Environ. Sci. Techno/. 1995, 29, 1407-1414
Analytical Methodology for the Determination of Freely Available Bleached Kraft Mill Effluent-Derived Organic Constituents in Recipient Sediments MICHAEL H . T A V E N D A L E , * ALISTAIR L. WILKINS, AND ALAN G. LANGDON Chemistry Department, University of Waikato, Private Bag 31 05, Hamilton, New Zealand
KEITH L. MACKIE, T R E V O R R . STUTHRIDGE, A N D PAUL N. MCFARLANE Forest Research Institute, Private Bag 3020, Rotorua, New Zealand
Methodology for the extraction, identification, and quantification of the freely available bleached kraft mill effluent constituents present in sediments is reported. A single Soxhlet Dean-Stark (SDS) extraction is followed by extract fractionation and quantification of the chlorophenolic constituents, resin acids, and base neutral resin-sourced cyclic hydrocarbons by gas chromatography mass spectrometry (GC/MS). The extractable organic halide (EOX) content is also determined. The methodology was evaluated for the recovery and reproducibility of selected target compounds, surrogate standards, and some isotopically carbon-13 and deuterium labeled compounds. The recoveries of resin acids, resin-sourced hydrocarbons, chlorophenols and chloroguaiacols were typically of the order 80-95%, while replicate analyses resulted in relative standard deviations of the order 5-15%. Application of the methodology to some recipient sediments adjacent to a N e w Zealand bleached kraft mill resulted in the detection of elevated concentrations of abietan-18-oic acid, retene, and de hydroa bietin.
Introduction Assessment of the fate of bleached kraft mill effluent (BKME) constituents in treatment systems and receiving waters has traditionally focused on the compounds present in the water column. Some important classes of BKME compounds display limited water solubility and moderate to high (11, and as a octanol/water partition coefficients (Kow) consequence, BKME recipient sediments are enriched with BKME constituents. Due to differing dissolved oxygen concentrations and bacterial activities,transformation and/ or mineralizationprocesses occurringin the sediment phase frequently vary from those occurring the aqueous phase. Therefore, studies of compound fate should examine both aqueous-phase and sediment-phase processes. Continued sedimentation of material with time may facilitate the characterization of prior effluent history by sediment core analyses (2). In addition, because a variety of organic substances accumulate in sediments, analyses of sediments can indicate evidence of environmental contamination in situations where the concentrations of the aqueous-phase contaminants may be below detection limits (3). Substances associated with solid media are generally categorized as freely available or bound (4). The freely available compounds are believed to exist in the interstitial water between particles or to be reversibly adsorbed on colloidal and particulate surfaces, while the bound constituents are considered to be compounds that are complexed to metals (5,s), conjugated by ether or ester linkages, and/or bound hydrophobically to humic acids (7). Fxtraction, identification, and quantification of freely available organic substances usually involve extraction with an appropriate solvent (or combination of solvents), while analysis of the bound constituents is possible using extraction protocols which release compounds from their conjugated form by hydrolysis. Methods involving hydrolysis may not quantitatively release all of the bound constituents (8)and may destroy compounds of importance. However, they are of value in the determination of compound speciation. In connection with our investigations of the environmental impact of a New Zealand pulp and paper mill, we had occasion to determine the levels of freely available organic substances present in recipient sediments downstream of the mill’s biologically treated BKME discharge. An important feature of such discharges originating from local pulp mills is the presence of appreciable levels of degraded resin-acid sourced hydrocarbons such as fichtelite (18-norabietane) and dehydroabietin (18-norabieta-8,11,13-triene) and saturated resin acids such as abietan- 18-oic acid and pimaran-18-oic acid (9). Methodology developed for the low molecular weight BKME sediment constituent analyses has recently been reviewed by Morales et al. (10,11). Previous methodology has focused on two main compound classes; the phenolic and chlorophenolic class and the fatty acid and resin acid class. In our studies investigating the transformation and * Corresponding author present address: New Zealand Forest Research Institute, Private Bag 3020, Rotorua, New Zealand. Telephone 64-7-347-5899; Fax: 64-7-347-9380; e-mail address:
[email protected].
0013-936X/95/0929-1407$09.00/0
@ 1995 American Chemical Society
VOL. 29, NO. 5, 1995 / ENVIRONMENTAL SCIENCE &TECHNOLOGY
1407
fate of resin acids, it became important not only to focus on the acidic compound classes but also to characterize the base neutral class. Methodology suitable for the extraction of the acidic compounds was considered inappropriate for the quantitative determination of the base neutral compounds. In particular, the historical procedures failed to remove a mixture of aliphatic hydrocarbons often encountered in the extraction of compounds from dried sediment. However, the base neutral constituents and the acidic components can be extracted simultaneously provided the solvent (or solvent combination) has sufficient polarity. If the acidic components are sufficiently resolved, either by fractionation, chromatographic procedures, or derivatization, from the base neutral components, the analysis of all three classes can be performed from a single extraction of the sediment as opposed to the traditional approach of using three separate extractions targeting each compound class. We now report the development and application of a protocol that facilitates the gas chromatographic (GC) resolution of previously unresolved saturated and unsaturated resin acids (such as abietan-18-oic acid and dehydroabietic acid) and affords a chlorophenolic fraction and a gel permeation chromatography (GPC)cleaned-up resinsourced cyclic hydrocarbon fraction in a single extraction of the sediment. The protocol also allows for the determination of generic parameters such as extractable organic halide (EOX), ameasure of thelipophilic chlorinated organic content.
