Environ. Sci. Techno/. 1995,29,2886-2892
Use of the Semipermeable Membrane Device as an in Situ Sampler of Waterborne Residues at
J O N A . L E B O , * R O B E R T W. G A L E , J I M M I E D . PETTY, DONALD E. TILLITT, JAMES N. HUCKINS, J O H N C . MEADOWS, C A R L E . O R A Z I O , KATHY R . E C H O L S , A N D D E N N I S J . SCHROEDER US.Department of the Interior, National Biological Service, Midwest Science Center, 4200 New Haven Road, Columbia, Missouri 65201
LLOYD E. I N M O N
US.Fish and Wildlife Service, Vicksburg Field Ofice, Vicksburg, Mississippi 39180
Semipermeable membrane devices (SPMDs) were used to passively sample aqueous polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) in Bayou Meto, AR. The two sites were upstream and downstream from the confluence with a tributary that delivers PCDDs and PCDFs to the Bayou. Following dialysis, cleanup, and fractionation, four replicate 17-9 SPMD samples from each site were analyzed by GUMS, and four were evaluated by H411E bioassay. Traces of only OCDD and HpCDDs were detected in samplesfrom the upstream site. The four samples from below the tributary contained averages of 1550 f 80 pg of 2,3,7,8-TCDD, 1640 f 80 pg of 2,3,7,8-TCDF, and lesser quantities of other congeners. The TCDD equivalents obtained by bioassay of replicate SPMD samples agreed well with results obtained by GC/MS. The quantities of 2,3,7,8TCDD and 2,3,7,8-TCDF sequestered by SPMDs at the downstream site were used to estimate the aqueous concentrations for both compounds as 2 pg/L.
Introduction Bayou Meto is a turbid, low-gradient stream that originates in the highlands of central Arkansas and empties into the Arkansas River 290 km to the southeast. Near Jacksonville, AR, about 40 km downstream from Bayou Meto’s source, a tributaryjoins BayouMeto from the North. This tributary,
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Rocky Branch Creek, is a point source of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs)-especially of 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD) and 2,3,7,8-tetrachlorodibenzofuran (TCDF)-to Bayou Meto. In 1971, the Vertac Company began manufacturing phenoxy acid herbicides (i.e., 2,4dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid) at a plant in Jacksonville. Due to extensive contamination with PCDDs and PCDFs, the Vertac site is ranked no. 18 on the U.S. EPA list of 1301 Superfund sites (I). Rocky BranchCreek flows throughthevertac site about 3 km above its confluence with Bayou Meto. Aquatic biota from Rocky Branch Creek and Bayou Meto have been found to contain elevated concentrations of PCDDs and PCDFs. A bullfrog (Ranacatesbeiana)sampled in 1984 from Rocky Branch Creek downstream from the Vertac site contained 103 pglg TCDD in its muscle tissue and 68 000 pg/g in its fat (21, and another sampled in 1985 contained 48 000 pg/g in its liver (3). A smallmouth buffalo (Zctiobusbubalus) sampled in 1987 from Bayou Meto near Jacksonvillecontained 203 pg/g TCDD and 21 pglg TCDF (as well as elevated concentrations of other PCDDs and PCDFs) on a whole-body, wet-weight basis (4, 5). This TCDD concentration was the highest found for any sampling site by the EPA in its National Study of Chemical Residues in Fish (5). Wood duck (Aixsponsa)eggs collected from Bayou Meto 9 km downstream from the confluence with Rocky Branch Creek contained concentrations of TCDD as high as 510 pg/g on a wet weight basis (6). TCDD exhibits endocrine- and reproduction-disrupting effects in fish and wildlife (7). Laboratory studies indicate that extremely low concentrations of TCDD can impair reproduction in fish, mammals, and birds (8). PCDD and PCDF residues have been implicated in the impairment of reproductive success in wood ducks nesting in Bayou Meto as far as 58 km downstream from the confluence with Rocky Branch Creek (9). As can be seen, concerns about the levels of PCDDs and PCDFs emanating from the Vertac site are justified. The elevated concentrations of these extremely toxic compounds are potentially detrimental to the well-being of humans living near Bayou Meto as well as to the biological health of the wetland resource as a whole. Bayou Meto is habitat to more than 64 species of fish (10). The wetland is year-round habitat for the wood duck as well as being important habitat for mallards (Anus platyrhnchos) and other migratory waterfowl during the colder months. The semipermeable membrane device (SPMD) and its use as a sampler of aqueous hydrophobic organic contaminants have beenextensivelycharacterized (11-16). The SPMDs used in the present work can be briefly described as segments of layflat polyethylene tubing with thin films of triolein sealed inside. The devices function as passive, in situ aquatic samplers that sequester dissolved hydrophobic contaminants from water. Perhaps the most important advantage SPMDs offer relative to conventional water sampling techniques (Le.,those that require passage of the water through reverse-phase sorbents ( I n ,polyurethane foam (18), or polystyrene-divinylbenzene copolymer resin (19) or those that entail partitioning of water with immiscible solvents (ZO)),is that SPMDs sample only
This article not subject to U S copyright. Published 1995 by the American Chemical Society.
