Hexane-Filled Dialysis Bags for Monitoring Organic Contaminants in Water Glen D. Johnson Pennsylvania Department of Environmental Resources, Bureau of Waste Management, Division of Special Investigations, Scientific Services Section, P.O. Box 2063 Harrisburg, Pennsylvania 17 105-2063 Solvent-filled dialysis bags can (1) detect lipophilic compounds that may otherwise evade detection, (2) provide time-integrated monitoring, (3) serve as biological surrogates when tissue sampling is impractical, and (4) substantially reduce laboratory effort. Bags consisting of regenerated cellulose dialysis tubing (1000 MW cutoff), containing 40 mL of n-hexane, are placed in protective cages which, in turn, can be suspended in surface water or lowered into a well. Exposure to a spring that is contaminated with Arochlor 1248 revealed that, under fairly steady state conditions, uptake is linear over 32 days, resulting in a 100-fold concentration. Bags were perfectly intact, and the contents did not require cleanup prior to analysis, even after exposure for 30 days in a biofouled pond. Fugacity-based bioconcentration kinetics, interpreted with respect to Fickian diffusion, may explain how this system works. Introduction In situ monitoring of organic compounds with biological surrogates is a technology receiving increasing recognition (1-3). Several systems have been proposed, all based on the theory that lipophilic compounds will passively diffuse from the water column into a lipophilic phase. These systems include hexane contained by either regenerated cellulose dialysis tubing (1)or by polyethylene membranes fitted in brass enclosures (2),as well as a thin lipid layer contained by polyethylene “layflat” tubing (3). Several advantages of such a system include the following: (1)Continuous time-integrated monitoring can be done. (2) Compounds that may otherwise go undetected may be concentrated to measurable levels in the solvent bags. (3) Laboratory analysis of the sample is much more rapid and less expensive than conventional water, sediment, or tissue analysis because the hexane matrix is directly injected into a GC without the need for sample preparation. Subsequently, the proposed system can be much more practical when exact concentrations in environmental media are not essential, such as with initial screening or contaminant source identification. (4) Bioavailability may be indicated. Whether a compound initially desorbs from a particulate or colloidal phase, or is truly aqueous, these bags reveal the potential of a compound to partition into a lipophilic phase. Notable limitations include the following: (1) Where exact concentrations must be known, such as for comparison to legal criteria, actual environmental media must still be sampled. Concentrations in water may, however, be predicted if sufficient data exists for modeling. (2) Although this method could probably be adapted to monitoring the acid-base/neutral class of priority pollutants,application to monitoring volatiles is not foreseeable. However, there is not a high demand for a simpler screening tool for volatiles since the conventional purge and trap method takes considerably less time than sample preparation for the other classes of priority pollutants. What follows is a general theory offering an explanation of how the system works. Results from monitoring Arochlor 1248 are then presented for a detailed time-series study of a spring that exhibits steady-state conditions and 0013-936X/91/0925-1897$02.50/0
for an open pond that exhibits biofouling conditions. The theoretical section does not need to be understood for those readers primarily interested in methods and application.
