Environ. Sci. Technol. 1992, 26, 1190-1197
Molecular Weight Distributions of Organic Halogens in Bleached Kraft Pulp Mill Effluents Jounl K. Jokela
*vt
and MlrJaSalklnoja-Salonent
Department of General Microbiology, University of Helsinki, Mannerheimintie 172, SF-00300 Helsinki, Finland and Environmental Unit, Department for Research and Development, University of Helsinki, Vapaudenkatu 6 A, SF-1 5 1 10 Lahti, Finland The molecular weight distribution of chlorinated compounds of bleached kraft mill effluent (BKME) was studied by aqueous and nonaqueous size exclusion chromatography (SEC) and by ultrafiltration. Ninety percent of the BKME halogenated organics dissolved into tetrahydrofuran used as a nonaqueous SEC eluent. The tetrahydrofuran-soluble component of the BKME had the same molecular weight distribution as the original BKME when both were analyzed by aqueous SEC. Over 85% of the chlorinated material of BKMEs studied by nonaqueous SEC was of low molecular mass (10 000 decreased to one-fourth upon 100-fold dilution with distilled water, indicating that the halogenated components occurred as micellar aggregates. We conclude that the molecular weight distribution of the BMKE chlorinated material obtained by nonaqueous SEC is more accurate than that obtained in aqueous media unless high dilution is applied.
Introduction Bleaching of paper pulp with chlorine-containing chemicals leads to the formation of large quantities of organic halogen compounds (for a review, see ref 1). As yet no technology exists to recycle these products into the process. Chlorinated organics in bleached kraft pulp mill effluent (BKMEs) are only partially removed by biological wastewater treatment (2-4). For instance, in Finland 9700 ton of organically bound halogen [measured as adsorbable organic halogen (AOX)] were discharged into the waterways in 1990,equivalent to 2.2 kg/ton of pulp bleached (5). The environmental fate of the organic halogen compounds discharged from pulp bleaching is poorly understood. Some of the low molecular weight compounds such as chlorinated phenols and benzenes are known to bioaccumulate in aquatic organisms (6),but these compounds represent only a few percent of the AOX discharged (7). The major part of the organic halogen discharged from the bleaching process is believed to be heterogeneous material of relatively high molecular weight and therefore biologically inert (8). The ecological behavior of this material, such as sedimentation, biodegradation, and bioaccumulation, can be expected to correlate to the molecular size and hydrophobicity. In this paper we describe the molecular weight distributions (MWDs) of pulp bleaching organic halogen compounds by aqueous and nonaqueous size exclusion chromatography (SEC) and by ultrafiltration. We were able to determine the halogen distribution by applying halogen detection to high-performance size exclusion chromatography (HPSEC). We show that the molecular sizes of halogen compounds were smaller than generally assumed; Department of General Microbiology.
* Department for Research and Development. 1190 Envlron. Scl. Technol., Vol. 26, No. 6, 1992
85-95% being smaller than 1000 g/mol.