Materials and Methods General Instructions. Sodium sulfate and potassium carbonate were heated to 450 "C in a muffle furnace and stored at 105 "C. All glassware was detergent washed and rinsed with acetone and hexane. Dean-Stark apparatus, Soxhlet extractors, Soxhlet thimbles, and continuous liquidliquid extractors were further cleaned prior to use by refluxing with hexane for 2-5 h. Solvents. Hexane (analyticalgrade, Ajax Chemicals) and 2-propanol (analyticalgrade, BDH) were redistilled in glass. Dichloromethane (analytical grade, Ajax Chemicals) was passed through a neutral alumina column (mesh 70-230, Merck) and redistilled in glass. Acetic anhydride was triply distilled prior to use. Acetone (analytical grade, Ajax Chemicals) was used for response factor, surrogate, and internal quantification standards. The purity of all solvents was checked by gas chromatographylmass spectrometry (GCIMS). Acid and base solutions used for pH adjustment were extracted with hexane. Excess solvent was removed by rotary evaporation. Sample Collection and Preservation. Surface sediments were collected using an aluminium dredge. Large material and overlying water were removed. The sediment was then homogenized and transferred to large wide-necked glass storage jars fitted with aluminium foil-lined tops and stored at 4 "C. SedimentExtraction. Homogenized wet sediment (1050 g) was weighed into a single walled cellulose thimble and placed in a Soxhlet Dean-Stark (SDS) extractor (12). Prior to extraction, surrogate standard solutions of anthracene-dla (100 pL x 1 mglmL), 2,3,6-trichlorophenol (1OpL x 0.1 mglmL), and 0-methylpodocarpic acid (50pL x 10 mglmL) in acetone were added to the surface of the sediment. The sample was then extracted for 24 h with hexane (100 mL). Azeotropically separated water was 1408 = ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 29. NO. 5, 1995
removed, 2-propanol (30 mL) was added to the hexane solution, and extraction continued for a further 48 h. Following extraction, the solvent phase was reduced to ca. 5 mL using a rotary evaporatorand transferred, with hexane rinsing, into a 50-mL volumetric flask and made up to volume with hexane. Extractable Organic Halide (EOX). An aliquot of the hexane extract (5 mL) was placed in a teflon capped test tube and washed twice with an acidified (pH 2) 0.01 M NaNO, solution (5mL). The hexane extract was dried over anhydrous sodium sulfate and reduced to 4 mL by rotary evaporation. The sample was transferred with hexane rinsing to a 5-mLvolumetricflaskcontaining n-hexadecane (100 pL) and made up to volume with hexane. The EOX was then determined using an Euroglas pyrolysis-microcoloumetric EOX instrument. Partitioning of Base Neutrals, Chlorophenols, and Resin Acids. The remaining hexane extract (45 mL) was transferred with hexane rinsing to a 250-mL separating funnel and washed with 0.1 M potassium carbonate (3 x 40 mL). The combined carbonate extracts were then extracted with hexane (25 mL). Emulsions were broken by centrifugation. The combined hexane extracts were dried over anhydrous sodium sulfate and analyzed for the base neutral compounds, while the carbonate extract was analyzed for chlorophenolicsubstances and for resin acids using 75% and 25%, respectively, of the carbonate extract. GPC Cleanup of Base Neutrals &tract. 22'-Difluorobiphenyl (10 pL x 1 mg/mL) was added as internal standard to the hexane extract, which was then reduced to near dryness and made up to 1 mL with hexanedichloromethane (1:l). Aportionofthe base neutralextract (0.5mL) was injected onto a 1 x 40 cm column packed with SX-3 Bio-Beads. The column was eluted using hexanedichloromethane (1:l) at a flow rate of 1 mLlmin and a pressure of 30-70 kPa. Solvent was degassed using a helium sparge and delivered using a Waters M-45 pump. A schematic diagram of the GPC apparatus is presented in Figure 1. The elution profile of the GPC columnwas initially established by GUMS analyses of successive 1-mlportions of column eluent from a standard sediment sample. The retention time of a retene standard was also determined using a Cecil 2012 UVwavelength detector operating at 254 nm. This information was used to establish the time at which fraction collection commenced. In a typical GPC separation, the first fraction (18mL) of eluent was discarded. The second fraction (50 mL) of eluent was collected and reduced to 1 mL using a rotatory evaporator. Elemental sulfur, if present, was removed from the extract by the addition of activated copper chips (13). After removal of the copper chips (with hexane rinsing), the extract was reduced to 1mL using a rotatory evaporator, and the extract was analyzed by GUMS as described below. Chlorophenolic Extract. A portion (75%) of the carbonate extract was made up to ca. 110 mL with 0.1 M potassium carbonate solution, and acetic anhydride (2 mL) was added. The solution was stirred vigorously using a magnetic stirrer for 10 min. The solution was adjusted to pH 6 using 10 M KOH and extracted for 16 h with dichloromethane in a 125-mLliquid-liquid extractor. 2,2'Difluorobiphenyl (10 pL x 0.1 mglmL) was added to the extractive solution as an internal standard, and the extract was reduced to ca. 1 mL using a rotatory evaporator. The extract was introduced onto a dry florid mini-column (Pasteur pipet), and the column was eluted with hexane-
Solvent
Gel Permeation Column Bio Beads Sx-3
Calibration use
FIGURE 1. Schematic diagram of gel permeation chromatography apparatus.
