Environ. Sci. Techno!. 1992,26,2028-2035
A Method for Estimation of Chlorinated Biphenyls in Surface Waters: Influence of Sampling Method on Analytical Results Joseph H. Hermans, Foppe Smedes,' Johannes W. Hofstraat,+and Wlm P. Coflnoz Tidal Waters Division, P.O.Box 207, NL-9750 AE Haren, The Netherlands
An accurate extraction and measurement procedure to determine chlorinated biphenyls (CBs) in surface waters was developed, and sampling techniques for removing suspended matter (SPM) were investigated. The procedure involved a 10-L batch liquid-liquid extraction directly from the sample bottle to prevent loss due to adsorption to the wall. Exhaustive extraction for recovery measurements was proposed, next to spikes, resulting in an extraction time of 10-45 h. A detection limit lower than 10 pg/kg and a coefficient of variation of 3-9% were obtained. Two sampling techniques to remove SPM were investigated: filtration and continuous-flowcentrifugation. Filter clogging and adsorption affected the accuracy of the filtration technique. Insufficient separation of low-density particulate organic matter in continuous-flow centrifuge determined the CB contents measured in centrifugates. Results using these techniques for distribution or bioavailability studies are questionable because freely dissolved CBs and dissolved organic matter (DOM) bound CBs cannot be distinguished. Centrifugation may be applicable to transport studies.
Introduction The chlorinated biphenyls (CBs) form an important class of contaminants. Because of their persistence, their residence time in the environment is high. The hydrophobic nature of these compounds gives rise to accumulation in sediments and biota. The toxicity of CBs differs for each congener and ranges from highly toxic to moderately toxic (1). The presence of CBs in the aquatic environment causes particular concern. Some contamination scenarios, for instance, indicate a highly negative impact on marine mammals (2, 3). A significant number of studies have been dedicated to CBs. Environmental monitoring programs in aquatic ecosystems almost inevitably include these compounds, with CB measurements mostly in sediments, on suspended particulate matter (SPM), and in organisms. These compartments are chosen because accumulative properties of the CBs produce concentration levels allowing accurate analytical determinations (4-8). Research concentrates on the mechanisms of the distribution of CBs in the environment, on uptake and elimination processes, and on biological effects. For these types of investigations, the availability of an accurate and reliable method for CB determination in the water phase is indispensable. A factor that complicates CB determinations in surface water is their intricate interactions with other substances that occur in this ecosystem. In the water phase, material is present in a wide range of sizes and with many different characteristics. The spectrum encompasses truly dissolved materials like proteins, humic acids, and fulvic acids, generally referred to as dissolved organic matter (DOM), and also colloids and suspended solids (9). In most cases, Present address: AKZO Research Laboratories Arnhem, Corporate Research Laboratory, P.O. Box 9300, NL-6800 SB Arnhem, The Netherlands. Present address: Institute for Environmental Studies, Free University, De Boelelaan 1115, NL-1081 HV Amsterdam, The Netherlands.