ARKANSAS I II
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Rocky Branch Creek
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FIGURE 1. Map of Bayou Meto in the Jacksonville area showing the relationship of the two sampling sites to the confluence with the point source, Rocky Branch Creek. The arrows signify the direction of water flow.
truly dissolved (bioavailable) contaminants (13, 14). By virtue of their design, SPMDs exclude contaminants that are associated with humic acids or sediments. In contrast to the conventional sampling techniques, a prefiltration step is unnecessary. Due to the large volumes of water passively sampled by SPMDs, unprecedentedly low quantitation limits can be attained. The primary objective of the study reported here was to supplement previously assembled information on the contaminant problem at Bayou Meto. The water at two sites in Bayou Meto was sampled in situ with SPMDs for aqueous PCDD and PCDF residues. All 2,3,7,8-chlorinated congeners and all C4-Cle PCDD and PCDF homologswere targeted for analysis. An integral part of the primary objective was to use SPMD-derived data to estimate aqueous concentrations of TCDD at a severely contaminated Bayou Meto site. SPMDs have not been extensively used for sampling PCDDs and PCDFs (I@,and a second objective of this work was to demonstrate their potential for monitoring these classes of contaminants. Dialyzates from environmentally exposed SPMDs have been shown to be readily amenable to evaluation by Microtox (21) and Mutatox (22,23). To fulfill a third objective of this study, dialyzates from SPMDs exposed at Bayou Meto were evaluated by an H4IIE bioassay procedure to demonstrate that the SPMD sampling technique is also complementary to this bioassay.
Experimental Section StudyDesign. SPMDs were deployed at two sites in Bayou Meto, AR,in August 1993 and were allowed to passively sample aqueous PCDD and PCDF residues for 28 d. The average water temperature over the sampling period was 32 "C. The geographic relationship of the sampling sites to the point source is shown in Figure 1. Site 1 is just upstream of the confluence of Rocky Branch Creek with Bayou Meto, and site 2 is immediately downstream from the confluence and is downstream from the outlets of both of Jacksonville's water treatment plants. The SPMDs consisted of 87 cm lengths (average weight 3.35 @ of 2.54-cm wide layflat polyethylene tubing (86pm average wallthickness) with 1.0mL (0.91g) oftriolein (1,2,3tri [cis-9-octadecenoyl]glycerol) heat-sealed inside. Shrouds fabricated from 0.9-mlengths of 0.3-m diameter galvanized steel culvert were used to protect the SPMDs during the 28-d sampling period. After installation of the SPMDs in the shrouds, the ends of the shrouds were covered with
protective metal screens. Two shrouds, each containing 16 SPMDs,were deployed at each of the two sampling sites. Bayou Meto is 10-15 m wide in the Jacksonville area; the two shrouds placed at each site were deployed near opposite shorelines. The shrouds were allowed to rest on the bottom of the Bayou and were securely tethered with braided steel cables to trees and other k e d objects. Because SPMDs have a propensity to sequester vaporphase contaminants (241, they were sealed inside solventrinsed virgin paint cans for shipment to and from the samplingsites. An additional can containingthe field blank SPMDs was transported to each sampling site. The two SPMDs constituting each field blank were exposed to air only while the corresponding water sampling SPMDs were being installed in or removed from their shrouds, and they were kept sealed in their cans throughout the interim. After the 28-d sampling period, the cans containing the field blank and Bayou-exposed SPMDs were shipped back to the laboratory in an ice-filled cooler and were kept in a freezer until analyses of the SPMDs began. Dialysis, Cleanup, and Fractionation. The SPMDswere thawed and subjected to an exterior cleanup that removed periphyton, adhering sediment particles, and carbonates from the polyethylene. This cleanup consisted of alternately rinsing the SPMDs with running tap water and vigorously brushing the polyethylene with a toothbrush, then briefly immersing the SPMDs in 1 M HC1, rinsing them with water, then acetone, and allowing them to air-dry. All SPMDs from the same shroud were regarded as identical. The 16 SPMDs from each shroud were randomly pooled into four 4-SPMD samples; each sample thus consisted of 3.64 g of triolein and 13.4 g of polyethylene. Four 4-SPMD samples from each site (two