diethyl ether (411 (10 mL1. The resulting elutant was reduced. initially by rotary evaporation and finally under astreamofdrynitrogengas, to ca. 1OOpL. Theconcentrate was then analyzed by GC/MS. Resin Acid Extract. A portion (25%) of the carbonate extractwasmadeup toca. llOmLwithdistilledwater,and the solution was adjusted to pH 9 with 5 M H2S04and extracted for 16hwithdichloromethane ina 125-mlliquidliquid extractor. 2,2’-Difluorobiphenyl(5OpLx 1 mg/mL) was added to the extractive solution as internal standard, and the extract was reduced to ca. 1 mL. An ethereal solution of diazoethane was added to the extract. After 10 min, excess diazoethane was removed using a stream of nitrogen. The extract was introduced onto a dry florisil mini-column (Pasteur pipet), and the column was eluted with hexane-diethyl ether (41) (10 mL). The resulting elutant was reduced, initially by rotary evaporation and finallyunderastreamofdrynitrogengas, to ca. 1OOpL. The concentrate was then analyzed by GC/MS. Gas Chrnmatography/Mass Spectrnmetry. Extracts were analyzed using a HP 5890 gas chromatograph interfaced to a HP 5971A mass selective detector. A 25 m x 0.2 mm HP Ultra 2 gas capillary column (film thickness 0.33 mml was used with helium as the carrier gas (linearvelocity of 30 cm s-9. Groh purge splitless injection (2 pL, 30 s splitlesstime) was performed usinga HP 7673 autoinjector. The GC was temperame programmed from 40 (2-minhold) to 120 “Cat 20 “C/min, then to 280 “C (22-min hold) at 5 W m i n (total analysis time 60 minl. AU compounds were identified and quantified by mass selective detection. Chlorophenolic compounds were analyzed using selected ion monitoring (SIM). The chlorophenolics were then quantified from the appropriate ion chromatograms. Response factors for the chlorophenolic Substances listed in Table 1 were determined using authentic compounds purchased from Helix Biotech Corporation. Base neutrals and resin acids were analyzed using spectral scanning (m/z40-500). Base neutral compounds were quantified from the resulting total ion current (TIC) chromatogram using a response factor of 1:1 relative to retene. Resin acids were quantified from appropriate ion chromatograms compiled from spectral scan data. Ion chromatogram response factors and retention times were determined usingauthentic resinacid standards purchased from Helix Biotech Corporation (Table 1). Resin acids for which no standards were available were quantified from the TIC chromatogram using a 1:l response factor relative
to dehydroabietic acid. The identification of these resin acids was assigned according to Zinkel et al. (141. The resin acid fraction also contains the fatty acid class of compounds, which may also be quantified from this extract. Information regarding the identification and quantification of fatty acids can be readily obtained in the literature and from authentic compounds.