Experimental Methods Samples. Mill A pulped pine using a X(CD)(EO)DED (X, xylanase; C, chlorine; D, chlorine dioxide; E, alkali; 0, oxygen) bleaching sequence. Mill B pulped pine + birch using a (CD)EDED bleaching sequence for pine and a DEDED sequence for birch. Mill C pulped (pine + spruce) and birch with a ratio of 2:l. Pulp was bleached using a O(CD)(EO)DEDsequence. Mill D pulped pine and birch with a ratio of 1:l. Two-thirds of the pine pulp was bleached with a sequence of (C,,D,)(EP)D(EP)D (P, peroxide) and one-third with a (C7,C3JEDDED sequence. Birch pulp was bleached using two separate lines (1 and 2) in a ratio of 2:1,respectively. Bleaching sequences were (Clo-20Da,go)(EP)H(EP)D(H, hypochlorite) (line 1) and (CIo-20Dao-g0)(EP)HD (line 2). BKME (pH 7.6) was sam pled after neutralizing and nitrogen amendment (prior to treatment). The samples were stored at 4 "C (short period) or frozen. Preparation of THF Samples. The samples for SEC with tetrahydrofuran (THF) as a mobile phase were freeze-dried with an Edwards freeze dryer (Model EF03, Edwards High Vacuum, Sussex, England) with the exception of BKME of mill B, which was acidified to pH 2 and then vacuum evaporated (Vac Speed, Savant Instruments, Inc., Hicksville, NY). The dry residues were extracted into THF with 52% (v/v) of concentrated nitric acid (65%) added in a bath sonicator (Bransonic 32, Branson Instruments Co., 10 min, mills A,C, and U) and platform shaker (2 h, all mills). The THF solution was filtered through a 0.45-prn nylon filter (Gelman acrodisk 13) before injection into the high-performance liquid chromatograph (HPLC). THF samples were stored at -20 "C. To remove traces of water from the THF extract, 0.5 g of 4-A molecular sieve (Sigma, St. Louis, MO) was added to 1mL of the THF extract, After the extract was shaken, the THF phase was collected and the molecular sieve washed 3 times with 0.5 mL of THF. Pooled THF was concentrated under a flow of nitrogen. Vapor Pressure Osmometry. Ten liters of BKME was concentrated with a rotating evaporator to 250 mL and then freeze dried. The residue was extracted with a total volume of 200 mL of THF (+1% concentrated HN%) 1n a bath sonicator. The THF solvent was evaporated mder a flow of nitrogen and the residue freeze dried. This dry residue was extracted with distilled THF by 15 rnin of ultrasonication. The suspension was centrifuged and fib tered. Number-average molecular weight (M,) wa-c mea. sured from the THF filtrate at 45 "C using b e n d for calibration with a Knauer vapor pressure osmo:neter [weight-average molecular weight (M,) range from 40 to ca. 25000 g/mol, KG Dr.-Ing. Herbert Knauer cO* GmbH., Berlin] following the manufacturer's instructions. Ultrafiltration. BKME of mill D (pH 7.6) was filtered through a Whatman glass microfiber filter (GF/A grade,
0013-936X/92/0926-1190$03.00/0
0 1992 Amerlcan Chemical soclew
s l e I. Properties of the BKMEs of the Mills A-Do organic carbon BKME from
wood species used
pH
in BKME, mg/L
mill A mill B mill C mill D
pine pine, birch pine, spruce, birch pine, birch
5.7 7.6 4.1 7.6
833 nd 541 376
soluble in THF mg/L % 554 nd 405 228
in BKME
67 nd 75 60
AOX, mg/L
halogen soluble in THF EOX AOX mg/L 70 mg/L % 123 97 125
123 20 64 33
100 21
52 32
77 nd 47 28
100
77 nd 89 87
0 BKME, bleached kraft pulp mill effluent; THF, tetrahydrofuran; nd, not determined; AOX, adsorbable organic halogen; EOX, THFsoluble halogen.
1.6 pm) to remove suspended solids (fiber, etc.). Twenty milliliters of BKME, 200 mL of 1:lO diluted (with distilled water) BKME, and 2000 mL of 1:lOO diluted BKME were partitioned using Amicon 1000 (UM 2) and 10000 (PM 10) molecular weight cutoff membrane filters, with an Amicon ultrafiltration cell Model 402 (Amicon Corp., Lexington, KY). Retentates were dissolved in 3 X 5 mL of distilled water. The pH of mill D BKME was also adjusted to 10.5 (with concentrated NaOH) and then handled as above. Size Exclusion Chromatography. The liquid chromatograph consisted of a Waters Model 510 HPLC pump (Millipore Corp., Waters Chromatography Division, Milford, MA) or a Micromeritics 750 solvent-delivery system (Micromeritics Instrument Corp., Norcross, GA), a Rheodyne 7125 injector (Rheodyne, Berkeley, CA) with a 1OO-kL loop or a Waters 700 Satellite WISP automatic injector, an HP 1050 Series multiple wavelength detector (Hewlett-Packard Co., Palo Alto, CA), a Waters 410 differential refractometer, and a Waters baseline chromatography workstation with GPC