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Environ. Sci. Technol., Vol. 26, No. 10, 1992
association of CBs with these substances will be energetically favorable (10, 1I ) . "Dissolved" hydrophobic contaminants like CBs are therefore considered to be subdivided into a freely dissolved and a DOM-bound fraction (12-20) * The analysis of CBs in the water phase is also difficult because the concentrations are extremely low due to the hydrophobic nature of these compounds. Concentrations in (sea)water in the low picogram per liter range have been reported for the most abundant congeners (21-25). Such low levels require dedicated analytical procedures. The analytical procedures described in the literature usually consist of an extraction step followed by a cleanup over some adsorbent and an analytical separation and determination by capillary gas chromatography with electron capture detection (26-28). Principal differences are only found in the extraction techniques, which are based on either liquid-liquid (LL) or liquid-solid (LS) extraction (29). Low detection limits are usually realized by taking large water samples. LL extraction procedures with samples up to 100 L and using reversed continuous-flow extractors have been published (29-32). With LS extraction, water extractions of even several hundreds of liters of sample have been reported (29). From LS extraction it is known that up to 95% of the DOM will not be retained on the extraction column (16,33). Therefore, this technique is often used to determine the freely dissolved concentration of hydrophobic contaminants like CBs (16,33). However, it should be realized that an undefined part of the DOM-bound contaminants will also be extracted. The extraction efficiency of DOM-bound contaminants with LS extraction depends on the flow rate of the sample through the extraction column (16). In this paper we focused on a batch LL extraction procedure to determine all CBs present in the water sampled. To prevent any lose of CBs by adsorption to the sample bottle, the sample was extracted directly from this bottle using water volumes up to 10 L. By concentrating the extract to 100 pL, it was possible to detect sufficiently low CB concentrations. Due to their hydrophobic nature, CB concentrations in water containing suspended matter are almost completely determined by this SPM. Distribution coefficients between SPM and solution have been reported to be in the order of lo5 (12,21,34).Removal of the SPM therefore is necessary. SPM, however, is an operationally defined parameter: in practice, the SPM is usually defined as all particles that are retained by a 0.45-pm filter. Hence, distinguishing between the SPM-bound and "dissolved" CBs is also operational. Numerous substances remain in the water phase after separation of SPM; these substances will be referred to as DOM in this article. It is important to consider the effect of different SPM-removal techniques on the dissolved CB content, Glass fiber filters, which have nominal l-pm pores but in fact range from 0.5 to 1.2 pm, are often used (8,21,22, 29, 30). Tangential-flow membrane filters with exact 0.45-pm pores are also available but have never been compared with glass fiber filters for CB sample preparation. Possible artifacts with filtration, such as clogging during filtration and adsorption of CBs to the filter, have
0013-936X/92/0926-2028$03.00/0
0 1992 American Chemical Society
been investigated. Another technique to remove SPM from water without the drawbacks of filtration is continuous-flow centrifugation (35). However, this separation technique is based on particle size as well as particle density, which might produce a sample with more particles relative to the filtration methods. The objective of the investigation presented in this paper is to present and evaluate an analytical method for the determination of CBs in water at the picogram per kilogram level and to assess the effects on measured CB contents of various filtration methods or continuous-flow centrifugation for the separation of SPM. The experimenta included in this study were as follows: CB recovery from natural waters by extraction; sample preservation by acidification; precision (reproducibility) of the procedure; detection limit of the procedure; sorption of CBs on filters; influence of clogging of the filters on the accuracy; comparison of results after filtration and after centrifugation of suspended particle removal methods; relation between CB content and DOC (UV/persulfate method).
