Arch. Environ. Contam. Toxicol. 51, 337–346 (2006) DOI: 10.1007/s00244-005-0162-0

Rapid Method for Determination of Dioxin-Like Polychlorinated Biphenyls and Other Congeners in Marine Sediments Using Sonic Extraction and Photodiode Array Detection J. Buzitis, G. M. Ylitalo, M. M. Krahn Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, United States Department of Commerce, 2725 Montlake Blvd. East, Seattle, WA 98112-2097, United States

Received: 30 June 2005 /Accepted: 11 January 2006

Abstract. A rapid method has been developed to measure dioxin-like polychlorinated biphenyl (PCB) congeners as well as other selected PCBs in sediment. The analytes were extracted from sediment by sonication with dichloromethane, and the PCBs were separated from interfering compounds on a gravityflow cleanup column packed with acidic, basic, and neutral silica gels eluted with 1:1 hexane:pentane (v/v). Subsequently, the dioxin-like PCB congeners were resolved from nonplanar PCBs and other chlorinated compounds by high-performance liquid chromatography (HPLC). Two important advantages of PDA over conventional UV detection are the ability to identify individual analytes by comparing their UV spectra with those of reference standards and the ability to establish the spectral homogeneity (purity) of the analytes by comparing spectra within a peak to the apex spectrum. The HPLC-PDA method was tested with reference and marine sediment samples. Concentrations of selected dioxin-like PCBs, selected nonplanar PCBs, and summed PCBs in sediments and National Institute of Standards and Technology standard reference materials determined by our rapid HPLC-PDA method were comparable with the levels in the same samples analyzed by alternative comprehensive methods (i.e., gas chromatography–electron capture detection or high-resolution gas chromatography–high-resolution mass spectrometry).

Organochlorine contaminants (OCs; e.g., polychlorinated biphenyls [PCBs], DDTs) are among the most widespread and refractory contaminants in the marine environment. OCs possess certain chemical properties (e.g., stability, low flammability, solubility in organic media) that not only make them practical industrial compounds but also highly persistent and bioaccumulative in aquatic animals (Beeton et al. 1979). Consequently, OCs are ubiquitous environmental contaminants and are the focus of many studies that demonstrate their toxicity to marine animals (Brinkman and deKok 1980; Dobson

Correspondence to: J. Buzitis; email: [email protected]

and van Esch 1993; Arctic Monitoring and Assessment Programme 2004). Because of the complex nature of marine sediments, many methods used to measure OCs in sediments are tedious, require large amounts of solvents for extraction and cleanup, and use costly and specialized detection methods (e.g., high-resolution gas chromatography [HRGC] with high-resolution mass spectrometry [HRMS] detection; MacDonald et al. 1997; Numata et al. 2003). A few methods (Szostek et al. 1999; Johnson et al. 2001) are currently being used to rapidly extract and analyze for selected OCs in sediments, but these rapid methods either do not provide specific information about the contaminants present (e.g., individual PCB congeners), or they employ expensive detection instruments. Thus, low-cost methods are needed to allow analyses of a large number of sediment samples in a short period of time. Some of the most toxic OCs routinely measured in samples from the marine environment include certain polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzo-p-furans (PCDFs), and the dioxin-like PCB congeners. Although the dioxin-like PCBs are not as toxic as some of the PCDDs and PCDFs (Van den Berg et al. 1998), these PCB congeners are often found in much higher concentrations in the marine environment than the dioxins or furans and may contribute the majority of the toxicity in marine samples (Koistinen 1990; Smith et al. 1990; Petreas et al. 1992; deBoer et al. 1993; Loganathan et al. 1995). Therefore, it is important to be able to measure these dioxin-like PCBs as well as other toxic PCBs in environmental samples, including sediments and tissues of biota. In the literature, numerous methods can be found for measuring PCBs in sediments or soils. Some are detailed methods that provide data for all 209 PCB congeners or have very low detection limits (Hartmann et al. 2004; Koh et al. 2004; Zhang et al. 2004; Nakata et al. 2005). These methods, however, usually have extensive extraction and cleanup procedures (e.g., 8 to 20 hours Soxhlet extraction and multiple cleanup columns), employ expensive instruments (e.g., HRGC-HRMS), or require large sample sizes (e.g., 10 to 200 g). In addition, rapid methods (e.g., ELISA assay, supercritical fluid extraction [SFE; microwave extraction] have been developed to measure PCB congeners and use far less complex extraction or cleanup

J. Buzitis et al.

338

steps and less-expensive equipment. However, some of these methods (e.g., ELISA assays) provide information on the total toxic equivalents but do not provide detailed concentration data for individual PCB congeners or other compounds that contribute to the TEQ values (Besselink et al. 2004). The microwave and automated solvent extractor methods (Numata et al. 2003; Aries et al. 2004) simplify the extraction process but continue to require extensive cleanup procedures and use expensive instruments for detection (HRGC-HRMS). The SFE methods (Szostek et al. 1999; Nilsson et al. 2002) simplify extraction and cleanup but again use expensive detectors or very large sample sizes (up to 200 g). For tissues of marine biota, a rapid method has been developed to measure dioxin-like PCBs and other selected OCs (including other PCB congeners, DDTs, and hexachlorobenzene) by high-performance liquid chromatography (HPLC) with photodiode array detection (PDA) (Krahn et al. 1994). Large numbers of tissue samples—even small tissue samples (0.3 to 5 g)—can be analyzed relatively quickly using this method. This rapid method has been shown to successfully resolve many individual congeners, including the dioxin-like congeners 77, 126, and 169. Summed concentrations of PCBs and DDTs can also be measured, and these sums have been shown to compare well with those determined in the same samples by gas chromatography (GC)– electron capture detection (ECD) or GC–mass spectrometry (MS). If concentrations of additional non–dioxin-like PCB congeners and pesticides are needed, a subsample of the tissue extract can be set aside and analyzed by GC-MS (Ylitalo et al. 2005). In this article, we describe a new method for the rapid analysis of dioxin-like and other PCBs in marine sediments that is essentially a modification of extraction and analysis methods described in Krahn et al. (1991 and 1994, respectively). In doing so, the advantages of rapid and cost-effective analyses have been retained, allowing a large number of samples, including ones with small sample sizes (<5 g), to be analyzed in a relatively short time (e.g., 14 field samples and 2 quality assurance samples in 24 hours). This modified HPLCPDA method was validated against three NIST standard reference materials (SRMs) and tested with marine sediments collected from urban and nonurban sites along the coasts of the contiguous United States and demonstrated that concentrations of the OCs determined by this rapid method agreed well with those determined by more laboriously and costly standard analytic methods (e.g., HRGC-HRMS and GC-ECD).