Theoretical Considerations Kinetics. Absorption of a lipophilic solute from water into an organic phase contained by a dialysis membrane may be limited by aqueous- and organic-phase diffusivities (4). The major controllable factors limiting diffusion would therefore be the organic phase (internal solvent) and the dialysis membrane. How these factors affect absorption may be explained by bioconcentration kinetics that can be interpreted with respect to Fickian diffusion. Bioconcentration, viewed as passive diffusion of stable solutes from the water column into an aquatic organism, has been successfully described (5-8) by two-compartment kinetics as dCL/dt = klCw - K2CL (1) where kl and k, are first-order uptake and depuration rate constants, respectively, Cw is the solute concentration in ambient water, CL is the lipid-phase concentration in the target lipid compartment of an aquatic organism, and t equals time. Based on the boundary condition that CL equals zero when t equals zero, eq 1 integrates to CL = Cwkl/k,(l - exp(-k$))
(2)
Organism uptake is therefore expected to be a first-order function of time until equilibrium is attained and CL = Cw(k1/k2). Two important simplifying assumptions of this model are that it does not incorporate the effects of food-chain input, as described by biomagnification kinetics (8, 9), and it considers the solutes in question to be recalcitrant. Therefore eqs 1 and 2 appear to describe an inanimate system like solvent-filled dialysis bags even more appropriately than an actual organism. By replacing solute concentrations with solute fugacities, Gobas and Mackay (5) described fish bioconcentration as V L ~ dfL/dt L = DAfw - f L )
(3)
where VL equals fish lipid volume (m3),2, equals the fish lipid fugacity capacity (mol m-3 Pa-’), t equals time (s), fw and fLare solute fugacities (Pa) in water and fish lipid, respectively, and Df is a transport parameter (mol Pa-’ s-’), including all resistances between the target fish compartment and water. Equation 3 then integrates to fL
= f d l - exp(-Dft/VLZL))
(4)
when subject to the same boundary conditions as eq 1. Fugacity, thought of as the “escaping tendency” of a solute from a particular phase (IO),essentially equals the solute’s partial pressure, adjusted for lack of ideality (12). Solute concentration is linearly related to fugacity by the fugacity capacity of a given phase for a particular solute, expressed as C = Zf (10). When a solute is partitioned among several phases (i.e., water and lipid) within a system, equilibrium is attained when the solute’s fugacity is equal in every phase (i,e., fw = fL). Equilibrium concentrations are therefore a linear function of fugacity capacities ( 1 0 , l l ) . Since fL = CL/ZL
0 1991 American Chemical Society
Envlron. Sci. Technol., Vol. 25, No. 11, 1991
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and f w = Cw/Zw, then eq 4 can be expressed in more conventional terms as
CL = CwZL/Zw(l- exp(-Dft/VLZL))
(5)
At equilibrium, ZL/Zw = CL/Cw, which is the familiar bioconcentration factor K b Assuming Zwis constant, the equilibrium biotic concentration is defined by the fugacity capacity of the target lipid compartment, ZL,for the solute in question. Kinetics, however, are not only controlled by ZL, but also by the lipid volume (VL) and the transport parameter (Df) since, when eq 5 is compared to eq 2 k2
= Of/ VLZL
(6)
Since the transport of a solute to and within the fish is expected to take place in a series of aqueous and lipid phases, Df can be shown to consist of a transport parameter for all aqueous phases, Dw, and all lipid phases, DL (5). Each of these transport parameters is further shown to equal its respective fugacity capacity times a flow term, Q (m3/s) (5). Therefore, Dw and DL equal QwZw and QLZL, respectively and eq 6 becomes
k2 = (QwZW/ZL+ QL)/VL
(7)
Equations 5-7 thus provide an insightful description of bioconcentration in fish, using parameters that are not difficult to obtain, and have biological significance. As will now be shown, these equations can also provide a theoretical foundation for describing the uptake kinetics and equilibrium values of a lipophilic solute that is absorbed by a solvent-filled dialysis bag. Gobas and Mackay ( 5 ) noted that the flow term, Q (m3/s),can be “... characterized by ... diffusivity, D (m2/s), divided by path length, 1 (m), times area, A (m2)”. Although these dimensions are very difficult, if not impossible, to measure in a living organism, they are simple measurements for a solvent-filled dialysis bag. The area, A, equals the total membrane surface area and path length, I , equals the membrane thickness. Also, since there is only one phase, such as hexane, into which the solute is transported, then eq 6 can be expressed as