Results and Discussion Method Development. Amethod has been developed that enables the identification and quantification of BKME constituents from the single extraction of a recipient sediment. Such an approach has the advantages of better sample homogeneity and a more rapid sample analysis compared to three separate extractions required for each of the target compound classes. The protocol utilizes a single Soxhlet Dean-Stark (SDSI extraction (121. followed by extract fractionation and the GUMS determination of chlorophenolic substances, resin acids, and base neutral resin sourced cyclic hydrocarbons with GPC removal of an unresolved complex mixture of non-target acyclic bydrocarbons. Quantitative analysis of nonpolar base neutral compounds requires extraction from dried material. SDS was therefore chosen as the preferred sediment extraction technique since this method azeotropically removes the water and exhaustively extracts for the range of target compounds in one step. Other methods in which the sediment is predried and subsequently Soxhlet extracted may incur significant loss of volatile constituents. The base neutral extract of BKME recipient sediments comprises a complex series of medium to high molecular weight hydrocarbons,aseriesof n-alkanes, and aco-eluting, unresolvable complex mixture (UCMI of branched chain and cyclic hydrocarbons, the majority of which are petroleum or biologicallysourced environmental contaminants. Identification and quantification of acidic compounds by gas chromatography were impracticable without prior removal of the UCM base neutral component since it coeluted with many of the resin acid-derived cyclic base neutrals and resin acid constituents. Resin acid-derived structures, such as fichtelite,were found to he degraded by use of sulfuric acid washing, and although flash column chromatography techniques using florisil, silica gel, or alumina were effective for the removal of polar high molecular weight organic material, they proved ineffective for the removal of the UCM. GPC, a technique used to VOL. 29. NO. 5.1995 I ENVIRONMENTAL SCIENCE 8 TECHNOLOGY
* 1409
TABLE 1
6as Chroma€egr@y Relative Retention Times and Characteristic Mass Ions of Compounds Examined in This Study RRT’
compound
quantitation ions [ d z (YO)]
Base Neutrals 1.742 2.050 2.102 2.216 2.385
188 (IOO), 187 ( 2 4 , 184 (13) i o 9 (1001, 191 (541,262 (46) 159 (1001, 241 (801,256 (24) 223 (loo), 238 (79), 181 (44) 219 (loo), 234 (711,204 (30)
2,4-dichlorophenol 2,3,6-trichlorophenoI 2,4,6-trichlorophenoI 2,3,4,6-tetrachlorop henol pentachlorophenol 4,5-dichloroguaiacol 3,4,5-trichloroguaiacol 4,5,6-trichloroguaiacol tetrachloroguaiacol 4,5-dichlorocatechol 3,4,5-trichlorocatechol tetrachlorocatechol 5-c hlorovanil Iin 6-chlorovanillin 5.6-dichlorovanillin
Chlorophenols (as Acetates) 0.989 1.149 1.226 1.471 1.774 1.413 1.609 1.666 1.819 1.569 1.779 1.968 1.483 1.505 1.734
162 (loo), 164 (65) 196 (loo), 198 (97) 196 (1001, 198 (97) 232 (100),230 (76) 266 (loo), 268 (651, 264 (63) 192 (1001, 194 (65) 226 (1001,228 (97) 226 (1001,228 (97) 262 (100),260 (76) 178 (1001, 180 (65) 212 (loo), 214 (97) 248 (loo), 246 (761, 250 (50) 186 (1001, 188 (34) 186 (1001, 188 (34) 220 (loo), 222 (65)
seco-I-dehydroabietic acidc seco-2-dehydroabietic acidC pimaric acid sandaracopirnaric acid abiet-13-en-18-oic acidc isopimaric acid palustric acid 0-rnethylpodocarpic acid abietan-18-oic acidc dehydroabietic acid abietic acid neoabietic acid 12-chlorodehydroabietic acid 14-chlorodehydroabietic acid 12,14-dichlorodehydroabietic acid
Resin acids (as Ethyl Esters) 2.360 2.379 2.486 2.507 2.510 2.550 2.556 2.575 2.580 2.592 2.659 2.746 2.837 2.882 3.120
146 (1001, 187 (441,330 (8) 146 (loo), 284 (151,330 (3) 121 (loo), 194 (21), 330 (9) 121 (loo), 257 (161,330 (111 121 (loo), 215 (741,332 (6) 241 (loo), 257 (581,330 (16) 241 (loo), 315 (621,330 (49) 227 (IOO), 316 (50) 163 (1001, 261 (231,334 (5) 239 (IOO), 328 (10) 256 (loo), 241 (611,330 (39) 135 (loo), 121 (34), 330 (27) 273 (loo), 275 (341,362 (13) 273 (1001, 275 (341,362 (11) 307 (100),309 (651, 396 (16)
anthracene-d1o fichteliteb dehydroabietinb tetra hydroreteneb retene
Compounds quantified using a response factor of 1:l relative to retene. Fichtelite: a Relative retention time (RRT) to Z,Z’-difIuorobiphenyl. 18-norabietane; dehvdroabietin, 18-norabieta-8.11.13-triene. Compounds quantified using a response factor of 1:l relativeto ethyl dehydroabietiate.