Materials and Methods Chemicals and Standard Reference Materials Stock solutions of the surrogate standard 1,2,3,4-tetrachlorodibenzop-dioxin (1,2,3,4-TCDD), HPLC recovery standard 1,7,8-trichlorodibenzo-p-dioxin (1,7,8-TriCDD), and individual PCB congeners were purchased from AccuStandard (New Haven, CT), all in hexane and all at concentration of 100 ng/lL. Potassium hydroxide and HPLC-grades of hexane, dichloromethane, and acetone were obtained from J. T. Baker (Phillipsburg, NJ). Concentrated sulfuric acid and HPLC-grade pentane were obtained from Fisher Scientific (Pittsburgh, PA). Silica gel 60 (70- to 230-mesh American Society for

Testing and Materials [ASTM]) was obtained from Bodman (Aston, PA) and sodium sulfate from Mallinckrodt (Paris, KY). Three sediment SRMs (1941, 1941a, and 1944) were purchased from the National Institute of Standards and Technology (NIST; Gaithersburg, MD). Basic silica gel (36% w/w potassium hydroxide–impregnated silica gel) and acidic silica gel (40% w/w sulfuric acid–impregnated silica gel) were prepared as described (Lebo et al. 1989; Smith et al. 1984; Smith et al. 1990).

Sediment Sample Collection Sediment samples were collected from various urban and reference sites along the United States Atlantic, Gulf of Mexico and Pacific coasts during the summers of 1992, 1994, and 1995. A total of 46 sediments collected from 23 sites were selected for analysis to give a wide range of contaminant levels and sampling locations. At each sampling site, surface sediment (top 2 to 3 cm) was collected with a modified Van Veen grab-sampler (0.1 m2) from three stations (Harmon et al. 1998). For each station, three grab samples were taken; the material was mixed thoroughly and composited. Samples for chemical analysis were stored at )20
C until analyses. The dry weight (dw) of sediments was determined by the method described in Sloan et al. (1993).

Determination of Percent Recoveries of PCBs and Method Detection Limits Using Spiked Sediments A 4-g sample of marine sediment (n = 7) collected from a reference site (Polnell Point, WA) was spiked with 10 ng each of the 15 PCB congeners (PCBs 77, 101, 105, 110, 118, 126, 128, 138, 153, 156, 157, 169, 170, 180, and 189) to establish method detection limits (MDLs) and the percent recoveries of each PCB congener by spike matrix addition. Previous GC-ECD analysis of this sediment showed that it contained nondetectable to trace levels of PCB congeners (unpublished data 1992). Extraction, cleanup, and HPLC-PDA analyses of the spiked sediment samples were conducted as described later. From these data, MDLs (ng/g dw) were calculated according to United States Environmental Protection Agency (EPA) code 40CFR, Part 136, Appendix B.

Extraction by Sonication Modifications were made to a sediment sonication method described in Krahn et al. (1991) to extract dioxin-like PCBs and other selected congeners from sediments. In the current study, sediment was thawed, mixed with a solvent-rinsed metal spatula, and weighed (approximately 4 g) into a tared 100-mL glass centrifuge tube to the nearest 0.01 g. Dichloromethane (20 mL) and the surrogate standard (1,2,3,4TCDD; 20 lL; 100 ng/lL) were added to each sample tube. Sodium sulfate (25g; fired overnight at 700
C) and 20 g copper activated with HCl (Krahn et al. 1988b) were then added to each sample tube. The mixture was stirred with a solvent-rinsed Teflon stir rod until homogeneous. The tubes were placed into an ultrasonic bath and sonicated for 15 minutes at 20
C. The tubes were removed from the bath and centrifuged at 1500 rpm for 2 minutes. Each extract was decanted into a solvent-rinsed 50-mL glass concentrator tube. Another 20 mL aliquot of dichloromethane was added to each sample tube, the mixture stirred with a Teflon rod, and the extraction step repeated. The sample was extracted a third time using 10 mL dichloromethane, and the three sample extracts were combined. A Teflon boiling chip was added to each tube, and the sample extract was concentrated to approximately 1mL on a Kontes tube heater.

339

Dioxin-Like PCBs and Other Congeners in Marine Sediments

Cleanup of Sediment Extracts The sample cleanup method for tissues, described by Krahn et al. (1994), was modified for cleanup of marine sediment extracts. The cleanup column (Fig. 1) was rinsed three times with 50:50 hexane:pentane (v/v) and air-dried before use. The following were added sequentially to each column (All World Scientific, Lynnwood, WA): a plug of glass wool (prefired in a muffle furnace at 400
C overnight and cooled to room temperature), neutral silica gel (2.0 g), basic silica gel (2.5 g), and acidic silica gel (15 g). The packed column was washed with 30 mL 50:50 hexane:pentane (v/v) to condition the silica. Each sample extract was quantitatively loaded onto the cleanup column by transferring the extract with a glass Pasteur pipet, rinsing the 50-mL concentrator tube with 1 to 2 mL dichloromethane, and adding the rinsate to the cleanup column. After the entire sample extract was loaded onto the acidic silica, 1 to 2 mL 50:50 hexane:pentane (v/v) was used to rinse the inside wall of the cleanup column and then allowed to stand for 15 minutes (stop-flow technique). The PCB congeners were eluted from the cleanup column with 45 mL 50:50 hexane:pentane (v/v) and collected into a solvent-rinsed 50-mL glass concentrator tube. Activated copper (1 g) and a Teflon boiling chip were added to the extract tube, and the extract was concentrated to approximately 1 mL on a Kontes tube heater. The HPLC recovery standard (1,7,8-TriCDD; 20lL; 100ng/lL) was added to each sample. The sample extract was transferred to a 2-mL glass vial, and the extracts were concentrated under nitrogen gas to 125 lL. The sample extract was transferred to a 4-mL amber vial with a 250-lL insert and analyzed by HPLC-PDA.