Experimental Section (A) Analytical Procedures. Reagents. All individual CBs were obtained from Promochem (Wesel, FRG). All organic solvents were pesticide analysis grade from Mallinckrodt (St. Louis, MO) except diethyl ether, which was from BDH (Poole, UK; Aristm quality). Neutral silica gel (Kieselgel60,230-400 mesh) was purchased from Merck (Darmstadt, FRG) and dried overnight at 180 "C prior to use. Basic alumina (Super I) was from ICN Biomedicals (Eschwege, FRG) and Chromosorb 60/80 mesh from Supelco (Bellefonte, PA). Sodium hydroxide and sodium sulfite were obtained from J. T. Baker (Phillipsburg, NJ). Sulfur-removing reagent was prepared after Japenga et al. (36)by dissolving 2 g of sodium hydroxide and 13 g of sodium sulfite in 60 mL of Milli-Q water and adding it to 210 g of basic alumina dried overnight at 180 "C. Subsequently, the reagent was dried under a gentle nitrogen stream in a flask at 180 "C until a water content of 10 f 0.5% was reached. The reagent was stored in a desiccator. Extraction. Before use, all glassware was rinsed with acetone, washed with a detergent solution, rinsed with demineralized water, and then kiln-fired at 250 "C. LL extraction was carried out in a 10-L glass sample bottle (Duran, Schott, NS 60/46) with a custom-made glass extractor by Elgebe (Leek, NL) placed on top of it, as depicted in Figure 1. The extraction solvent pentane evaporates when the flask is heated, condenses into the condenser, and is transported through a glass tube to the bottom of the sample bottle where it is dispersed into the water with a Teflon-coated magnetic stirrer. Pentane is collected again at the top of the bottle and flows back into the flask together with the extracted components. By prolonged application of this procedure, the extracted components are concentrated in the pentane solution. The speed of the stirrer and of the evaporation were set so that a fine pentane dispersion in the water and an almost flowing pentane stream from the condenser were sustained throughout the extraction process. Extracts were concentrated on a water bath with a slightly modified Kuderna-Danish apparatus (test tube and globe are in one piece) equipped with a modified Snyder column, all custom-made by Elgebe. Cleanup. Water, if present, was removed from the extract by freezing it with liquid nitrogen and decanting the extract into another flask. After 1mL of isooctane was added as a keeper, the remaining extract was concentrated to 1 mL by Kuderna-Danish evaporation and a gentle stream of nitrogen. Next, the extract was eluted over a
L
Glass tube
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-
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glass sample bottles and transported to the laboratory. Filtration. For filtration, water samples were tapped from the shipboard closed water system in 100-L stainless steel pressure containers obtained from De Jong Gorredijk (Gorredijk, NL). The filtration took place by discharging the sample from the container through a Teflon tube by means of 0.8 atm nitrogen pressure. Glass fiber filters (GF No. 6) were obtained from Schleicher & Schuell (Dassel, FRG) and membrane filters (Versa Flow Capsule) from Gelman Sciences (Ann Arbor, MI). Before use, glass fiber filters were kiln-fired at 250 “C overnight. A stainless steel filter holder from Schleicher & Schuell for 29-cm-diameter glass fiber filters was used. The filter holder was sequentially cleaned with Milli-Q water, acetone, and again Milli-Q water after each filtration. Membrane filters were used without further cleanup. The filtrate was collected in 10-L glass bottles and taken to the laboratory. (C) Analytical Experiments. Recovery with Spikes. Ten liters of North Sea water was sampled by the filtration method using a glass fiber filter and then extracted with the batch continuous extractor. After removal of the pentane layer, the water was spiked with 5 ng of individual CBs dissolved in 10 pL of methanol. The sample was stirred for 1 h and then extracted for 7 h, during which the flask with pentane was replaced at l-h intervals (exhaustion method). The CB contents were determined in every subextract. For all following experiments, samples were spiked with 500 pg of CB155 in 50 pL of methanol 1 day before extraction. Recovery without Spikes. Water from the Western Scheldt having a salinity of 4.5% was collected in February 1989 by filtration and centrifugation. Extraction by the exhaustion method was done for 2 days by refreshing the flask with pentane at doubled time intervals starting with 0.5 h for the filtrate (0.5, 1,2, etc.) and starting with 1h for the centrifugate (1,2,4, etc.). The extracts were then analyzed for CBs. Sample Preservation. Four water samples having a salinity of 3% and pH =8 were sampled by centrifugation in April 1989 from the river Rhine. Two samples were preserved by acidification to pH 2 with nitric acid. Immediately after sampling, one pair of samples (pH 2 and 8) was extracted using the exhaustion method at time intervals of 1,2,4,8,16, and 32 h, respectively. The other pair of samples was extracted similarly after 1 month of storage at room temperature. Precision. Duplicate water samples were collected by centrifugation in August 1989 from seven different points in the Ems estuary and acidified to pH 2 with nitric acid. Samples were extracted over 3 days, cleaned-up, and then analyzed according the procedures described earlier. Ten samples were extracted a second time to check extraction efficiency. Coefficients of variation (CVs) were estimated from the duplicate measurements by using the relative differences between these duplicates according to (39)