option software or a Luxor 806 microcomputer. For aqueous SEC, TSK G4000PW and G3000PW columns (Toyo Soda Manufacturing Co., Tokyo, Japan; particle size 10-13 pm) were connected in series and eluted at 1mL/min with 0.025 M NaHC03-polyethylene glycol (0.5 g/L, MW 6000)-NaOH buffer, pH 10.5 (9). Polystyrene sulfonate standards of narrow MWD and lignin model compounds were used for calibration as described elsewhere (9). Injection volume was 10-50 pL. For nonaqueous SEC, Ultrastyragel 10000 A + 1000 A + 500 A 100 A styrene-divinylbenzene copolymer colF n s (30 X 0.8 cm id., Millipore Corp.) were connected 1l1 series and eluted at 1 mL/min flow rate with THF (Rathburn Chemicals LTD., Walkerburn, Scotland, WLC-grade, unstabilized). Light absorption was recorded at broad (225-445 nm) and narrow (275-285 nm) bandwidths with a reference wavelength of 450-550 nm. For calibration, PS standards of narrow MWD were used (IO) Supplemented with a PS 498 000 standard (polydispersity 1.20, Pressure Chemicals, Pittsburg, PA) and with three b i n model compounds as described elsewhere (11). The calibration curve was fitted using a third-degree function. Correlation coefficient of the calibration was >0.999. The injection volume was 10-50 pL. The 75 small molecular weight compounds of which the elution behavior was tested are indicated in the Results Section. Halogen Measurement, Adsorbable organic halogen (AOX) of BKME was measured with a halogen analyzer ECS 1000 equipped with a Boat-Control unit (Euroglass BV, Delft, The Netherlands) according to Standard SCAN-W 9:89 (22). The chromatographic fractions eluted with THF (1min) weremanually collected after the H P 1050 detector into glass vials, then transferred into quartz or porcelain cups,
+
Table 11. Average Molecular Weights (M, and M,) of the Organic Halogens and 225-445-nm Absorbing Compounds in T H F Extracts Made from BKMEs of the Mills A-D and Recovery Percent of Organic Halogens from the Nonaqueous SEC
BKME from mill A mill B mill C mill D
organic halogens M, M,
compounds absorbing 225-445 i m M, M,
190 230 280 250
220 230 270 210
330 440 550 540
515 860 750 760
C1 from columns,a % 71 80 88 100
It Sum of chlorine in THF SEC fractions collected as percent of that injected.
and evaporated under a flow of nitrogec. The halogen content of the residue in the cup was titrated microcoulometrically after combustion in the halogen analyzer. For the assay of inorganic halogen contents of the THF solutions, the THF was evaporated, the residue dissolved in water, and inorganic halogen assayed by direct injection (bypassing combustion) into the microcoulometric cell of the halogen analyzer. Carbon Measurement. Organic carbon was measured with TOC-5000 total organic carbon analyzer (Shimadzu, Japan) using sodium hydrogen phthalate (total carbon) and sodium hydrogen carbonate plus sodium carbonate (inorganic carbon) for calibration. BKME-carbon in THF extracts was measured after evaporation of the THF in N2 flow and then with the freeze dryer. The residue was dissolved in water, and the carbon content was measured with TOC-5000 analyzer. Chemicals. The chemicals were obtained from the local suppliers unless another source is indicated.
Results Molecular Weight Distribution of Organochloiine Compounds in BKME. Molecular weight distributions of THF extracts of different BKMEs were analyzed using HPSEC with THF as the eluent. HPSEC using the organic solvent was preferred over aqueous SEC because we found that recovery of relevant chlorinated model compounds (some chlorophenols) was extremely poor with TSK-PW columns, indicating strong adsorption to the column matrix. Table I summarizes the properties of the BKMEs studied. MWDs of the chlorine compounds in the different BKMEs are presented in Figure 1. Data obtained on the average molecular weights are compiled in Table 11. The peak of the MWDs of the THF-soluble halogen compounds occurred at 200-300 in all the wastewaters tested (Figure 1). The MW calibration was based on lignin model compounds and narrow molecular weight distribuEnviron. Scl. Technol., Vol. 26, No. 6, 1992
1191
1,s r
0
c
i
X
1,34
e
4:
s i U
13
1,14
I
,m ,74 10
100
1000
loo00
I
I
07
-1
10
100
10
100
loo00
101
1WW
100000
K:
I
t
.13
4:
,I1
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I 10
low
Molecular weight (g/mor)
100
Flgure 2. Comparison of MWDs detected with R I and by absorbance at 225-445 nm (top panel) and with absorbance at 275-285 and 225-445 nm (lower panel).
