trifluralin into each flask. Aliquots were taken from time to time, centrifuged (3000g) for 10 min, and filtered (0.45 µm Millipore Millex HV) before HPLC analysis. A constant temperature of 25 °C was maintained throughout the experiment. Analysis of Pesticides. All samples were analyzed in an HPLC instrument equipped with Waters Model 712 WISP autosampler, two Waters Model 510 pumps, and a Waters Model 996 Photodiode Array Detector. For trifluralin analysis on rhamnolipid solutions an isocratic method with acetonitrile-H3PO4 (0.01 N) (1.6:0.4 v:v) was employed at a flow rate of 2 mL/min through a Novapak C18 (8NVC184µ) column. Trifluralin analysis in TX-100 solutions required a 1.35:0.65 v:v acetontrile-H3PO4 mobile phase at the same flow rate through the same column. The absorbance of trifluralin was measured at 275 nm. Atrazine was quantified in both surfactant solutions with an isocratic method acetonitrileH3PO4 (0.01 N) (1:1 v:v). Atrazine’s absorbance was measured at 228 nm. An isocratic method of methanol-H3PO4 (0.01 N) (1.5:0.5 v:v) was used for coumaphos analysis on rhamnolipid solutions, whereas the mobile phase was modified to 1.4:0.6 v:v methanol-H3PO4 (0.01 N) for coumaphos analysis on TX-100. Coumaphos absorbance was measured at 320 nm. The three pesticides were detected on a linear range at concentrations as low as 0.05 mg/L (equivalent to 0.15 µM for trifluralin, 0.23 µM for atrazine, and 0.138 µM for coumaphos). Development of the Kinetic Model. A pseudo-first-order kinetic model was developed to describe the solubilization

TABLE 1. Chemical Structure and Properties of Selected Surfactants

a

Obtained experimentally after measuring surface tension using the method described by Mata-Sandoval et al. (23).

TABLE 2. Aqueous and Micellar Solubilization Obtained Parameters for Trifluralin, Atrazine, and Coumaphos in the Presence of Triton X-100 or Rhamnolipid Mixture surfactant rhamnolipid mixture trifluralina

Ksub (mmol pest/mol surf) 3.8 Ksupra (mmol pest/mol surf) 485. Ksupra/Sw (mol surf/L) 54009 Ksupra/Ksub 12.76 kOWM (µM-1 min-1) 1.082 ( 0.009 × 10-2 -1 kOWM (min ) 1.18 ( 0.046 × 10-5 a

Triton X-100

atrazinea coumaphosa 12.42 12.85 73 1.03

0.09 2.49 4308 27.7

trifluralina 2.5 99.7 111025 39.9 9.36 ( 0.43 × 10-3 2.02 ( 0.18 × 10-3

atrazinea coumaphosa 4.57 10.7 60.8 2.34

0.09 5.07 8772 56.3

Pesticide.

of an organic pollutant from the true aqueous phase into the micellar phase of a surfactant. The process can be visualized as a two compartment box model as shown in Figure 1. The model considers that the rate of micellar solubilization of the compound depends on the concentration of the micellar surfactant available for binding HOC molecules. Additional considerations incorporated into this model are as follows. (1) The transport of the contaminant from the solid phase to the true aqueous phase is a fast step (aqueous saturation is considered at t ) 0 min), and its concentration is always equal to the maximum possible at the CMC. A constant surface area of the pesticide in contact with the aqueous phase has been guaranteed to keep a uniform rate of pesticide transport from the solid phase to the true aqueous phase. Such rate law states that the dissolution is controlled by the rate of diffusion of contaminant from the solid to the aqueous phase (13). Since the agitation and temperature of the system is controlled during the experiment, the pesticide rate of diffusion depends on the total area of solid-aqueous interphase. Preliminary experiments (results not shown) proved that the maximum amount of trifluralin dissolved into the 90 mL of surfactant solution of TX-100 of 3200 µM represented

less than 1.5% of the initial amount of pesticide (1.94 mmol) introduced into the system. Therefore, the total solid pesticide surface area in contact with the liquid can be considered as constant throughout the experiment. (2) The limiting step of the solubilization is the transport of the pollutant molecules from the true aqueous phase into the surfactant micellar phase. The process is considered reversible. The solubilization of a contaminant into the surfactant micellar phase is proposed as described by the following reaction