clean up environmental samples based on molecular size exclusion (13,successfully partitioned the aromatic and resin acid derived cyclic base neutral compounds from the straight chain alkanes and UCM. However, the resin acids were only partially resolved from the UCM base neutral component. Quantitative separation of resin acid and phenolic constituents from the neutral compounds was achieved by extraction of the acidic components from hexane into aqueous base. The resin acids and phenolics were then subsequently extracted from the aqueous base into dichloromethane. The base neutrals components (UCM and cyclic compounds) which remained in the hexane were then separated by GPC. The effect of the GPC cleanup is illustrated in Figure 2. Method Evaluation. A series of extraction and recovery trials were undertaken with a homogenized recipient sediment sample. The extraction, fractionation, and GCI MS analyticalprotocol depicted in Scheme 1was developed. The performance and reproducibility of the method were assessed using a surface sediment collected from the recipient immediately downstream of the discharge from a local bleached kraft pulp mill. The mill’sBKME discharge had been subjected to extensive biological treatment (16). The sediment contained readily detectable levels of chlo1410
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 29, NO. 5,1995
rophenols, fatty acids, resin acids, and neutral compounds. Analyses of upstream and downstream sediment samples demonstrated that the bull< of the fatty acids identilied in the recipient sediment originated from sources other than the BKME discharge, hence their detection was of little significance in assessing the impact of the mill’s BKME discharge on the local environment. Accordinglya detailed account of the protocol’s performance towards fatty acids (which was in general comparable to that established for resin acids) is not given here. A frequently encountered problem in environmental analyses is the incomplete recovery of target compounds from the sediment matrix. The performance of the methodology, excluding matrix effects, was determined by adding solutions of selected compounds to the hexane extractive solution after the SDS extraction step. The recoveriesof the selected compounds through the carbonate partitioning and derivatization steps were also assessed (Table 2). AU of the selected compounds displayed acceptable (>75%) recoveries. Only neoabietic and 12,14-dichlorodehydroabietic acids, both of which are typically minor BKME constituents, exhibited recoveries below 80%. In a like manner, when extractive solutions were spiked with a
A.
Abundance 3000000 -
Time ->
25.00
30.00
35.00
40.00
45.00
50.00 min B.
3000000
2400000 1800000
1200000 600000 0 Time ->
25.00
35.00
30.00
40.00
45.00
50.00 min
FIGURE 2. GC/MS total ion current chromatograms of base neutral extract prior to (A) and after (6) GPC cleanup. SCHEME 1
Flow Diagram of Analytical Protocol
I Sediment
1 Soxhlet Dean-Stark Extraction I
w Subsample
+, -Fraction
I
t
of Compound Classes
I
-1
to the resin acids, while the pKa of many of the chlorophenolic compounds differs substantially from the pKa of 2,3,6-trichlorophenol. Limitations of this type can however be overcome using carbon- 13or deuterium labeled specimens of target compounds. When specimens of 2,4,6trichl~rophenol-’~Cs, 2,3,4,6-tetra~hlorophenol-~~C~, penta~hlorophenol-’~C6, and dehydroabietic-6-d2 acid (synthesized in our laboratory) were applied at concentrations similar to the native compound concentrations, recoveries ranged from 74 to 90% for the labeled chlorophenols, and for the labeled resin acid, 91%recovery was observed. Since the recoveries of 0-methylpodocarpic acid (87.5%) and dehydroabietic-6-4 acid (91.3%) are similar (Table 3), it can be reasoned that 0-methylpodocarpic acid is a suitable representative surrogate compound for resin acid analyses. Furthermore, if a recovery of 80% or greater is applied as the criterion for method acceptance, 2,3,6-trichlorophenol and anthracene-& are also acceptable surrogate standards for chlorophenols and for resin cyclic hydrocarbons (such as retene and dehydroabietin), respectively. The recoveries of a variety of chloroguaiacols, chlorocatechols, and chlorovanillinswere also determined (Table 3). While chloroguaiacols (with the possible exception of tetrachloroguaiacol)were adequatelyrecovered from spiked sediment samples,chlorocatecholsand chlorovanillinswere poorly recovered. It is apparent from the relative standard deviations (Table 3) that the quantification of the chlorovanillin class is limited by the highlyvariablerecovery.Their poor recovery and reproducibility reflect problems with the in situ carbonate/acetic anhydride acetylation step. Liquid-liquid extraction facilitates the quantitative extraction ofthe derivatized and nonderivatized compounds. Both vanillin and acetylated vanillin appeared in the phenolic fraction, indicating incomplete acetylation of the vanillin class. Morales et al. (11)have reported a technique bywhich complete acetylation of vanillic, and related phenolic aldehydes can be achieved by pyridine/ acetic anhydride acetylation. This technique would require the extraction