10
100
loo00
lo00
1oooM)
b -
2 5
F4
3 2
0' 0
-lar
wipm (dmor)
Flgure 1. MWDs of the chlorine-containing and UV-absorbing compounds of BKMEs from different mills (A-D) using different wood species and bleaching sequences. The mlecular weight averages M, (CI) and M, (CI) were calculated from the chlorine distributions.
tion polystyrenes. 85-95% of the solvent- (THF-) soluble halogen of the BKMEs eluted at MW loo% indicate the presence of compounds in the THF extract that were not adsorbed by active carbon at all or only incompletely, such as highly hydrophilic halogen cornpounds and inorganic chloride. When THF was e\ SPOrated from the extracts, the residue redissolved in water, AOX analysis was repeated, and recoveries of around 80% were obtained compared to the original AOX assnYed directly on the untreated wastewater. These results ' 3 h W that 80% (or more) of the AOX contained in the B.KME
i41 MillB
5 1.2:
A
Mill D
B
THF soluble part of the BKME
p 1,l < 1,000
i4 a
+
1-
I
Molecular weight (g/mol)
Molecular weight (g/rnol)
FbuW 3. MWDs of BKME (mill B) and the THF-soluble part of it a w e d by aqueous SEC using 0.025 M NaHC0,-polyethylene glycol (0.5 g/L, MW 6000)-NaOH buffer, pH 10.5, as the eluent. The peak at ca. 100 g / m i orlginated from the solvent used when lt was evaporated before redlssolvlng the residue in water.
[o
< 1,000
1:l
1:lO
01000-10000
1:lOO
1:l
> 10,Ooo
1:lO
I
1:lW
dilution factor
Flgw 4. Fractbnatbn of AOX and TOC of BKME by uttrafittratbn wkh membranes with a nomlnal cutoff of 100 and 10000. BKME of mlll D waa used, containing 24 mg of AOX/L and 280 mg of TOC/L, pH 7.6. The figure shows the distributions obtained for the BKME and for k dilutions made up with distilled water. In all cases 5 mg of BKME TOC was applied (active membrane dlameter 70 mm). wa8 recovered in the THF
extract, and is thus represented
in the MWDs shown in Figure 1. Figure 3 shows the MWDs obtained when both the untreated BKME and the THF extract prepared from it were analyzed by aqueous SEC, using lignin model compounds a d narrow molecular weight polystyrene sulfonates for calibration. It shows that the MWDs obtained were isomorphic over a 200 g/mol molecular mass range, with psaks located at 950 g/mol. The results thus prove that 10-20% of the AOX, which possibly was not included in the THF extract (Table I, last column), was not of significantly higher molecular size than the THF-soluble part. The peak (950) of the MWD of THF extract (UV-vis 226-445 nm) in aqueous SEC (Figure 3) is clearly higher than that obtained (350) by nonaqueous SEC for the same Werial (Figure 1, mill B). Molecular Size Fractionation of BKME by U1trafiltration. Ultrafiltration is commonly used to frach a t e samples according to molecular size and to get a W h picture of the amounts of molecules of different molecular sizes. In order to compare ultrafiltration with nonaqueous SEC system, we partionated BKME into three fractions using filters with a nominal cutoff at 1OOO ad 10000 and analyzed the MWDs of T H F extracts of fractions by SEC. Figure 4 shows that the partitioning by ultrafiltration *&e total organic carbon (TOC)and AOX in the BKME h e strongly dependent on the strength of the wastewater *the time of the filtration. The amounts of organic
Flgure 5. MWDs of the ultrafiltered BKME fractions. BKME (mlll D, pH 7.6) was fractionated by ultrafiltration (nominal cutoff 1000 and 10000)at 1:lO dilution made In distilled water.