OW + M T OM where OW represents the pollutant in the aqueous phase, M is the surfactant micellar phase, and OM is the pollutant bound to the micelles. The rate of micellar solubilization of the pollutant is represented by the following differential equation

d[OM] ) [OW][M]kOWM - [OM]kOMW dt VOL. 34, NO. 23, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Schematic diagram of the solubilization of a hydrophobic organic compound (HOC) in an aqueous system containing a surfactant. where [OW] and [OM] are respectively the concentrations of the pollutant in the true aqueous phase and in the micellar pseudo-phase, and [M] is the concentration of micellar surfactant free of pollutant. [M] can be expressed in terms of pollutant solubilization enhancement as

[M] ) [Csat] - [OW]cmc - [OM]

(2)

where [OW]cmc is the maximum concentration of pollutant in the aqueous phase at the CMC, and [Csat] is the maximum concentration of pollutant in both micellar and aqueous phases at a surfactant concentration above its CMC. Both [OW]cmc and [Csat] are calculated using the equations

[Csat] ) ([Csurf] - [CMC])Ksupra + [OW]cmc

(3)

[OW]cmc ) [CMC]Ksub + [Sw]

(4)

where [Csurf] is the surfactant concentration above the CMC, [CMC] is the surfactant critical micelle concentration, and [Sw] is the pollutant aqueous solubility in the absence of surfactant, Ksub is the surfactant maximum aqueous solubilization capacity at concentrations below the CMC, and Ksupra is the surfactant maximum micellar solubilization capacity above the CMC. The analytical solution for eq 1 leads to the following expression

[

[OM] ) [OM]t)0 -

C1 C1 exp-C2t + C2 C2

]

(5)

where [OM]t)0 is the initial concentration of pollutant in the micellar phase, and C1 and C2 are constants defined by the expressions:

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C1 ) kOWM[OW]cmcKsupra([Csurf] - [CMC])

(6a)

C2 ) kOWM[OW]cmc + kOMW

(6b)

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The values of C1 and C2 were obtained by fitting the experimental data to eq 5 using nonlinear regression analysis. The aqueous-micelle solubilization kinetic rate constant (kOWM) and the micelle-aqueous transfer rate constant (kOMW) were finally calculated from eqs 6a and 6b.

Results Figure 2 shows the solubilization curves at equilibrium for the three pesticides at different concentrations of rhamnolipid mixture or Triton X-100. Pesticide solubilization showed linear dependence on the concentration of either surfactant. A distinctive change of slope below and above the CMC of both surfactants was observed for trifluralin and coumaphos. However, atrazine solubilization curves and specially the one for rhamnolipid showed a continuous solubilization enhancement below and above the CMC (Figure 2B). As seen in Figure 2, pesticides were increasingly solubilized into the aqueous micellar phase at concentrations above the CMC. Having a lower CMC than Triton X-100 (Table 1), the rhamnolipid mixture showed an onset of trifluralin and coumaphos micellar solubilization at a lower concentration than its synthetic counterpart. However, Triton X-100 micellar phase proved to be more effective than rhamnolipids in solubilizing both pesticides. The onset of atrazine solubilization in the rhamnolipid mixture is continuous below and above the CMC, whereas for Triton X-100 the apparent solubility of this pesticide is only enhanced once the surfactant CMC is surpassed. The biosurfactant showed on the other hand a slightly higher micellar solubilization enhancement of atrazine. On a mol basis, Triton X-100 was able to solubilize approximately twice as much trifluralin and coumaphos as rhamnolipids, whereas rhamnolipids could solubilize only 13% more atrazine than the synthetic surfactant. As seen from Figure 2A,C, no significant aqueous solubilization enhancement of trifluralin or coumaphos (more hydrophobic) was observed at concentrations of either

FIGURE 2. Maximum micellar and aqueous solubilization of trifluralin (A), atrazine (B), and coumaphos (C) in the presence of rhamnolipid mixture (filled symbols) and Triton X-100 (open symbols). Symbols represent experimental data. Lines stand for the linear model fit. Error bars represent standard deviation from duplicates. surfactant below their CMCs. This fact is also verified by the low values of Ksub for both pesticides in both surfactants presented in Table 2. However, Figure 2B and values of Ksub show that atrazine (more hydrophilic) solubility was enhanced even at concentrations below the CMC of the surfactants. This behavior is more evident for the rhamnolipid curve. During the kinetic solubilization experiments, aqueous and micellar phase concentrations of trifluralin in both surfactants were monitored over time. The experimental results and model fit curves are shown in Figure 3. Trifluralin concentration values at the plateau of each curve in Figure 3A,B confirm that Triton X-100 has a higher capacity of enhancing trifluralin solubilization than the rhamnolipid mixture. The kinetic rate constants kOWM and kOMW obtained after optimizing the solubilization model were compared to assess the efficacy of both surfactants in solubilizing trifluralin. Values of 1.088 × 10-2 and 1.076 × 10-2 uM-1‚min-1 for kOWM and 1.21 × 10-4 and 1.15 × 10-4 min-1 for kOMW were obtained after trifluralin solubilization in rhamnolipid solutions of 400 and 1600 µM, respectively. Values of kOWM for Triton X-100 at these same surfactant concentrations were equal to 9.05 × 10-3 and 9.66 × 10-3 uM-1‚min-1, and for kOMW they were equal to 2.15 × 10-3 and 1.89 × 10-3 min-1. As noticed, each pair of rate coefficient values at the two concentrations of either surfactant was very similar. This was expected, since the proposed model considers that the kinetic constants are independent of the surfactant concentration and dependent only on parameters such as the mixing speed, the temperature, and the total surface area of