HPLC Calibration Standards and Internal Standards Five multilevel calibration standards were prepared in hexane and contained the following compounds: 1,7,8-TriCDD; 1,2,3,4-TCDD; and PCB congeners 77, 101, 105, 110, 118, 126, 128, 138, 153, 156, 157, 169, 170, 180, and 189. The linear range for the HPLC-PDA was determined by using a five-point calibration curve created from these standards where the injected on column mass of PCBs ranged from 1 to 625 ng, and internal standard on the column mass was held constant at 600 ng. The midlevel standard (1 ng/lL; 25lL injection) was used as a single-point calibration to quantitate all PCB concentrations. The surrogate standard used to quantitate analyte concentrations was 1,2,3,4-TCDD, and the recovery standard used to quantitate the percent recovery of surrogate standard was 1,7,8-TriCDD.

HPLC-PDA Analyses The sediment extracts were analyzed for dioxin-like PCBs and other selected congeners on Cosmosil PYE analytic columns (4.6 mm · 250 mm; 5 lm particles; Nacalai Tesque, Inc. Kyoto, Japan; obtained through Phenomenex, Torrance, CA) cooled to 16
C and measured by ultraviolet (UV) photodiode array (PDA) detection (Krahn et al. 1994). Concentrations of summed PCBs (SPCBs) were calculated using the method reported in Ylitalo et al. (1999). Levels of individual PCB congeners and summed PCBs were reported as ng/g dw.

Fig. 1. Diagram of the gravity-flow cleanup column used to isolate PCBs from sediment extracts

response factor of any PCB congener that exceeded € 15% of the initial midlevel calibration value or if there was a major modification to the HPLC system. Acceptance criteria for analyses of NIST SRM 1944 were similar to those NIST uses for its Intercomparison Exercises (i.e., concentrations of individual analytes were within +35% from the upper and –35% from the lower limits of the 95% confidence interval of NIST-certified concentrations). Other data acceptance criteria were replicate analyses of selected samples had to be within 35% of the relative standard deviation (RSD), and surrogate recoveries in all samples had to be within 60% to 130%.

PCB Determination by GC-ECD Sediment samples were extracted and quantitated using the GC-ECD method reported previously (Krahn et al. 1988a; Krahn et al. 1988b; Sloan et al. 1993). Identification of individual PCBs was confirmed using GC-MS. The SPCB values were calculated by summing the concentrations of 17 PCB congeners (18, 28, 44, 52, 66, 101, 105, 118, 128, 138, 153, 170, 180, 187, 195, 206, and 209) and multiplying the sum by two (Lauenstein et al. 1993). The SPCB concentrations were reported as ng/g dw.

Quality Assurance PCB Determination by HRGC-HRMS Quality-assurance (QA) procedures included analyses of NIST SRMs, method blanks, and replicate samples using certified calibration standards. The HPLC-PDA was calibrated with a series of multilevel PCB standards. The multilevel curves were repeated if the daily, single-point calibration (the midlevel multilevel standard) had a

As part of a comparability study, three sediment samples collected from an urban site (Puget Sound, WA) were analyzed by the current HPLC-PDA sediment method as well as by Axys Analytical Services employing EPA method 1668 using HRGC-HRMS.

J. Buzitis et al.

340

Table 1. Mean percent recoveries (€ SD) and MDLs for 15 PCB congeners measured by HPLC-PDA sediment method using spike matrix additiona Congneers

Mean € SD (n = 7) (%) Recovery

Dioxin-like PCB congeners 77 105 118 126 156 157 169 189 Non–dioxin-like PCB congeners 101 110 128 138 153 170 180 Summed PCBs

RSD

MDL ng/g, dw

99 96 100 86 98 97 98 100

€ € € € € € € €

3.5 3.7 5.2 4.0 3.4 2.6 4.1 5.4

3.6 3.9 5.1 4.6 3.5 2.7 4.2 5.2

0.58 0.59 0.77 0.60 0.49 0.40 0.61 0.79

100 96 120 100 110 100 100

€ € € € € € €

2.2b 2.2 3.4c 3.0b 2.0b 3.6 2.2

2.2 2.3 2.9 2.9 1.7 3.4 2.1

0.35 0.78 0.42 0.48 0.35 0.56 0.34 3.5 € 3.0

a Spike matrix samples were prepared by adding 10 ng each PCB congener 4 g of clean reference sediment. b Previous analyses by GC-ECD measured detectable concentrations of these analytes in the sediment sample. c A known coelution on HPLC-PDA interfered with this component.

Statistical Analysis Concentrations of PCBs were log transformed to increase the homogeneity of variances. The relations of individual PCB concentrations determined by HPLC-PDA and GC-ECD were assessed by analysis of variance and simple linear regression (Zar 1984). All statistical analyses were completed using JMP Statistical Software (SAS Institute, Cary, NC).