halogen (AOX) and carbon (TOC) in the >loo00 retentate decreased by approximately 40% each time the BKME was diluted 10-fold with distilled water. This was not due to the clogging of the filters, as the same amount of original wastewater was filtered each time; the difference was in the amount of distilled water only. It thus seems that the BKME organic constituents form associations in aqueous solutions which are gradually dissociated upon dilution. The association could not be reversed by an increase of pH since we obtained an essentially identical partitioning of the BKME by ultrafiltration at pH 7.6 and 10.5 (not included in Figure 4). This may explain why aqueous SEC analysis gives higher MWDs than the nonaqueous SEC even at pH 10.5 (compare Figure 1 to Figure 3, mill B). Figure 5 shows the MWDs of the THF extracts prepared from ultrafiltration fractions 10 OOO. The MWDs had an ascending order, indicating that fractionation by size had indeed occurred. The peaks of the 10 000 ultrafiltration fractions were located at 350,800, and 2500, respectively (225-445 nm, Figure 5). Molecular weights of the fractions 1OoO-lOOOO and >loo00 were lower than predicted by the nominal cut-off values of the respective membranes. To find out whether there are intermolecular associations between BKME constituents in THF solution, the experiment shown in Figure 6 was performed. BKME (mill D)was fractionated by nonaqueous (THF) SEC and each fraction was rechromatographed by the same system. If the MWD in the primary chromatogram was significantly affected by intermolecular attraction, the fractions should show peak broadening upon rechromatography. The results (Figure 6A) revealed that the peaks obtained were not much broader than those obtained with the narrow molecular weight polystyrenes used for calibration. The molecular weights obtained upon rechromatography were identical within ca. f10% to those of the primary chromatogram, indicating no major impact of intermolecular associations (Figure 6B). Solvent Solubility of Halogenated BKME Components. T H F was chosen as the solvent because it solubilized virtually all the AOX contained in the BKME (Table I) and was suitable for subsequent chromatography. Our routine preparation of the T H F extracts of BKMEs gave solutions containing a trace amount of water, which did not interfere with subsequent chromatography using Ultrastyragel columns. However, if the traces of water were removed prior to chromatography,much of the higher molecular weight material was precipitated. This is demonstrated in Figure 7. The chromatogram shows that Environ. Sci. Technol., Vol. 26, No. 6, 1992
1103
\
Table 111. Low Molecular Weight Model Compounds Tested for Elution in SEC no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
compound
sourcea
no.
R M M An R An An An An M M M F F M F H H M L F L L L L L L L L L M M M F F F L F
39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75
dichloromethane trichloromethane tetrachloromethane 1,2-dichloroethane l,l,l-trichloroethane 1,1,1,2-tetrachloroethane 1,1,2,2-tetrachloroethane 1,l-dichloroethene 1,2-trans-dichloroethene trichloroethene tetrachloroethene acetic acid monochloroacetic acid dichloroacetic acid trichloroacetic acid stearic acid 4,10-dichlorostearic acid tetrachlorostearic acid benzene 1,2-dichlorobenzene 1,3-dichlorobenzene 1,4-dichlorobenzene 1,2,3-trichlorobenzene 1,2,4-trichlorobenzene 1,3,5-trichlorobenzene 1,2,3,4-tetrachlorobenzene 1,2,3,5-tetrachlorobenzene 1,2,4,5-tetrachlorobenzene pentachlorobenzene hexachlorobenzene phenol 2-chlorophenol 4-chlorophenol 2,3-dichlorophenol 2,4-dichlorophenol 2,5-dichlorophenol 2,6-dichlorophenol 3,4-dichlorophenol
compound 3,5-dichlorophenol 2,3,6-trichlorophenol 2,4,6-trichlorophenol 3,4,5-trichlorophenol 2,3,4,54etrachlorophenol 2,3,4,6-tetrachlorophenol 2,3,5,6-tetrachlorophenol pentachlorophenol catechol 4-chlorocatechol 3,5-dichlorocatechol 3,4,6-trichlorocatechol tetrachlorocatechol guaiacol (2-methoxyphenol) 3,5-dichloroguaiacol 3,6-dichloroguaiacol 4,5-dichloroguaiacol 4,5,6-trichloroguaiacol tetrachloroguaiacol 3-chlor~syringol~ 3,5-dichlorosyringol trichlorosyringol 2,3,5,6-tetrachlorocymenec biphenyl 2-chlorobiphenyl 2,3-dichlorobiphenyl 2,4,54richlorobiphenyl 2,2’,4,64etrachlorobiphenyl 2,2/,3,4,5’-pentachlorobiphenyl 2,2’,4,4’,5,6’-hexachlorobiphenyl 2,2’,3,4,5,6,6/-heptachlorobiphenyl 2,2’,3,3’,4,5/,6,6’-octachlorobiphenyl 2,2/,3,3/,4,4’,5,5’,6,6’-decachlorobiphenyl dehydroabietic acid chlorodehydroabietic acid dichlorodehydroabietic acid 3-formyl-2,4,5-trichlorothiophene