k2 = DL/VLZL = QLZL/VLZL = QL/ VL = DA/lVL
(8)
Combining eqs 2, 5, and 8 then reveals a model for describing the uptake of a lipophilic solute from water by an organic solvent filled dialysis bag as
CL = CwzL/zw(l - exp(-DA,t/ VLjm))
(9)
where the subscript m refers to the dialysis membrane, and although hexane is presently the solvent of choice, the subscript L is used to indicate that this model is expected to apply to any lipophilic solvent. Note that D, without a subscript, equals diffusivity (m2/s), as opposed to the transport parameter Dp The proposed model, eq 9, is simply a bioconcentration formula with a new interpretation of the rate constant, k2. Once a value for k2 = DA,/l,VL is obtained, diffusivity, D, can be easily solved for since A,, ,I and VL are straightforward measurements. For solvent-filled dialysis bags, diffusivity pertains to solute partitioning between the water and organic solvent within the membrane pores. Diffusivity in both the aqueous and organic phases is a function of solute and solvent properties, which can be interpreted by the Stokes-Einstein equation as D = (R/a)T/Gwn 1898 Environ. Scl. Technol., Vol. 25, No. 11, 1991
(10)
where R is the universal gas constant, a is Avogadro’s number, T is the absolute temperature, r is the solute radius, and n is the solvent viscosity (12). Equation 10 was actually developed for large particles, but has proved applicable to molecules (12). At constant temperature, diffusivity would therefore be inversely related to both the size of the solute molecules (or solvated molecules) and solvent viscosity which, for our concern, is actually two solvents-water and hexane. For very lipophilic compounds (K, 10‘9, the overall diffusion rate is expected to be limited by aqueous-phase diffusion ( 4 , 5 , 7). Derivation from Fickian Diffusion. Equation 9 can also be derived from Fick’s first law, which describes diffusion of solutes across an inert membrane by N
dn/dt = DA,/l,(C1
- C,)
(11)
where dn/dt is the diffusive rate of a solute across a membrane of area A and thickness 1, D is the diffusivity (m2/s) for a particular solution, and C1and C, are solute concentrations on both sides of the membrane (12). As seen in eq 11, equilibrium is achieved when the concentrations are equal since the formula pertains to the same solvent on both sides of the membrane. In order to express diffusion of a solute from water to a lipophilic solvent, solute fugacities can be used in place of concentrations, resulting in dfL/dt = DArn/lrn(fW - f d
(12)
which, if expressed with respect to volume of the lipophilic solvent, VL, becomes dfL/dt = DAm/knVL(fw - fL)
(13)
Equation 13 is equivalent to eq 3, the initial differential equation that defines bioconcentration as a function of solute fugacities. The solution to eq 13, when expressed in concentration form, is therefore eq 5 except that Of/ VLZL now equals DA,/l,VL, as expressed in eq 9. Predicting Water Concentration. Equation 13 may potentially be used for estimating an unknown solute concentration in ambient water. If solute concentrations in the hexane bags are converted to fugacities, then the slope of the linear region of an uptake curve equals dflldt. Substituting this value in eq 13, and rearranging, allows for the following solution of the solute fugacity in water as a function of the solute fugacity in a lipophilic solvent:
fw = (dfL/dt)/(DArn/1mVL) + f L
(14)
Converting fugacities to concentrations and solving for Cw then equals
CW = Zw[(dfL/dt)/(DAm/lmVL) + CL/ZLI (15) According to Mackay and Paterson ( I O ) , ZWequals the reciprocal of Henry’s law constant, H, for a particular solute. Also, with use of the approach by these authors for calculating the fugacity capacity of octanol, a value for ZL can be obtained by 1 / u L 7 L P 1 where u L is the molar volume of the lipophilic solvent saturated with water, 7~ is the activity coefficient of the solute in the lipophilic solvent, and P is the vapor pressure of the liquid solute a t the system temperature. Therefore, if these properties can be obtained, the solute concentration in ambient water can theoretically be estimated by Cw = [(dfL/dt)/(DA/lVL)