Results The percent recoveries (mean € SD) of the 15 PCB congeners added to clean marine sediment (from Polnell Point, WA) are listed in Table 1. The mean recoveries ranged from 86% to 120%, and the RSDs (n = 7) for all PCB congeners were £ 5.2%. Percent recoveries of PCBs 101, 138, and 153 were likely >100% because trace levels of these analytes had been previously measured by both HPLC-PDA and GC-ECD in this sediment (data not shown), but the sediment did not contain detectable levels of the other congeners as determined by both of these methods. The high percent recovery of PCB 128 (120%) was most likely caused by coelution with another PCB congener or an unknown interfering compound on the Cosmosil PYE column, as indicated by poor peak purity and spectral library match on the PDA. Table 1 also shows the MDLs as calculated using EPA method in code 40CFR Part 136, Appendix B. To validate the HPLC-PDA sediment method, replicate samples of three NIST sediment SRMs (1941, 1941a, and

1944) were analyzed for PCBs by the HPLC-PDA sediment method, and the results were compared with NIST-certified or published values as well as with values obtained by GC-ECD (Krahn et al. 1988a; Krahn et al. 1988b; Sloan et al. 1993). In general, the PCB data (based on dw) obtained by HPLC-PDA were in good agreement with the NIST-certified values as were the GC-ECD results (Table 2). However, some congeners and pesticides are known to coelute on the HPLC system (e.g., PCBs 90/95/99/101/149; PCBs 87/153/dieldrin/transnonachlor; PCBs 170/194; and PCBs 123/128) (Krahn et al. 1994). We report the concentrations of these coelutions as if the concentration were from only the major congener of interest (e.g., PCBs 101, 128, 153, and 170). This causes the concentrations measured by HPLC-PDA to be higher than those measured by GC-ECD or other detailed methods. As listed in Table 2, NIST also measured several of these coeluting congeners for the SRMs. When available, the values reported by NIST were summed and compared with the HPLC-PDA values. The GC-ECD method did not report these additional congeners. SRMs 1944 and 1941a reported 10 congeners that also were analyzed by HPLC-PDA, and SRM 1941 reported 7 congeners. The concentrations measured by HPLC-PDA showed that 8 of the 10 congeners for SRMs 1944 and 1941a were within the QA limits set for both the HPLCPDA and GC-ECD methods. For SRM 1941, 5 of the 7 congeners were within the QA limits for both methods (see Quality Assurance in Materials and Methods). The non-ortho– substituted PCBs (e.g., 77, 126, and 169) in the SRMs were not detected by the HPLC-PDA sediment method nor were they reported for either the GC-ECD method or by NIST. The chromatogram in Figure 2 (SRM 1944) shows good separation between the dioxin-like PCB congeners and other chlorinated compounds. Forty-six field sediment samples were analyzed by both HPLC-PDA and GC-ECD to further demonstrate the performance of this rapid HPLC-PDA sediment method for PCB quantitation. Eight of the 15 congeners measured by both methods were found in all 46 samples. In general, concentrations of these 8 congeners, as well as summed PCB values, were correlated over a wide range of analyte concentrations between both methods (Fig. 3). Despite some observed bias between the methods, especially at low analyte concentrations (<10 ng/g dw), levels of various PCBs measured by HPLCPDA and GC-ECD were significantly correlated. For example, concentrations of PCBs 138 (r2 = 0.804; p < 0.0001), 105 (r2 = 0.614; p < 0.0001), 128 (r2 = 0.667; p < 0.0001), and SPCBs (r2 = 0.912; p < 0.0002), as determined by HPLC-PDA, were significantly correlated with the concentrations measured by GC-ECD. Concentrations of PCB 170/194 determined by HPLC-PDA and PCB 170/190 by GC-ECD were also significantly correlated (r2 = 0.129; p < 0.0083), but this relation was not as strong as the previously mentioned PCBs, presumably because of the coelution of different congeners with PCB 170 by the two methods. Concentrations of all 15 individual PCB congeners and SPCBs measured by both HPLC-PDA and GCECD in a subset (n = 5) of the 46 field sediments collected from various sites are listed in Table 3. In addition, levels of selected PCB congeners, as well as SPCBs in three sediment samples determined by HPLC-PDA, were compared with values measured by HRGC-HRMS (EPA method 1668) (Table 4). Concentrations of most PCBs

2 9.0 2.1 5.1 16 1.9 5.0 160

90 € 6.0

€ € € € € € € €

230 79 23 54 200 23 40 1,700

0.7 3.5 9.4

2.8 5.0

< 0.52 28 € 56 € < 0.48 7.1 € 43 € < 1.2 35 €

b

b

21 € 2.2 57 € 1.5 75 € 4.5 2.7 € 0.3 36 € 0.7 1,400 € 3.6 51.6 100 € 0.7

b

b

b

€ € € € € € €

b

b

220 63 8.5 62 110 22 44

b

b

66 € 1.8

b

3.8 1.7 0.1 1.3 2.6 0.6 0.6

6.5 € 0.3

b

24 € 0.6 58 € 1.9

b

b

b

17 € 1.3 46 € 1.0

NIST-Certified Valuesd

€ € € € € € € €

5.9 1.6 03 1.1 4.5 0.4 1.3 33

93 € 1.5

81 27 9.5 17 66 9.3 18 460

c

< 0.51

c

< 0.23 7.5 € 0.6 19 € 0.7 < 0.21 1.7 € 0.1

HPLC-PDAd (n = 3)

GC-ECDd (n = 3)

HPLC-PDAd (n = 8)

€ 1.2 € 2.0 € 0.8 € 2.0 € 1.3 € 55 50.3 88 € 2.8

3.5 23 33 4.2 16 420

b

22 € 4.4

b

b

b

b

b

4.5 € 0.8 14 € 3.1

b

GC-ECDd (n = 2)

€ € € € b

b

25 24 7.3 14

b

b

1.8 0.5 0.26 0.3

44 € 0.7

b

b

b

b

b

5.8 € 0.23 15 € 0.7

b

Accepted NIST Valuesa,d

€ € € € € € € €

5.7 4.2 2.1 2.2 5.3 1.8 2.5 47 98 € 1.8

47 16 6.5 10 35 6.4 7.9 380

c

< 0.56

c

< 0.25 5.3 € 1.3 13 € 3.8 <0.23 0.80 € 0.03

HPLC-PDAd (n = 3)