FIGURE 3. Trifluralin concentration over time in rhamnolipid micellar phase (A), Triton X-100 micellar phase (B), and aqueous phases of rhamnolipid mixture and Triton X-100 at concentrations below the CMC (C). Symbols represent experimental data for rhamnolipid (filled) and Triton X-100 (open) concentrations. Lines stand for model estimates. Error bars represent standard deviation from duplicates. the solid phase in contact with the aqueous phase. As previously stated, the procedure for the kinetic experiments was carefully designed to control such parameters and procure their steadiness. The average values of kOWM and kOMW are presented in Table 2. Solubilization of trifluralin was also followed over time in solutions of both surfactants at concentrations below their CMC (Figure 3C). The results showed that trifluralin saturation of the aqueous pseudophase is a fast process that happens within the first seconds of the experiment.

Discussion Trifluralin and coumaphos solubilization results contrast with the solubilization enhancement of hydrocarbons and PAHs reported by other authors (13, 14) where rhamnolipids and other biosurfactants observed higher Ksupra values than some synthetic surfactants. During solubilization experiments in organic/aqueous two phase system, Kanga et al. (13) reported higher Ksupra values for naphthalene and methyl substituted derivatives in glycolipids from Radococus H13-A than in Tween-80. The glycolipid mixture could solubilize up to 6 times more naphthalene than the synthetic surfactant. Thangamani et al. (14) proved that on a mol basis, rhamnolipids were able to solubilize almost 22 times more hexadecane than dodecyl benzene sulfonate (DBS) in similar organic/aqueous two phase systems. The authors justify the higher solubilizing capability of biosurfactants to the greater volume of their micellar phase when compared to that of their synthetic counterparts. Having lower CMCs than most VOL. 34, NO. 23, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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of synthetic surfactants and micelles of larger size and smaller aggregation numbers, biosurfactants are capable of solubilizing larger amounts of organic compounds in the micellar phase. In previous experiments, we tested apparent solubilization of trifluralin in solutions of several rhamnolipid mixtures produced by P. aeruginosa UG2 during the growth on different carbon sources (8). It is important to note that the composition of the rhamnolipid mixture was not altered by the nature of the carbon source. We were careful to maintain similar total concentrations of the different rhamnolipids mixtures during these comparative experiments. A high solubilization enhancement of trifluralin (approximately 120 mmol pest/mol surf) was observed by the rhamnolipid mixture produced when corn oil was used as carbon source, whereas those mixtures produced with hydrophilic carbon substrates were less effective in promoting the apparent solubility of the pesticide (13-37 mmol pest/mol surf). In the case of the rhamnolipid produced with corn oil, the impurities accounted for approximately 11% of the total mass, whereas the impurities for the other mixtures ranged between 1 and 21%. This suggests that both the nature and the percentage of hydrophobic impurities remaining in the rhamnolipid mixture after its purification may improve its solubilization properties by acting as cosolvents. The degree of purity and nature of impurities in a biosurfactant are two possible explanations for disparities between our solubilization results and the ones of other authors. For instance, while some authors obtained the glycolipid as a purified lyophilysate from other source but do not specify the method for its production, extraction, or purification (14), other sources (13) used the culture supernatant containing the impure biosurfractant directly on their experiments without any further purification. For instance, one possible explanation for the higher values of Ksub in rhamnolipids (compared to that in Triton X-100) for trifluralin and atrazine is that impurities may be acting as effective cosolvents even at surfactant concentrations below the CMC. It is important to state also that the molecular structures of pesticides are complex and include combinations of diverse functional groups compared to the uniform conformation of hydrocarbons or PAHs used as solutes in experiments by other authors (13, 14). van der Waals forces probably account for most of the interactions between hydrocarbons or PAHs and the hydrophobic core of surfactant micelles, whereas additional dipole:dipole, dipole-induced, and (or) hydrogenbonding interactions between pesticide molecules and either the hydrophilic surface or the hydrophobic core of micelles might be important. For instance, trifluralin’s electronegative nitro groups may have repelling interactions with the hydrophilic micellar surface where the ethoxy groups of Triton X-100 or the hydroxy and ester groups in rhamnolipids are located. On the other hand, the more hydrophobic “trifluoro” and the dipropyl groups of trifluralin could probably interact more strongly through wan der Vaals forces with the micellar core constituted by either the alkylphenol groups in Triton X-100 or the fatty acid chains of rhamnolipids. In atrazine, the polar amino groups (distributed more evenly in the molecular structure) may tend to interact more with the hydrophilic surface of micellar structures, whereas the rather small isopropyl and ethyl groups will interact with the inner core of the micelles. The ester group in the coumarin structure and the ether groups in the phosphorothioate group of coumaphos may have stronger interactions with the micellar surface through dipole:dipole, dipole-induced, or even hydrogen bonding, while the diethyl groups and some sectors of the coumarin structure might tend to interact with the hydrophobic core of micelles. The more uniform molecular structure and type of interactions between hydrocarbons and surfactant micelles 4928