+, I
solution of selected compounds immediately prior to the florisil filtration step, recoveries were typically of the order 85-99Yo. Sediment matrix effects were investigated by determining, in triplicate, the recovery of the three surrogate standards, added as acetone solutions, directly onto the sediment immediately prior to extraction (Table 3). Sediments were extracted within 10 min of surrogate addition as when equilibrium is achieved; phenolic surrogates may become bound (17). Anthracene-dlo,2,3,6-trichlorophenol, and 0-methylpodocarpic acid were chosen as surrogate standards since these compounds were not present in the recipient sediments and because they behave in a representative manner to the three target compound classes. The results presented in Table 3 should however be interpreted with caution since the carboxyl group of 0-methylpodocarpic acid has a C-4 configuration different
VOL. 29, NO. 5 , 1995 / ENVIRONMENTAL SCIENCE &TECHNOLOGY
1411
TABLE 2
TABLE 3
Recoveries of Selected Compounds by the Method in the Absence of the Sediment Matrix
Recoveries of Selected Compounds Spiked onto the Sediment
pattition/ derivatiration YO recovery
compound
florisil cleanup YO recovery
Base Neutrals retene squalene
99.4 96.2
nle nle
90.5 88.1 91.6 92.7 89.8 85.3 85 99.2 92.8 93.5 93 94.2 94.0 95.4
86.8 89.9 92.7 91.2 95.9 97.1 97.7 96.2 94.8 95.8 89.7 97.4 98.7 91.3
94.6 92.0 90.2 84.4 104.2 81.2 70.6 88.2 88.6 56.6
95.9 94.1 93.8 92.1 88.7 95.7 88.7 95.8 93.5 92.3
Chlorophenolics 2,4-dichlorophenol 2,4,6-trichlorophenoI 2,3,4,6-tetrachlorophenol pentachlorophenol 4,5-dichloroguaiacol 3,4,5-trichloroguaiacol 4,5,6-tric hloroguaiacol tetrachloroguaiacol 4,5-d ic h Iorocatec h o I 3,4,5-trichlorocatechol tetrachlorocatechol 5-chlorovanillin 6-chlorovanillin 5,6-chlorovanillin Resin Acids pimaric acid sandaracopimaric acid isopimaric acid palustric acid dehydroabietic acid abietic acid neoabietic acid 12-chlorodehydroabietic acid 14-chlorodehydroabietic acid 12,14-dichlorodehydroabieticacid
Surrogate Standards anthracene-dlo 95.6 2,3,6-trichlorophenol 99.4 0-methylpodocarpic acid 96.2
nle 91.3 98.4
a Spiking levelsapproximately 1Opginto hexane. n/e: Florisilcleanup was not examined for these compounds
of the underivatized phenolics into the organic phase (liquid-liquid extraction at pH 6 ) and then acetylation as outlined (11). Since the levels of chlorovanillins occurring in the local sediments were low and the major classes of phenols were adequatelyacetylated by the in situ procedure, the more forcing acetylation protocol was not deemed necessary. It could however be substituted for the procedure utilized in this study. The poor recovery of chlorocatechols is most likely a consequence of their retention and/or degradation on the sediment matrix. Binding of chlorocatechols to Fe(II1) and Al(II1) in sediments has recently been examined (18). Alternatively,it may be that the chlorocatecholsare unstable in the carbonate solution prior to acetylation (19). Since chlorocatecholswere efficiently recovered when introduced postextraction (Table 3), the possibility that they were lost during the fractionation and derivatization stages of the method can be discounted. The reproducibility of the methodologywas determined by performing atriplicate analysis on the recipient sediment. We consider that relative standard deviations (RSD) of less than 20%provide an adequate level of reproducibility. The data presented in Table 4 demonstrated that the reproducibility was acceptable for most compounds. The base neutral compounds exhibited less than 5% RSD while chlorophenolic compounds and resin acids displayed RSDs 1412
ENVIRONMENTAL SCIENCE &TECHNOLOGY / VOL. 29, NO. 5 , 1995
compound
spike level % recovery
YO RSDa
Surrogate Standards anthracene-dlo 10,uglg 2,3,6-trichlorophenoI 100 nglg 0-methylpodocarpic acid lOpg/g
96.0 80.7 87.5
3.7 10 13.4
Isotopically labeled Compounds 2,4,6-tri~hIorophenoI-'~C6 50 nglg 2,3,4,6-tetra~hIorophenol-~~C,j50 ng/g penta~hlorophenoI-~~c6 50 nglg dehydroabietic-6-d2 acid IOOpglg
84.0 73.7 90.1 91.3
1.7 5.5 2.6 0.9
110.4 93.2 86.1 67.6 13.0 21.4 9.3 0.7 1.4
12.2 9.7 9.8 6.7 172 161 194 8.9 10.3
Selected Phenolics 4,5-dic hloroguaiacol 100 nglg 3,4,5-trichloroguaiacol 100 nglg 4,5,6-trichloroguaiacol 100 nglg tetrachlorguaiacol 100 ng/g 5-c hlorovanilli n 100 ng/g 6-chlorovan il lin 100 nglg 5.6-dichlorovanillin 100 nglg 4,5-dichlorocatechol 100 nglg 3,4,5-tric hlorocatechol 100 ng/g tetrachlorocatechol 100 ng/g
0
a RSD, relative standard deviation; standard deviation of analyses expressed as % of average compound recovery.