sourceG
L F F L L T T L M J J J J F J
J J J J J J J J M Ac Ac Ac Ac Ac Ac Ac Ac Ac H H H H
~ A cAccuStandard , Inc., 25 Science Park, Suite 687, New Haven, CT 06511. An, Analabs, 80 Republich Dr., North Haven, CT, 06473. E, Ega-Chemie, Steinheim, Germany. F, Fluka AG, Buchs, Switzerland. H, Helix Biotech Corp., No. 215-7080 River Rd, Richmond, BC, Canada. J, Department of Chemistry, University of JF@kyl& J p b k y k Finland. L, local commercial supplier. M, E. Merck AG, Darmstadt, Germany. R, Rathburn Chemicals LTD., Walkerburn, Scotland. T, Tokyo Kasei Kogyo Co., Ltd., 3-1-13, Nihonbashi-Honcho, Chuo-Ku, Tokyo 103, Japan. Syringol, 2,6-dimethoxyphenol. Cymene, isopropyl-4-methylbenzene.
-
molecules higher than 300 g/mol were virtually absent. The above results show that completely dry THF could not be used to dissolve BKME organics. The presence of water in the extract, however, may lead to dissolution of inorganic chloride. We therefore tested for the solubility of inorganic chlorides in the THF. The test was performed by shaking an excess of solid chloride salt for 2 days in THF. It was found that NaCl dissolved 5), a loss of linear correlation can be observed ( 8 , I I ) . Several factors can cause the observed deviation from linearity: reduced membrane permeation (412-14); decreased bioavailability (15, 16);loss of similarity in phase properties of the fish lipids and l-octanol with respect to organic chemicals (17, 18);a substantial influence of biotransformation on the elimination kinetics (13, 19, 20); nonequilibrium determinations of bioconcentration factors (21). Gobas et al. (22) concluded that reduced lipid solubility and reduced bioavailability are the most likely factors contributing to the loss of the linear correlation of nonmetabolizing chemicals. Considerable deviations from the observed linear correlations between BCF and KO,have also been observed for chemicals with log KO,< 5 (19,23-26). These deviations are probably caused by relatively high biotransformation rates (26-28). Several biotransformation processes have been shown to occur in fish at rates that are thought to be sufficient to affect the elimination rates of chemicals significantly (29-31). Some examples of studies describing the extent of bioconcentration of chemicals that are susceptible to biotransformation are discussed below. The data from these studies are combined and presented in Figure 1. The BCF values are compared with the relationship for inert chemicals, as calculated from the data of Mackay ( 4 ) . BCF values of most organophosphorus pesticides in guppy agree well with calculated values on the basis of the equation for inert chemicals (28) (Figure 1). The three most hydrophobic chemicals and also two of the lower hydrophobic organophosphates have relatively low BCFs. The elimination rate constants of these compounds were found to be substantially higher than expected on the basis of their log KO,,which may be related to a relatively higher biotransformation rate. In vitro studies supported the idea that the organophosphates with relatively low BCF values are biotransformed more rapidly (32). BCF values of some mononitrobenzene derivatives in guppy, which have log KO,values between 1.89 and 3.09, correlated well with the octanol-water partition coefficients (24). The BCFs of more hydrophobic chloronitrobenzenes in rainbow trout, Oncorynchus mykiss, as given by Niimi et al. (2.5) did not show any correlation with the calculated octanol-water partition coefficient of the compounds. From Figure 1it is obvious that the deviation of the BCF values from the calculated values on the basis of the
0 1992 American Chemical Society
Envlron. Sci. Technol., Vol. 26, No. 6, 1992
1197