€ 2.0 € 1.7 € 1.8 € 1.1 € 2.0 € 32 48.3 91 € 5.1

2.9 16 20 4.7 10 310

b

18 € 1.4

b

b

b

b

b

3.1 € 0.4 9.5 € 1.9

b

GC-ECDd (n = 23)

NIST SRM 1941a

b

b

32 € 1.1 9.5 € 0.85 1.9 € 0.32 13 € 0.97 27 € 0.74 4.8 € 0.34 5.8 € 0.58

b

b

b

0.93 € 0.14

b

3.6 € 0.27 10 € 1.1

b

NlST-Certified Valuesd

b

NIST values are accepted for PCBs in this SRM because it was developed for PAH analyses. Not reported. c Peak detected at analyteÕs retention time on HPLC-PDA did not match a PCB library spectra; therefore, it was not reported. d Coeuting compounds were present that affected congener quantitation. See individual congeners for coelutions. When reported by other methods, cocluting congeners were summed. e Results are reported as: HPLC-PDA ¼ 105/167/79. f Results are reported as: HPLC-PDA ¼ 157/127. g Results are reported as: HPLC-PDA ¼ 101/90/95/99/149; GC-ECD ¼ 101/90; SRM 1944 ¼ 101/90/95/99/149; SRM 1941 ¼ 101/90/66/95; SRM 1941a ¼ 101/95/99/149. h Results are reported as: HPLC-PDA ¼ 110/129. i Results are reported as: HPLC-PDA ¼ 128/123/unknown coeluting compound. j Results are reported as: GC-ECD, SRM 1944, SRM 1941, SRM 1941a ¼ 138/163/164. k Results are reported as: HPLC-PDA, SRM 1941a ¼ 153/87/dieldrin/transnonachlor; GC-ECD 153/132; SRM 1944 ¼ 153/87/transnonachlor; SRM 1941 ¼ 153/dieldrin/transnonachlor. l Results are reported as: HPLC-PDA ¼ 170/194; GC-ECD, SRM 1944, SRM 1941a ¼ 170/190; SRM 1941 ¼ 170/190/194. m Results are reported as: GC-ECD ¼ 180/unknown coeluting compound. n SPCBs measured by HPLC-PDA were calculated using method described in Ylitalo et al. 1999. o SPCBs measured by GC-ECD were calculated using method described in Sloan et al. 1993.

a

Dioxin-like PCB congeners 77 105e 118 126 156 157f 169 189 N–dioxin-like PCB congeners 101g 110h 128i 138j 153k 170l 180m Summed PCBsn,o DW (%) Recovery Surrogate Standard (%)

Cogneners

NIST SRM 1941

NIST SRM 1944

Mean concentration € SD (ng/g, dw)

Table 2. Concentrations of selected PCB congeners measured by HPLC-PDA or GC-ECD and National Institute of Science and Technology certified values in three NIST SRMs

Dioxin-Like PCBs and Other Congeners in Marine Sediments 341

J. Buzitis et al.

PCB 156

PCB 77

342

17

5

10

23

SS

RS

PCB 118

PCB 128

21

19

PCB 170 PCB 105

PCB 138 PCB 180

PCB 153 PCB 110

AU

15

RS

15

20 Time (min.)

25

30

35

RS Recovery Standard SS Surrogate Standard

Fig. 2. HPLC chromatogram at 220 nm obtained from NIST SRM 1944 sediment analyzed by HPLC-PDA sediment method (see Materials and Methods section for details)

measured by both methods were in good agreement. The percent difference between the two methods ranged from 0% to 40% for each of the congeners measured by both HPLC-PDA and HRGC-HRMS. The mean percent difference for each sample overall was 10% for S11 and S12 and 20% for B2. PCB 77 was measured in all the samples by both methods, but the levels measured by HRGC-HRMS were somewhat higher than those determined by HPLC-PDA. In contrast, PCB 126 was measured by HRGC-HRMS but was not detected by HPLC-PDA. PCB 169 was not detected in any of the samples by either method.

Discussion An HPLC-PDA sediment method was developed to rapidly analyze a large number of sediment samples for dioxin-like and other selected PCB congeners in a relatively short time and at a lower cost than more detailed methods (e.g., GC-MS). Using this HPLC-PDA method, one person could extract, cleanup, and perform PCB congener analysis on 16 samples in a 24-hour time period. In comparison, the GC-ECD method used for the validation of the HPLC-PDA method would require 85 hours to analyze the same samples. The HPLC-PDA method also uses a much smaller sample mass (<5 g) than other reported methods (10 to 200 g) (Nilsson et al. 2003; Numata et al. 2003; Besselink et al. 2004) yet yields congenerspecific data as well as SPCB values. The data can then be used directly to assess contamination or be used to prioritize samples for further characterization by other methods. This allows flexibility so that accurate data are available rapidly to allow decisions to be made in near real-time (e.g., for site characterization and remediation planning).

The HPLC-PDA sediment method accurately measures concentrations of individual dioxin-like PCB congeners as well as several additional non–dioxin-like PCBs that are not possible to determine using some detailed analytical methods. For example, dioxin-like PCBs 77, 126, 156, 157, 169, and 189 have known coelutions on the DB5 GC column in the GC-ECD method. Therefore, this HPLC-PDA method can more accurately measure concentrations of dioxin-like PCB congeners so that toxic equivalent values in sediments can be determined. Another advantage of the sediment HPLC-PDA method is that the spectral data obtained by the photodiode array detector, together with the associated software, allows the identity and purity of each analyte peak to be determined (Krahn et al. 1994). Peak identity is confirmed by matching the spectra of the analyte peak with that of a standard in a reference library. Peak purity is obtained by comparing all spectra across the analyte peak to that of the apex spectrum. Both of these comparisons allow the analyte peak to be identified and measured accurately. Although some of congeners have coelutions (e.g., PCBs 90/95/99/101/149) that affect the purity of the peak for that individual congener, this does not affect the sum of PCBs values. When this occurs in a field sample, the congener value is flagged and reported as an estimate. Several challenges were encountered while developing the HPLC-PDA sediment method. For example, aromatic compounds (ACs) and sulfur, which are frequently found in marine sediments, can potentially interfere with PCB quantitation on the HPLC-PDA system. Sediments from urban sites contained much higher concentrations of ACs than were encountered for most tissues of marine biota collected from the same or nearby sites. Although interferences and biogenic material (e.g.,