between 10 and 20%. The RSDs for the chlorocatechols and chlorovanillins were poor. The difficultiesin analyzing chlorocatechols were discussed earlier. The variation in the chlorovanillin data was considered to be caused by incomplete acetylation. The poor reproducibility of palustric acid was due to its proximity to the detection limit and its potential for isomerization. A notable feature of the protocol is its ability to baseline resolve abietan-18-oic acid and dehydroabietic acid using a nonpolar HP Ultra 2 (or equivalent) methylsilicone gum capillary column. While the methyl or pentatluorobenzyl esters of these acids are inadequatelyresolved or co-eluted on nonpolar columns, the corresponding ethyl esters were readily resolved by GC andlor GUMS analysis using a nonpolar column. Previous studies (20,21)have resorted to the use of mass spectroscopic ion ratios andlor peak height ratios to quantify the methyl esters of these resin acids. Detection Limits. Using a 40-g wet weight sediment sample, which typically afforded a 12-gdry weight sample, and using a minimum signal to noise ratio of 3:1, the method's detection limits were 0.5, 100, and 100 nglg for chlorophenolic compounds (SIM acquisition), resin acids, and base neutrals (scan acquisition), respectively,on a dry weight basis. Should the need arise, the sensitivitytoward resin acids and base neutrals can be greatly improved to a detection limit similar to that for obtained for chlorophenolic compounds, by acquiring the GC/MS data in SIM mode (see Table 1 for appropriate ions). Application to Representative Recipient Sediments. Results for three sediment samples are presented in Table 4. The sediment samples were collected from the basin of a BKME treatment lagoon receiving alkaline extractionstage effluent and foul condensate (site 1). This treatment lagoon contained a low dissolved oxygen content of ca. 1 mg/L. The second sediment sampled was collected from the effluent drain midway between the basin and the effluent's outfallpoint (site2). And the third sediment sampled,which was collected from immediately downstream of the outfall
Pulp and Paper Mill
Aerated Lagoons sewer
Mechanically Aerated Lagoon System
Hydroelectric-Lake
*
Aerators
0
Sampling Sites
FIGURE 3. Location of sediment sampling sites. TABLE 4
Application and Reproducibility of Sediment Methodologf recipient sediment compound
site 1 n=l
site 2 n= 1
EOX (pg of Cl/g dry weight)
site 3 n=3 av concn 186
Yo RSD
23.8 45.4 30.9 13.4
4.1 4.4 5.8 4.5
17.9 24.3 160.0 22.3 42.6 8.0 21.1
0.6
13.8 13.8 17.1 13.8 14.5 16.0 15.3 33.0
nd nd 4.5 6.0 5.2
56.2 80.7 15.7
4.0
Base Neutrals @g/g Dry Weight) fichtelite dehydroabietin tetrahydroretene retene
9717 15424 167 nd
47.8 10.7 35.7 20.6
Chlorophenolics bg/g Dry Weight) 2,4-dichlorophenol 2,4,6-trichlorophenoI 2,3,4,6-tetrachlorophenol pentachlorophenol 4,5-dichloroguaiacol 3,4,5-trichloroguaiacol 4,5,6-trichloroguaiacol tetrachloroguaiacol 4,5-dichlorocatechol 3,4,5-trichlorocatechol tetrachlorocatechol 5-chlorovanillin 6-chlorovanillin 5,6-chlorovanillin
nd 410 nd 1045
41 nd 173
330
Resin Acids (Icg/g Dry Weight) seco-I-dehydroabietic seco-2-dehydroa bietic pimaric acid sandaracopimaric acid abiet-13-en-18-oic acid isopimaric acid palustric acid abietan-18-oic acid dehydroabietic acid abietic acid neoabietic acid 12-chlorodehydroabietic acid 14-chlorodehydroabietic acid 12,14-dichlorodehydroabietic acid
other resin acids
1050 503 686 32 463
8.3 2.6 0.7 1.3
11312 1552 56
12.8 3.4 0.9
56 117 2862
0.5 0.6 17.8
23.2 10.7 23.7 2.1 39.4 10.3 3.1 94.9 56.7 33.6 4.3 0.7 5.2 2.5 43.4
17.6 19.2 16.6 17.4 18.3 14.9 31.6 21.2 15.3 16.3 5.9 13.5 12.5 20.3
Av concn, average concentration. RSD, relative standard deviation. Standard deviation of analyses expressed as % of average compound concentration. nd, concentration below detection limit. a
VOL. 29, NO. 5, 1995 /ENVIRONMENTAL SCIENCE & TECHNOLOGY
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(site 31, was used as our recipient sediment sample to evaluate the methods performance and reproducibility. Site locations are presented in Figure 3. Single sample analyses were undertaken for the site 1 and 2 samples. At each of the sites the dominant resin constituent was abietan-18-oic acid (11 312, 13, and 95 pglg dry weight sediment, respectively). This compound, which has been rarely reported in the literature, is not a constituent of the effluents entering the treatment system (21, 22). It is however a significant component of the treated BKME being discharged from the treatment system (21). Abiet-13-en-18-oicacid, which like abietan-18-oic acid is not a constituent of the effluents entering the treatment system, is a probable intermediate in the biological degradation of abietic acid, the dominant resin acid constituent entering the treatment system (21). A wide variety of other resin acids including pimaric acid, sandaracopimaric acid, dehydroabietic acid, seco-1- and seco2-dehydroabietic acids, and oxidized andlor hydroxylated resin acids such as 7-oxo-dehydroabietic acid were also detected. Substantiallevels of fichtelite and dehydroabietin were also observed in the treatment basin sediments. Lower levels of these compounds and the more heavily aromatized analogues, tetrahydroretene and retene, were identified in site 2 and 3 sediment samples (Table 4). A variety of chlorophenolic substances, among which trichlorophenol and pentachlorophenol were dominant, were also detected. This study confirms the necessity to characterize the resin acid and base neutral fractions of sediments since it is apparent the level and structures present are of significance and importance in establishing fate and impact of BKME constituents in the recipient environment.