cv = 4
(1)
where k is the number of duplicate measurements and d is the relative difference of a duplicate measurement. Detection Limit. Two approaches were followed to obtain an estimate of the detection limit. First, eight empty sample bottles were washed with 400 mL of pentane. The solvents were treated as real extracts, and after cleanup, the CB concentrations were measured. Second, the liquid-liquid extraction procedure was applied to 20 water samples that already had been extracted. The ex2030
Envlron. Scl. Technol., Vol. 26, No. 10, 1992
tracts were subsequently subjected to the complete analytical procedure to determine their CB contents. Detection limits were calculated as three times the standard deviation of these “blanks” (40). (D) Sampling Experiments. Adsorption to Filters. Chromosorb (60-80 mesh) was loaded with CBs by adding 1mL of a CB standard containing 100 pg/L of individual CBs in isooctane to a column containing 0.5 g of Chromosorb. The isooctane was evaporated to the air, and after 3 days the column was eluted with Milli-Q water flowing at 350 mL/min. Starting with 50 mL of eluent, each subsequent elution fraction was doubled in volume from the previous, until a total of 9 L of water was eluted. The fractions were collected into volumetric flasks placed directly under the column. To determine the extent of adsorption to filters, the same procedure was repeated with either a membrane filter or a glass fiber filter placed in series with a CB-loaded Chromosorb column. Extractions were carried out by stirring each fraction for 2 h, using 1 mL of isooctane for the 95%) of CB155. For unequilibrated spiked water samples (preextracted), recoveries of the added CBs were >90% within I hour of extraction (data not shown). For unspiked samples, extraction times appeared to be much longer, according to the slope of the extraction curves shown in Figure 2. The higher chlorinated congeners especially needed an extraction time of 10-45 h, indicating a stronger affmity of higher chlorinated CBs to DOM (43). The fast extraction time with the unequilibrated spiked samples might be explained by the use of already extracted water and the short equilibrium time after spiking (only
a 250
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40
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60
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Figure 3. Influence of acidification and 1-month storage on the extraction curve of CB138. -c-
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Table I. Calculated CVs from Seven Duplicate Measurements for CBs and HCB in Ems Centrifugates, According to Formula (See Text)
CB155
10
20
30
40
50
60
70
CB HCB 18 31 28 52 44 101
range, pglk 50-100 50 20-80 50-150 50-100 25-50 50-100
cv
CB
4.0 8.7 28.5 6.0 3.2 5.1 15.9
118 153 138 187 180 170
range, pg/k 50-100 50-200 50-200 25-100 25-100 10-50
cv 7.2 8.1 7.3 4.2 6.3 6.8
80
extraction time (hr) Figure 2. Extraction curves for some unspiked CB congeners, HCB, and one equilibrated spiked CB (CB155) with (a) filtered and (b) centrifuged Western Scheidt water (contents of CB155 in centrifugate are in picograms absolute).
1h). For spiked samples, equilibration times nearly equal
to extraction times were essential to realize a more similar behavior between CB spikes and CB contaminants as confirmed by the extraction curves of CB155 (Figure 2). Investigations applying reversed continuous-flow extractions showed recovery of spikes lower than 50% after a 1-h extraction time (32,45). It is therefore unlikely that frequently applied extraction times of 0.1-1 h with LL extractions (30,31)will extract all of the CBs present (35). Freely dissolved CBs will be fully extracted, but the fraction of the DOM-bound CBs which will be extracted strongly depends on the experimental conditions. In addition to inefficient extractions after sampling, the adsorption of CBs to the wall of the sample container can also cause significant losses (30, 32, 45). The batch LL extractor described in this work does not suffer from adsorption problems as the extraction is performed in the sample container. Neither are there problems with extraction efficiency because, in principle, extraction time can be indefinitely long. The average recovery of 1-day-equilibrated spikes of CB155 for all experiments with complete extractions was 96 f 12% (n = 35). Therefore, for a given sample, applying this exhaustive extraction technique gives more reliable analytical results. Sample Preservation. It was observed that samples that were initially visually clear showed turbidity after storage, probably due to biological activity. This phenomenon especially occurred in centrifuged samples. Turbid samples often showed malfunctioning of the extractor, due to an improper separation of the water and