343

Dioxin-Like PCBs and Other Congeners in Marine Sediments

Fig. 3. Comparison of values (ng/g dw) obtained for 46 field sediment samples analyzed by both HPLC-PDA and GC-ECD

lipids) could be removed from tissues using a small-bore (9mm diameter) gravity-flow cleanup column according to the method of Krahn et al. (1994), ACs in extracts of urban sediments could not be adequately removed using the same-size cleanup column. As a result, the amount of each silica gel layer in the cleanup column was increased by a factor of five, requiring the use of a larger bore column with a 22-mm diameter. Although ACs were adequately removed in most sediments using this cleanup modification, not all ACs were removed from highly contaminated sediments. Sediment extracts eluted much faster through the larger-bore cleanup columns compared with the smaller-diameter tissue columns. Therefore, interfering compounds were not in contact with the sulfuric acid phase long enough to completely digest them, allowing ACs to elute off the cleanup column. Thus, the dwell time of the extract on the acidic silica gel was increased to 15 minutes by using a stop-flow technique (see Materials and Methods for details), and then the analytes were eluted from the column with the remaining volume of eluent. Using this technique, ACs were completely removed from all sediment extracts, therefore producing an extract suitable for HPLCPDA analysis.

Sulfur was also often found in relatively high concentrations in marine sediments. Although the stop-flow technique removed the ACs from sediment extracts, it did not eliminate sulfur interferences. To remove the sulfur, activated copper was added during the concentration step after cleanup. The additional copper effectively scavenged residual sulfur from the extract, thus removing the interfering sulfur peak (retention time 7 to 9 minutes) from the chromatograms. For sediment samples, poor recoveries (<20%) were obtained for certain pesticides (e.g., p,p¢-DDT, o,p¢-DDT), as well as for the surrogate standard (1,7,8-TriCDD) when initially attempting to use the same extraction solvent (50:50 pentane/ hexane, v/v) that was used in the tissue HPLC-PDA method (Krahn et al., 1994). As a result, dichloromethane was tried as an alternative extraction solvent for sediments to determine if recoveries could be increased. The change in solvent did increase pesticide recoveries, but it did not increase the recovery of the surrogate standard. Because 1,7,8-TriCDD was fully eluted from the smaller-bore cleanup column used for the tissue method, its failure to elute from the large-bore sediment column could be attributed to either the increase in the amount of silica in each layer of the cleanup column or the stop-flow

J. Buzitis et al.

344

Table 3. Concentrations of selected dioxin-like PCBs and other PCB congeners determined by HPLC-PDA and GC-ECD in marine sediments collected from various regions of the U.S Concentration (ng/g, dw) Arroyo City Arroyo Colorado, TX Congeners Dioxin-like PBC Congeners 77 105 118 126 156 157 169 189 Non–dioxin-like Congeners 101 110 128 138 153 170 180 Summed PCBs Recovery surrogate standard Sample size (g) DW (%) a b c

Seal Beach San Pedro Bay, CA

Gould Canal Biscayne Bay, FL

Quincy Bay Boston Harbor, MA

Miami River Biscayne Bay, FL

HPLC-PDA GC-ECD HPLC-PDA GC-ECD HPLC-PDA GC-ECD HPLC-PDA GC-ECD HPLC-PDA GC-ECD <0.25 < 0.21 < 0.2 < 0.23 < 0.2 < 0.18 < 0.57 < 0.26 < 0.2 < 0.21 < 0.22 < 0.22 < 0.21 < 0.2c < 0.19 2.5 84 4.06 49.0

a

<0.2 < 0.3 a a a a a

0.31 a

0.21 0.31 < 0.3 0.21 < 0.2 3.0 100 10.1 47.8

< 0.23 0.96 1.7 < 0.21 < 0.18 < 0.17 < 0.51 < 0.24 3.0b 1.8 1.2b 1.9 3.2b 0.45c 2.0 37 82 4.03 76.1

a

0.40 2.0 a a a a a

2.0 a

0.40 2.0 3.0 0.70 1.0 30 97 10.2 74.9

< 0.4 3.6 8.6 < 0.37 0.45 < 0.28 < 0.89 <0.4 21b 14 2.7b 5.0 12b 2.7c 3.3 110 85 4.03 32.0

a

< 0.35 12 23 < 0.31 2.2 < 0.24 < 0.74 < 0.33

1.5 7.3 a a a a a

8.6 a

1.2 9.0 10 1.6 3.2 140 80 10.2 31.4

49b 17 7.9b 18 41b 8.0c 12 280 86 4.03 38.6

a

8.0 21 a a a a a

17 a

6.0 30 36 6.0 18 400 84 10.2 37.8

a

< 0.18 19 35 < 0.16 3.6 16 < 0.4 < 0.18

12 29 a a a a a

130b 36 23b 35 120b 34c 46 780 84 4.08 57.7

46 a

4.4 54 84 1.9 41 760 75 10.2 55.4

Not reported. Coeluting PCBs were present that affected the HPLC quantitation (increased the value; see Materials and Methods, HPLC-PDA analyses). HPLC-PDA results are reported as PCBs 170/194.