+ C L ( U L ~ L P ) ] /(16) H
Although uncertainty increases with each property that must be estimated, even an approximation of CW would
be very valuable when a solute occurs a t concentrations below the detection limit associated with conventional water sampling. Furthermore, since a critical assumption for calculating Zw as 1/H is that the solute is truly dissolved in water (IO),the estimated Cw represents the most bioavailable form of the solute. Effects of Particulate Matter. The discussion so far has pertained to the transfer of a solute from a true aqueous phase into a lipophilic phase. In reality, however, lipophilic contaminants in natural waters are often adsorbed to particulate matter (13-16), resulting in implications for interpreting the water fugacity capacity term, Zw. Whereas the fugacity capacity of water for a truly dissolved solute is simply the inverse of Henry's constant for that solute (IO), the fugacity capacity of a sorbed phase has been shown t o equal 2, = K @ / H (17) where Kp is the particulate/water partition coefficient, p is the particulate matter density, and H is Henry's constant for the compound in question (10). In many instances, Zw will therefore actually be Z,. Parameter Estimation. Parameters of the proposed model, eq 9, may be estimated hy fitting experimental data to C, = Cwa(l - exp(-bt)) (18) where a and b are statistical estimates of the parameters ZL/ZWand DA,/VL1,, respectively. If eq 18 is fit by an iterative nonlinear regression method, an appropriate seed value for parameter a (therefore ZL/Zw)is the l-octanol/water partition coefficient, K , (13,which is readily available for most compounds of concern (18). A seed value for parameter b may, however, be more arbitrary. If empirical data are accurately described by eq 9, several practical applications may result. First, the model describes controllable (nonenvironmental) sources of variability that must be maintained as constant as possible in order to compare results among bags. These sources include purity and volume of the internal solvent, along with membrane surface area and thickness. Second, the model can be used to predict theoretical equilibrium values so that observed equilibrium can be compared for measuring the 'efficiency" of these bags. Also, when it is impossible to reach true equilibrium, the model may be used to predict the time required to reach equilibrium or some fraction of time thereof. Third, since equilibrium concentration in the bag is approximated by Cw(ZL/Zw),the effects of solvents other than hexane may be evaluated prior to experimentation by obtaining the fugacity capacity of these solvents (ZL). Fourth, this model can be used to compare the uptake kinetics of a hexane-filled dialysis bag to an actual organism. Thermodynamics. The driving force behind diffusion and concentration of a lipophilic compound from ambient water into an organic solvent can be expressed as the free energy of transfer. Considering the solvent bag and ambient water as a system, the free energy is expressed as the reversible work done on this system (12),quantified as AGO = -RT In K (19) where AGO is the Gibbs free energy of transfer (J/mol), R is the universal gas constant, T i s absolute temperature, and K is the ratio of solute concentration in the solventfilled bag over that in water (CL/Cw). The solute concentration in the organic solvent can then be expressed as
CL = Cw exp(-AG"/RT)
(20)
Oialysir Tubioqil000 m.r. CvfOIli Fllad with 40 d n-tiaana
Suing
Figure 1. Cutaway of two 40-mL hexane-filled dialysis bags in prctective cage.
When K equals the ratio at equilibrium, AGO can be thought of as the partitioning potential a t the given temperature. Equations 19 and 20 also apply to bioconcentration by simply replacing the organic solvent/water equilibrium factor, K, with a bioconcentration factor, Kb. The two equilibrium values are therefore related to each other by the free energies of their respective concentration processes, a concept known as a linear free energy relationship (19).