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TABLE 3. Chemical Structure and Properties of Selected Pesticides

a

This work.

b

Reference 26.

could enable the possibility of correlating in a reliable way the extent of their micellar solubilization by using parameters such as the hydrocarbon aqueous solubility, Kow, and the surfactant properties such as the hydrophilic:lipophilic balance (HLB), the aggregation number, or the CMC. For example, studies by Lunning Prak et al. (27) showed that solubilization rates (k) of n-alkanes in the micellar phase of nonionic surfactants had close correlation with the micellewater partition coefficients (Km,w), the solute molar volume, and the surfactant HLB or ethoxylate chain length. However, the complex interactions betweeen pesticide functional groups (absent in hydrocarbons and PAHs molecular structures) and surfactant micelles could lead to solubilization results difficult to correlate to the parameters mentioned before. This is the case of some of the results that are presented next and discussed. Below, our results on micellar solubilization of pesticides are contrasted with those of others authors (13, 14, 28-30) and with trends expected after analysis of the theory of surfactants micellar structure and pesticides physicalchemical properties. As expected from the Kow values of the three pesticides (see Table 3), trifluralin showed the highest affinity for the micellar phases of the two surfactants. Being less hydrophobic and more soluble in water, atrazine experienced a higher initial solubility at surfactant values below the CMC; however, the solubilization enhancement on the micellar phase was, as expected from the Kow values, much lower than that of trifluralin (3.8 times lower in rhamnolipid and 9.3 times lower in Triton X-100). Due to its properties, coumaphos was expected to show an intermediate Ksupra value between those of trifluralin and atrazine but surprisingly showed the lowest solubilization enhancement of the three compounds. Lopes et al. (28) determined partition coefficients (Ksupra/sub) of coumaphos and other coumarin derivatives between several surfactant micellar phases and water using fluorometric methods. In three surfactant solutions (SDS, polyoxyethylene lauryl ether C12E10, and DMPC zwitterionic phospholipid), coumaphos molecules tended to bind to the more polar surface of the micelles rather than to the nonpolar core. This might explain why coumaphos (compared to trifluralin and atrazine) experienced such a low solubilization enhancement in both surfactants despite having a high Kow value. Although no similar type of research has been reported so far for atrazine or trifluralin, we believe that the low solubilization enhance-