Table 4. Concentrations of dioxin-like PCBs and other PCB congeners in sediment samples analyzed by HPLC-PDA and HRGC-HRMS Concentration (ng/g, dw) S11 08/24/99 Congeners Dioxin-like PCB congeners 77 105 118 126 156 157 169 189 Non–dioxin-like PCB congeners 101 110 128 138 153 170 180 Summed PCBs a

S12 08/26/99

B2 08/22/99

HPLC-PDA

HRGC-HRMS

HPLC-PDA

HRGC-HRMS

HPLC-PDA

HRGC-HRMS

2.2 21 70 < 0.28 7.3 1.9 < 0.69 0.53

3.3 21 72 0.096 8.5 1.6

1.8 21 68 < 0.3 6.3 1.6 < 0.72 0.44

2.5 19 69 0.1 7.2 1.4

1.4 15 53 < 0.34 4.2 1.2 < 0.81 0.38

1.5 12 44 0.049 5.1 0.80

260b 100 29b 62 160b 25c 38 1,400d

a

0.52 a a a a a

14 35 720e

240b 93 23b 56 150b 21c 32 1,200d

a

0.48 a a a a a

14 28 680e

180b 93 29b 45 100b 20c 35 1,000d

a

0.56 a a a a a

13 33 380e

Not reported. Coeluting PCBs were present that affected the HPLC quantitation (increased the value; see Materials and Methods, HPLC-PDA analyses). c HPLC-PDA results are reported as PCBs 170/194. d SPCBs measured by HPLC-PDA were calculated using method described in Ylitalo et al. 1999. e SPCBs were calculated by summing Aroclor equivalents (EPA Method 1668). b

Dioxin-Like PCBs and Other Congeners in Marine Sediments

technique. After testing the two internal standards (i.e., 1,7,8TriCDD, 1,2,3,4-TCDD) individually, it was found that 1,7,8TriCDD was retained more strongly by the larger silica phases of the large-bore cleanup column. In contrast, 1,2,3,4-TCDD eluted quantitatively within the 45-mL collection volume. Therefore, to obtain good surrogate recoveries, the roles of the two internal standards were reversed from the PCB tissue method (i.e., 1,2,3,4-TCDD was used as the surrogate standard, and 1,7,8-TriCDD, which was added to the sample after the cleanup step, was used as the recovery standard). In summary, a rapid, cost-effective method has been developed to measure dioxin-like PCBs, SPCBs, and other PCB congeners in sediments. There was good agreement between the data from the HPLC-PDA method and that obtained from the GC-ECD or HRGC-HRMS methods. Identity and purity of all analyte peaks could also be determined with the HPLC-PDA sediment method. Finally, dioxin-like PCB congeners could be analyzed with sufficient accuracy and sensitivity to enable TEQ concentrations to be determined.

Acknowledgments. We appreciate the technical assistance or advice of Donald Brown, Jennie Bolton, Daryle Boyd, Richard Boyer, Catherine Sloan, Larry Hufnagle, Sena Camarata, and Carol Pitzel. We thank Erika Hoffman from the United States EPA for providing data from HRGC-HRMS analyses, Catherine Sloan and Jennie Bolton for providing GC-ECD data, and Jim Meador for assistance with the statistical analysis and interpretation. We appreciate the helpful suggestions and manuscript review by Doug Burrows and Dave Herman.

References Arctic Monitoring and Assessment Programme (2004) AMAP assessment 2002: Persistent organic pollutants in the Arctic. Arctic Monitoring and Assessment Programme, Oslo, Norway, p xvi Aries E, Anderson DR, Ordsmith N, Hall K, Fisher R (2004) Development and validation of a method for analysis of ‘‘dioxin-like’’ PCBs in environmental samples from the steel industry. Chemosphere 54:23–31 Beeton AM, Allen AR, Andren AW, Cristol SJ, Durfee RL, Freeman AM et al. (1979) Polychlorinated biphenyls. National Research Council, Washington, DC Besselink HT, Schipper C, Klamer H, Leonards P, Verhaar H, Felzel E, et al. (2004) Intra- and interlaboratory calibration of the DR Calux bioassay for the analysis of dioxins and dioxinlike chemicals in sediments. Environ Toxicol Chem 23:2781– 2789 Brinkman UAT, deKok A (1980) Production, properties and usage. In: Kimbrough RD, (ed) Topics in environmental health. Halogenated biphenyls, terphenyls, naphthalenes, dibenzodioxins and related products. Amsterdam, The Netherlands, Elsevier/North Holland Biomedical Press, p 1 deBoer J, Stronck CJN, Traag WA, Meer JVD (1993) Non-ortho and mono-ortho substituted chlorobiphenyls and chlorinated dibenzop-dioxins and dibenzofurans in marine and freshwater fish and shellfish from The Netherlands. Chemosphere 26:1823–1842 Dobson S, van Esch GJ (1993) Polychlorinated biphenyls and terphenyls, 2nd ed. Environmental Health Criteria 140. World Health Organization, Geneva, Switzerland Harmon MR, Gottholm BW, Robertson A (1998) A summary of chemical contaminant levels at benthic surveillance project sites (1984–1992). NOAA Technical Memorandum NOS ORCA 124: 2. United States Department of Commerce, Silver Spring, MD