Although the energetic (enthalpy) and ordering (entropy) factors contributing to free energy may be very different between liquid/liquid partitioning and bioconcentration processes, the overall free energy of transfer appears to be very similar (20). Compounds that concentrate in an organic solvent fded dialysis bag are therefore expected to have a high bioconcentration potential where, of course, molecular size is not limiting. Methods Preparation of Apparatus. For constructing each dialysis bag, approximately 12.5 cm of regenerated cellulose dialysis tubing (Spectra Por 6, MW cutoff 1ooO,8-mm flat width) is fded with 40 mL of pesticide-grade n-hexane and sealed a t each end with 8.8-cm Spectra Por enclosures. A 2.5-cm metal binder clip (standard office supply) is then clamped over each enclosure to secure the seal and provide a means to link the bags together (see Figure 1). An oversized enclosure is necessary to ensure a proper seal. Also, 40 mL of hexane was chosen to ensure a sufficient recovery volume in case any samples required concentration prior to analysis. A string of bags is placed in a protective metal cage made of hardware cloth, as displayed in Figure 1. These cages can then be suspended in surface water by attaching an anchor and buoy at opposite ends of the cage, or they may be lowered into a groundwater monitoring well. Environ. Sci. Technol., VoI. 25. No. 11. 1991
1809
Table I. Laboratory Controls
treatment Arochlor 1248 Arochlor 1260 blank
concn after 5 days, pg/L water dialysis bags 0.60-0.47 0.49-0.45 0.0.0
69.0-81.0 16.0-22.0 0.0.0
AU hardware is washed with soapy hot water, then rinsed with charcoal-filtered, deionized water, and finally rinsed with hexane. Sample Recovery. After a cage is withdrawn from water, the contents of each bag are drained into separate glass vials for shipment and storage. Concurrent water samples for PCB analysis are taken in 1/2-galglass jars. Chemical Analysis. PCBs were analyzed with a Varian 4600 GC/ECD, according to EPA method 608 (21),with the exception of sample preparation for hexane media. All hexane samples were shot directly into the GC without the need for cleanup. For the springhouse study, ambient inorganic water chemistry was measured every hour for the duration of the experiment with Hydrolab Datasondes. The Datasondes were overlapped for 10 days to check for consistency between machines and were also checked against field measurements taken a t each sampling time with a YSI Model 33 conductivity and temperature meter and a Corning Model 103 pH meter. For the pond study, field chemistry was monitored with the same meters and inorganic laboratory analyses were performed according to EPA methods (22). Data Analysis. The Gauss-Newton method of nonlinear least squares (23) was used to fit data from the springhouse study to a nonlinear model. Fixed-effects analysis of variance was used (23) to characterize analytical uncertainty associated with use of Datasondes to monitor ambient water quality. Controls. Two bioassay chambers were filled with 20 L of deionized, charcoal-filtered water and one chamber was spiked with 5 pg/L each of Arochlor 1248 and 1260. After duplicate bags in each tank were exposed for 5 days, the negative control revealed no contamination while the positive control indicated that the system works (see Table I). Field blanks also revealed no contamination. Application. Six cages, each containing three hexane bags, were suspended in the concrete encasement of a spring that was completely enclosed by a springhouse. Triplicate bags and duplicate water samples were taken a t each sampling time for evaluating the uptake kinetics over 32 days while monitoring the ambient water concentrations. The performance of these bags under more extreme open field conditions was tested by placing two cages, each containing duplicate bags, in a hypereutrophic pond, followed by sampling duplicate bags and duplicate water samples after 10 and 30 days of exposure.
160
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1000 Environ. Sci. Technoi., Voi. 25, No. 11, 1991
180 JULIAN DAY
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200
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Figure 2. Ambient water quality indicators measured during the kinetic study, presented as the daily mean pius/minus one standard deviation from 24 hourly measurements each day. H
0
Experimental Results Springhouse Study. A preliminary experiment in March 1989 showed that hexane-filled dialysis bags concentrated Arochlor 1248 by 25-fold after 5 days of exposure in a spring that maintained steady PCB concentrations. These results, along with observed very steady ambient water quality, supported the use of this spring for conducting a more detailed kinetic study which, in turn, was conducted during June and July 1990. For the kinetic study, ambient water quality indicators were measured hourly with Datasondes. The results, reported in Figure 2, show that the overall ranges for the indicators are very minimal over 32 days, even though
170
10
20 DAY
30
40
Figure 3. Uptake of Arochior 1248 by hexane-filled dialysis bags: H, concentration in hexane; W,concentration in water: line, predicted concentration in hexane from eq 21.
differences between the Datasondes inflate these ranges. Analysis of variance for the 10-day overlap period showed a significant difference ( a = 0.0001, p = 0.0000) between the Datasondes for all three indicators, even after the effect of "days" was factored out to correct for serial correlation. Although this analytical uncertainty should be acknowledged, it does not represent a serious problem because the statistical differences are due to very small variability within each Datasonde.