samples from each shroud) were subjected to an analytical scheme that culminated in analysis by gas chromatography/mass spectrometry (GCIMS), and four 4-SPMD samples from each site (two samples from each shroud) were subjected to a less elaborate scheme that culminated in H41IE bioassay. One of the four SPMDs that constituted each sample that would be analyzed by GUMS was opened, spiked with 600 pg of each of 15 13C-labeledPCDDand PCDF procedural intemalstandards, and then resealed. (None of the samples destined to undergo bioassay were spiked with surrogates.) The four SPMDs constituting each sample were placed in a glass canning jar, 900 mL of hexane was added, and the jar was capped with an aluminum foil-lined lid. To minimize the quantities of lipids and polyethylene oligomers in the dialyzates, the dialyses were conducted at 18 "C (Le.,at subambient temperature [251).The analytes were allowed 48 h to dialyze into the hexane. The dialyzates were concentrated by rotary evaporation and then were subjected to a sequence of two reactive/ chromatographic cleanups previously described (26) and summarized below. The 2-cm i.d. glass columns used in the first cleanup were packed with, from bottom-top, potassium silicate (27);sulfuric acid-impregnatedsilica gel; and coarse silica gel impregnated with sulfuric acid. Dichloromethane was the eluant for the first chromatographic cleanup, For the second cleanup, 1-cmi.d. columns were packed with, from bottom-top, silica gel; potassium silicate; and sulfuric acid-impregnated silica gel. The concentrated eluates from the first cleanup were applied, and the analytes were eluted with 3%(v/v)CHZC12 in hexane. The eluates from the second reactivelchromatographic cleanup were treated with copper to remove elemental VOL. 29, NO. 11, 1995 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 2887
sulfur, then were concentrated by rotary evaporation. The concentrated solutions were fractionated by size exclusion chromatography (SEC). A Phenogel column (22.5-mm i.d. x 250-mm; 10-pm particles; 100-Aporesize; Phenomenex, Torrance, CA)was used for the SEC; the mobile phase was 20% (v/v) CHzC12 in hexane pumped at 4.0 mLlmin. The first 72 mL of the eluates from the SEC column was discarded the collected fractions extended from 72 to 240 mL of the eluates. The collected fractions from SEC were concentrated by rotary evaporation. Further fractionations were not performed for those samples destined to be evaluatedbyH4IIE bioassay; the solutions were transferred to vials, isooctane (keeper solvent) was added, and the solutions were evaporated to 150-pL volumes. (The H4IIE bioassay procedure is described below.) Samples to be analyzed by GUMS were subjected to two additional fractionation procedures described elsewhere (2628) and summarized below. The concentrated eluates from the SEC fractionation were transferred to autoinjector vials for fractionation on a porous graphitic carbon column (4.6-mm i.d. x 100-mm Hypercarb; Keystone Scientific, Bellefonte, PA). The fractionation procedure was developed to isolate PCDDs and PCDFs from PCBs, chlorinated naphthalenes, chlorinated diphenylethers, and other potential interferents (28). The chromatographic apparatus used in the carbon fractionation included a Perkin Elmer Series 410 pump and a Perkin Elmer ISS 200 autosampler (Perkin Elmer, Inc., Norwalk, CT), a Foxy 200 fraction collector (Isco, Inc., Lincoln, NE), and aValco N60 six-port, air-actuated switching valve Walco Instruments Co., Inc., Houston, TX). The mobile phase was pumped at 2.5 mLlmin throughout the multi-segment gradient program; the column was equilibrated with hexane at the initiation of the program. Upon sample injection, hexane was pumped for 4 min, at which time an 8-min linear gradient to 40% (vlv) toluene in hexane was begun. From 12 until 32 min, 40% (vlv) toluene in hexane was pumped isocratically. At 32 min, the direction of the flow through the column was reversed, and toluene was pumped isocratically for 60 min. The eluate resulting from the first 30 rnin of this reverse-flow toluene segment of the solvent program was collected by the fraction collector as the PCDDlPCDF fraction. In preparation for the next sample injection, the direction of the flow through the column was switched back to forward, and the mobile phase composition was changed via gradient to hexane. The collected eluates from the porous graphitic carbon column were concentrated by rotary evaporation and then were subjected to final fractionations on columns of alumina (26). The alumina (3.6g of basic alumina in 5-mL serological pipettes) was prewashed with 5% (vlv) CH2C12 in hexane, the concentrated PCDD/PCDF solutions (in nonane) were applied, and the alumina was washed with 12 mL of 5% (vlv)CH2C12 in hexane. The resultant eluate was discarded, and the PCDDs and PCDFs were eluted with 15 mL of CHZClz. Eluates collected from alumina were transferred to conical autoinjector vials. Into each vial was spiked 1.0 ng of l3C12-l,2,3,4-TCDD,the instrumental internal standard. The solutions were evaporated with streams of nitrogen to about 10 pL volumes. Analysis by GCIMS. GC/MS analysis was performed using an HP 5890 Series I1 GC interfaced to a Finnigan4023 quadrupole MS. An HP 7673 autosampler was used 2888
ENVIRONMENTAL SCIENCE &TECHNOLOGY / VOL. 29. NO. 11, 1995
to inject 2 of 10pL of the purified dialyzates. The analytes were chromatographed on a 60-m x 250-pm i.d. (0.25-pm film thickness) DB-5 capillary column with an initial hold of 1 min at 120 "C, followed by a ramp to 210 "C at 20 "Clmin, then to 300 "C at 3 "Clmin, with a final hold of 15 min. Mass windows were established for each of the homolog groups and were monitored sequentially during the temperature program. Within each mass window, three or four ions were measured for positive identification of each analyte. H4IIE Bioassay. The H4IIE bioassay procedures were as reported by Tysklind et al. (29). The H4IIE rat hepatoma cells were seeded in 96-well microtiter plates at 7000 cells/ well in 250 pL of D-MEM culture media (30). After a 24-h incubationperiod, the cells were dosedwith sample extracts or with standards in 5-pL volumes of isooctane. The cells were exposed, in quadruplicate, to six different concentrations of the samplesin a4-fold dilution series. The samples' responseswere calibrated against those of TCDD standards for the determination of TCDD equivalents (TCDD-EQ). TCDD standards were dosed at eight concentrations: 0; 0.069; 0.206; 0.617; 1.85;5.5; 16.7;and 50 pglwell. Ethoxyresorufin-0-deethylase (EROD) activity in the H41IE cells was measured kinetically (29)in a fluorometric plate reader (Cytofluor 2300, Millipore Corp., Bedford, MA). Linear portions of the dose-response curves were used to compare the relative potencies of the samples with those of the standards of TCDD. The determination of TCDD equivalents (TCDD-EQ)was by slope-ratio assay (311, as previously described (32).Variance estimates were based on an additive model of variance (31) and were calculated as previously described (30, 32).
Results and Discussion GC/MS Analysis. The results of the analyses by GUMS of the Bayou Meto SPMDs are presented in Tables 1, 2, and 3. Samples from both protective shrouds deployed at each site contained PCDD and PCDF residues that were very similar, in both the qualitative and quantitative sense. This similarity was also true for the two field blank SPMD samples. Although unexpected, the similarity of the residues and concentrations enabled presentation of the data from the SPMDs in Tables 1 and 3 as average concentrations that include SPMDs from both shrouds at each site and both field blank SPMDs. Of the 18 PCDDl PCDF congeners (17 of which are 2,3,7,8-chlorinated) routinely quantitated at the Midwest Science Center (MSC), only those shown in Table 1 were present in the SPMDs from Bayou Meto. The quantities given in Table 1 are expressed as pg of PCDD or PCDF congener sequestered/ 17 g (combined weights of triolein and polyethylene) of SPMD or as pgl8.5 g of SPMD (for the field blanks). The instrumental internal standard, 13C12-1,2,3,4-TCDD, served to compensate for variations in solution volumes, thus enabling the analytical precision evidencedin Tables 1 and 2. The isotope dilution method was used to quantify the PCDD and PCDF levels; therefore, the values given in Table 1 are inherently corrected for procedural losses incurred during dialysis and during all procedural steps thereafter. The 13C-labeledsurrogates shown in Table 2 were present to aid in the identification and quantitation of all of the native congenersshown in Table 1except 1,2,4,7,8-PeCDD. No 13C-labeledsurrogate for this compound was present, and quantitation of this congener was based on an external standard. (Native 1,2,4,7,8-PeCDDwas historically included
TABLE 1
Concentrations (pgl4-SPMD Sample) of Specifically Targeted PCDDs and PCDFs in SPMDs from Bayou Meto and Results from H411E Bioassay (TCDD=EQISample) upstream of tributary (site 1; n = 4)
downstream from tributary (site 2 n = 4)
field blanks ( n = 2)
(I-TEQjd
(I-TEQ)
(I-TEQ)