ment of atrazine by both surfactants might be related to a similar behavior. Atrazine’s distribution of polar groups is more uniform in its molecular structure, and this characteristic might limit the interaction of this pesticide with the polar micellar surface. This might explain why the solubilization enhancement of atrazine is not important above the CMC and the magnitudes of Ksub and Ksupra are not that different, specially when interacting with rhamnolipids. On the other hand, trifluralin polar and nonpolar groups are more localized in specific regions of the molecule. This could permit to fit more trifluralin molecules inside the micellar structure with the polar groups facing the surface and the hydrophobic structure interacting with the inner core. Following the procedure described by Miller (29) of normalizing the Ksupra values with respect to the aqueous solubility (see Table 2), we found that indeed there is a greater relative enhancement of apparent solubility for coumaphos than atrazine and that the obtained normalized values correlate well with the trend of the Kow of the pesticides: trifluralin > coumaphos > atrazine (see Tables 2 and 3). The parameter Ksupra/Ksub also presented in Table 2 is a ratio of the relative affinity of the pesticide for the micellar portion of the surfactant and the surfactant monomers in the aqueous phase. Such parameter follows as well the same trend of the Kow values for the pesticides. However, even when Miller (29) found an inverse relation between the solubilization enhancement of organic compounds and their corresponding aqueous solubilities, we have found that coumaphos having a lower aqueous solubility than trifluralin observes as well a lower relative enhancement of apparent solubility. We believe that this irregular behavior is still related to the unique interactions of coumaphos with the micellar phase mentioned by Lopes et al. (28). Our experimental results showed that only atrazine observed slightly higher Ksupra values in the rhamnolipid micellar phase than in the synthetic surfactant. Coumaphos and trifluralin still showed higher solubilization enhancement in Triton X-100. According to Kanga et al. (13) and Thangamani and Shreve (14), glycolipids tend to form micelles with a less dense packing of monomers due to the large size of the hydrophilic group, which includes the sugar moieties and the carboxylic groups on the lipid end. Being anionic, the carboxylic groups tend to repel each other and limit the micellar aggregation number. This theoretically should result in less dense rhamnolipid micelles with large volumes able to accommodate a greater amount of HOCs as compared to Tween 80 and DBS, respectively. Under neutral pH conditions Tween 80 micelles have a formula weight of 76 700 (aggregation number ) 59 and m.w. ∼ 1300) (30), DBS micelles of approximately 39 000 (aggregation number ) 112, m.w. ) 348.5) (14), and Triton X-100 of 87 500 (aggregation number of 140, m.w. ) 625) (30). Triton X-100 micelles are therefore expected to be larger than those of the other synthetic surfactants and more effective in enhancing the solubility of organic pollutants. Yet, we do not have values of rhamnolipid formula weights or aggregation numbers to compare them with those of Triton X-100. It is also well documented that for rhamnolipids, these parameters depend strongly on variables such as pH and ionic strength among others (31). Since the values of kOWM for trifluralin in both surfactants were very close, we can assume that trifluralin transport from the bulk aqueous phase into the micelles happens at similar rates, and the pesticide molecules take about the same time to align themselves in both surfactants micellar core. In turn, kOMW average value was significantly lower for trifluralin in rhamnolipid than in Triton X-100. This may suggest that once the pesticide molecules are attached to each surfactant micellar core, they diffuse out to the bulk of water at a lower rate in the case of the rhamnolipid as compared to Triton X-100.

Solubilization kinetic experiments at surfactant concentrations below the CMC (Figure 3C) showed that trifluralin saturation of the true aqueous phase in the presence of surfactant monomers is a fast process. Such results validate the model assumption that the contaminant molecules are transferred rapidly from the solid phase to the true aqueous phase contributing to its instantaneous saturation. The truly limiting step is therefore the much slower mechanism of diffusion of the molecules from the bulk of the solution to the interior of the micellar phase. The results of this work are relevant for future studies where the application of surfactants in the solubilization of contaminants is coupled to their eventual biodegradation. Some sources postulate that microbial uptake of target contaminants happens mostly outside of the micelles in the true aqueous phase. The direct uptake from the micellar phase is very limited and greatly influenced by the nature of the surfactant (its toxicity) and by the affinity between the contaminant molecules and the micellar phase (15, 32, 33). Even when Triton X-100 can enhance the micellar solubilization of a contaminant, its toxic effect on biodegrading microorganisms may limit the microbial uptake of the target compound to that portion truly dissolved in the bulk of water. Instead, because rhamnolipids are microbial in origin, they might be expected to help to promote, or at least not hinder, the possible access of microorganisms to the pollutant portion attached to the micellar phase. The eventual disappearance of a target compound in a completely mixed system might depend on three important rates: (1) the rate at which the molecules can diffuse out of the micellar phase, (2) the rate of microbial uptake of the pollutant from the true aqueous phase, and (3) the rate of microbial uptake of the pollutant directly from the micellar phase. The rate constants kOMW and kOWM and the equilibrium constants Ksub and Ksupra obtained in the present work can be helpful tools for a better understanding of the steps that control the biodegradation of a pollutant in water when a micellar phase is involved.

Acknowledgments The authors want to thank the Department of Environmental Biology at the University of Guelph, for supplying the P. aeruginosa UG2 cultures. Our acknowledgments to the U.N.A.M. (Universidad Nacional Auto´noma de Me´xico), CONACYT (Consejo Naconal de Ciencia y Tecnologı´a), and The Fulbright Comission for their economical support on the development of this work. The authors want to acknowledge the National Science Foundation Environmental Engineering Program (Grant BES-9702603) and the U.S. Department of Agriculture for funding part of this research.

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Received for review March 20, 2000. Revised manuscript received August 23, 2000. Accepted September 12, 2000. ES0011111