Along with relative steady water quality, the ambient concentration of PCBs only fluctuated between 7.6 and 11.6 ppb (see Figure 3), which is less variable than one may expect of a laboratory-controlled flow-through system ( I ) . Maintaining relatively steady ambient conditions is critical for two reasons: (1)understanding how absorption kinetics are affected by properties of the solvent-filled dialysis bags and (2) estimating the time required to attain equilibrium. This is because transferring a solute from water to an organic phase is a diffusion process, which is, in turn, dependent on the ambient conditions, especially temperature (20) and the solute concentration (9,12). For this reason, equilibrium studies performed with static systems where the ambient concentration drops toward zero during the experiment (3) only reveal how a given mass of solute partitions between different solvents, but cannot reveal the time required for equilibrium in the environment where ambient contamination has a steady source. The pattern of PCB uptake by the dialysis bags, as seen in Figure 3, is clearly linear within 32 days. This same pattern was reported by Sodergren (1)when a flow-through system maintained a relatively steady water concentration of PCBs; however, since data were reported on a logarithmic scale, Siidergren's results have been misinterpreted as meaning the bags approached steady state within 8 days ( I , 3). The predicted values in Figure 3 result from fitting the data to eq 18, which, after only four iterations, converged to CL = Cw1061.2(1- exp(-0.003t)) (21) where t equals days and Cw equals 9.29 pg/L, the arithmetic average over 32 days. Residuals from this model appeared to be normally distributed and revealed no serial correlation. The parameter estimates in eq 21 can be further interpreted by eq 9. Since 32 days turns out to only incorporate the linear region of the uptake curve, the required time to equilibrium can only be estimated. When 99% of the equilibrium concentration is achieved, then 1 - exp(-0.003t) in eq 23 would equal 0.99. Solving for t then equals 1516 days, or -4.2 years, meaning the actual time to equilibrium may be impossible to measure directly. Other solute/solvent combinations, or smaller solvent volumes, may result in much shorter time periods for attaining equilibrium. Pond Study. Hexane bags were suspended in a pond (-0.15 acre) that receives overflow from the spring used in this study and is known to be contaminated with -1 pg/L PCBs. This simple experiment was to address several questions: (1) How will the bags work in a lentic system with very minimal flow; (2) will the bags remain durable, and therefore reliable, when exposed to a harsh environment; and (3) how will the bags perform when exposed to a much lower ambient concentration that is around the analytical detection limit? The second question raised here was rather rigorously tested because the pond is hypereutrophic, contains a high degree of suspended and dissolved material, and supports a population of snapping turtles. Results are reported in Table 11, where we see the bags not only absorbed PCBs, but also yielded an increasing concentration over time. While the hardware was biofouled and covered with sediment within 10 days, the dialysis membrane itself had only a film of sediment covering -15% of its surface area after 30 days. Such durability was also reported by Sodergren ( 1 , 2 4 ) ,whose 3-4-mL bags showed no degradation or periphyton growth after 6 weeks of exposure in a eutrophic river ( I ) and, most remarkably, had no degradation after 1year of exposure
Table 11. PCB Concentrations in Water and Dialysis Bags, with Ambient Water Quality, for Pond Study
dav Arochlor 1248, pg/L hexane waterb
0
10
30
naa naa
68.0-74.0 1.15-1.16
110.0-110.0 0.43-0.54
Water Quality Field Measurements temp, "C 21 23 17 60 61 55 cond, pmhoslcm
PH DO," mg/L Water Quality TOC,' mg/L susp solids, mg/L set. solids, mg/L TDS,' mg/L alkalinity
6.3 6.6
5.3 9.8
5.4 9.4
Laboratory Measurements 22.0 44.0