Acknowledgments
(2) Paasivirta, J.; Knuutinen, J.; Maatela, P.; Paukku, R.; Soikkeli,J.; SHrkka, J. Chemosphere 1988, 17, 137-146. (3) Lytle, T. F.; Lytle, J. S. Chemosphere 1987, 16, 171-182. (4) Remberger, M.; Allard, A. S. ; Neilson, A. H. Appl. Environ. Microbiol. 1986, 51, 552-558. (5) McBride, M. B.; Wesselink, L. G. Environ. Sci. Technol., 1988, 22, 703-708. (6) Kung, K. H. S.; McBride, M. B. Environ. Sci. Technol. 1991, 25, 702-709. (7) Rav-Acha, Ch.; Rebhun, M. Water Res. 1992, 26, 1645-1654. (8) Remberger, M.; Hynning, P. A.; Neilson, A. H. Environ. Toxicol. Chem. 1988, 7, 795-805. (9) Wilkins, A. L.; Panadam, S. Appifa, 1987, 40, 208-212.
(10) Morales, A.; Birkholz, D. A.; Hrudey, S. E. Water Environ. Res. 1992, 64, 660-668. (11) Morales, A.; Birkholz, D. A.; Hrudey, S. E. WaferEnviron. Res. 1992, 64, 669-681. (12) Laamparski, L. L.; Nestrick, T. J. Chemosphere 1989, 19, 27-31. (13) Bates, T. S.; Carpenter, R. Anal. Chem. 1979, 51, 551-554. (14) Zinkel, D. F.; Zank, L. C.; Wesolowski, M. F. Diterpene Resin Acids; U.S. Department of Agriculture, Forest Service, Forest Products Laboratory: Madison, WI, 1971, pp Al-A7 and C3C32.
(15) Norstrom, R. J.; Simon, M.; Mulvihill, M. J. Int.J. Environ. Anal. Chem. 1986, 23, 267-287 (16) Stuthridge, T. R.; Campin, D. N.; Langdon, A. G.; Mackie, K. L.; McFarlane, P. N.; Wilkins, A. L. Water Sci. Technol. 1991, 24, 309-317. (17) Remberger, M.; Allard, A.-S.; Neilson, A. H. Appl. Environ. Microbiol. 1986, 51, 552-558. (18) Remberger, M.; Hynning, P. A.; Neilson, A. H. Environ. Sci. Technol. 1993, 27, 158-164. (19) Abrahamsson, K.; Xie, T. M.I. Chromatog. 1983,279, 199-208. (20) Brownlee, B.; Fox, M. E.; Strachan, W. M. J.; Joshi, S. R. 1. Fish. Res. Board Can. 1977, 34, 838-843. (21) Zender, J. A.; Stuthridge, T. R.; Langdon, A. G.; Wilkins, A. L.; Mackie, K. L.; McFarlane, P. N. Water Sci. Technol. 1994, 29, 105-121 (22) Stuthridge, T. R. D.Phi1. Thesis, University of Waikato, New Zealand, 1990.
The financial support (D. Phil. Fellowship) to M.H.T. by N.Z.F.P. Pulp and Paper Ltd is gratefully appreciated.
Received for review January 7, 1995. Accepted January 17, 1995.
Literature Cited
ES940300E
(1) Suntio, L. R.; Shiu, W. Y.; Mackay, D. Chemosphere 1988, 17, 1249-1290.
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@Abstractpublished in AdvanceACSAbstmcts, February 15,1995.