pentane phases as a result of emulsion formation. Acidified samples appeared to be free from any emulsion and from turbidity after prolonged storage. Centrifugates, with and without added acid, extracted immediately after sampling showed no differences in extraction curves (Figure 3). After 1-month storage, CB extraction from the acidic sample was much faster than from the untreated sample (Figure 3). The untreated sample was not even fully extracted after the longest extraction time (80h). The effect of acidification on extraction rate was more significant for the higher than for the lower chlorinated CBs. The slower CB extraction rates for the untreated, stored, sample can be explained by the enclosure of CBs due to the continued biological activity in the sample, which makes them less accessible to extraction. In acidic samples biological activity is prevented, and after some time, CBs will become more available for extraction, possibly due to breakdown of DOM (35). Acidification is therefore recommended for a proper and faster extraction of CBs. Precision. Seven replicate samples from the Ems were analyzed and the CVs were calculated as described above. The measured concentration ranges and CVs of unspiked CBs are presented in Table I. CVs were between 3 and 9% for all CBs, with the exception of CB31 and CB101. For CB31, the higher error was caused by bad separation efficiency of this compound in the GC system. For CB101, an overall blank increment was noticed. The recovery of CB155 was 96 f 7% (n = 14), and successive extraction indicated that recovery of unspiked CBs was well over 95%. Detection Limit. The detection limit was considered to be 3 times the standard deviation of the blank value. As it is hardly possible to obtain a real “blank” water sample in view of the extremely low concentrations that can be measured, two approaches were followed to obtain an estimate of the detection limit. In the first approach, the extraction was excluded, while in the second approach, Environ. Sci. Technoi., Voi. 26, No. 10, 1992
2031
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Table 11. Determined Detection Limits for HCB and Some
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Flgure 4. Desorption curves for eiutlon with MIIIi-Q water of (a) CB28, (b) CB101, and (c) CB180 from a single 2.5-g Chromosorb column directly and from a simllar Chromosorb column with a glass fiber fitter in serles. The Chromosorb column was loaded with 100 ng of Individual CBs.
any contamination of the sample vessel was removed prior to the blank extraction. In Table I1 are given the detection limits for all CBs as obtained by both approaches. The detection limits are well below the targeted 10 pg/kg level for most CBs. (B) Sampling. Adsorption to Filters. CBs released from CB-loaded Chromosorb columns are predominantly freely dissolved (the concentration of organic matter in Milli-Q was considered to be negligible). Chromosorb appeared to release CBs immediately after water elution, as depicted by the filled symbols in Figure 4 for CB28, CB101, and CB180, representing low-, medium-, and high-chlorinated compounds, respectively. These figures 2092
3.7 12 7 12
2.5 3.3 2.3
4.4 10 3.4 8
3.0 5 3.3
"In picograms per kilogram of water as 3 times their standard deviations in 8 blank measurements without, and 20 blank measurements with the extraction procedure, respectively (see text). Table 111. HCB (pg/kg), CB (pg/kg), and DOC (mg/L) Contents in Centrifuged Water (centr) and the First Two Filtered 10-L Fractions with Glass Fiber (gf) and Membrane (mb) Filters (Means of Duplicates) gf filtrate
E
118 153 105 138 187 180 170
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detectn limit, pg/kg without with extrctn extrctn (n = 8) (n = 20)
Envlron. Scl. Technol., Vol. 26, No. 10, 1992
CB
5-15 L
HCB 18 31 28 101 118 153 105 138 187 180 DOC
170 190 70 110 130 40 70 30 60 20 20 7.3
15-25 L 160
210 80
110 100 20 40 20
40 5 10 6.2
mb filtrate 5-15 L 15-25L 70 30 10 20 30 5 40 10 40 5 20 5.5
120 (1.2) 40 (3.4) 10 (1.2) 20 (2.3) 30 (3.4)