345

Hartmann PC, Quinn JG, Cairns RW, King JW (2004) Polychlorinated biphenyls in Narragansett Bay surface sediments. Chemosphere 57:9–20 Johnson JC, Van Emon JM, Clarke AN, Wamsley BN (2001) Quantitative ELISA of polychlorinated biphenyls in an oily soil matrix using supercritical fluid extraction. Anal Chim Acta 428:191–199 Koh C-H, Khim JS, Kannan K, Villeneuve DL, Senthilkumar K, Glesy JP (2004) Polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) and 2,3,7,8-TCDD equivalents (TEQs) in sediment from the Hyeongsan River, Korea. Environ Pollut 132:489–501 Koistinen J (1990) Residues of planar polychloroaromatic compounds in Baltic fish and seal. Chemosphere 20:1043–1048 Krahn MM, Morre LK, Bogar RG, Wigren CA, Chan S-L, Brown DW (1988a) High-performance liquid chromatographic method for isolating organic contaminants from tissue and sediment extracts. J Chromatogr 437:161–175 Krahn MM, Wigren CA, Pearce RW, Morre LK, Bogar RG, MacLeod WDJ, et al. (1988b) Standard analytical procedures of the NOAA National Analytical Facility. New HPLC cleanup and revised extraction procedures for organic contaminants. NOAA Technical Memorandum NMFS F/NWC-153. United States Department of Commerce, Silver Spring, MD Krahn MM, Ylitalo GM, Joss J, Chan S-L (1991) Rapid, semi-quantitative screening of sediments for aromatic compounds using sonic extraction and HPLC/Fluorescence analysis. Mar Environ Res 31:175–196 Krahn MM, Ylitalo GM, Buzitis J, Sloan CA, Boyd DT, Chan S-L, et al. (1994) Screening for planar chlorobiphenyl congeners in tissues of marine biota by high-performance liquid chromatography with photodiode array detection. Chemosphere 29:117–139 Lauenstein GG, Cantillo AY, Dolvin SS (1993) NOAA national status and trend program development and methods. Sampling and analytical methods of the National Status and Trends Program, National Benthic Surveillance and Mussel Watch Projects: 19841992. Volume I. Overview and summary of methods. NOAA Technical Memorandum NOS ORCA 71: 34-38. United States Department of Commerce, Silver Spring, MD Lebo JA, Zajicek JL, May TW (1989) Large-scale preparation of potassium hydroxide-modified silica gel adsorbent. J Assoc of Anal Chem. 72:371–373 Loganathan B, Kannan K, Watanabe I, Kawano M, Irvine K, Kumar S et al. (1995) Isomer-specific determination and toxic evaluation of polychlorinated biphenyls, polychlorinated/brominated dibenzo-p-dioxins and dibenzofurans, polybrominated biphenyl ethers, and extractable organic halogen in carp from the Buffalo River, New York. Environ Sci Technol 29:1832–1838 MacDonald DD, Ikonomou MG, Rantalaine A-L, Rogers IH, Sutherland D, Van Oostdam J (1997) Contaminants in white sturgeon (Acipenser Transmontanus) from the upper Fraser River, British Columbia, Canada. Environ Toxicol Chem 16:479–490 Nakata H, Hirakawa Y, Kawazoe M, Nakabo T, Arizono K, Abe S-I, et al. (2005) Concentrations and compositions of organochlorine contaminants in sediments, soils, crustaceans, fishes and birds collected from Lake Tai, Hangzhou Bay and Shanghai city region, China. Environ Pollut 133:415–429 Nilsson T, Bøwadt S, Bjçrklund E (2002) Development of a simple selective SFE method for the determination of desorption behavior of PCBs in two Swedish sediments. Chemosphere 46:469–476 Nilsson T, Sporring S, Bjçrklund E (2003) Selective supercritical fluid extraction to estimate the fraction of PCB that is bioavailable to a benthic organism in a naturally contaminated sediment. Chemosphere 53:1049–1052 Numata M, Yarita T, Aoyagi Y, Takatsu A (2003) Microwave-assisted steam distillation for simple determination of polychlorinated

346

biphenyls and organochlorine pesticides in sediments. Anal Chem 75:1450–1457 Petreas MX, Wiesmuller T, Palmer FH, Winkler JJ, Stephens RD (1992) Aquatic life as biomonitors of dioxin/furan and coplanar polychlorinated biphenyl contamination in the Sacramento-San Joaquin River Delta. Chemosphere 25:621–631 Sloan CA, Adams NG, Pearce RW, Brown DW, Chan S-L (1993) Northwest Fisheries Science Center organic analytical procedures. In: Lauenstein GG, Cantillo AY (eds) Sampling and analytical methods of the National Status and Trends Program, National Benthic Surveillance and Mussel Watch Projects: 19841992. Volume IV. Comprehensive descriptions of trace organic analytical methods. NOAA Technical Memorandum NOS ORCA 71:53–97. Silver Spring, MD, United States Department of Commerce Smith LM, Stalling DL, Johnson JL (1984) Determination of part-pertrillion levels of polychlorinated dibenzofurans and dioxins in environmental samples. Anal Chem 56:1830–1842 Smith LM, Schwartz TR, Feltz K, Kubiak TJ (1990) Determination and occurrence of AHH-active polychlorinated biphenyls, 2,3,7,8-tetrachloro-p-dioxin and 2,3,7,8-tetrachlorodibenzofuran in Lake Michigan sediment and biota. The question of their relative toxicological significance. Chemosphere 21:1063–1085

J. Buzitis et al.

Szostek B, Tinklenberg JA, Aldstadt JH (1999) A simple method for the quantitative microextraction of polychlorinated biphenyls from soils and sediments. Chemosphere 38:3131–3139 Van den Berg M, Birnbaum L, Bosveld ATC, Brunstrom B, Cook P, Feeley M, et al. (1998) Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife. Environ Health Perspect 106:775–792 Ylitalo GM, Buzitis J, Krahn MM (1999) Analyses of tissues of eight marine species from Atlantic and Pacific coasts for dioxin-like chlorobiphenyls (CBs) and total CBs. Arch Environ Contam Toxicol 37:205–219 Ylitalo GM, Buzitis J, Boyd DT, Herman DP, Tilbury KL, Krahn MM (2005). Improvements to high-performance liquid chromatography/photodiode array detection (HPLC/PDA) method that measures dioxin-like polychlorinated biphenyls and other selected organochlorines in marine biota. In: Ostrander GK (ed) Techniques in aquatic toxicology. Volume 2. Boca Raton, FL, Taylor and Francis, pp 449–464 Zar JH (1984) Biostatistical analysis. 2nd ed. Prentice Hall, Englewood Cliffs, NJ Zhang Z, Huang J, Yu G, Hong H (2004) Occurrence of PAHs, PCBs and organochlorine pesticides in the Tonghui River of Beijing, China. Environ Pollut 130:249–261