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Quantification of resin acids in CTMP (chemithermomechanical pulping) effluents using an enzyme-linked immunosorbent assay
~
Pergamon
Waf. Sci. Tech. Vol. 35, No. 2-3, pp. 93-99,1997. Copyright © 1997 IAWQ. Published by Elsevier Science Ltd Printed in Great Britain. 0273-1223/97 $17'00 + 0'00
PH: S0273-1223(96)OO919-5
QUANTIFICATION OF RESIN ACIDS IN CTMP (CHEMITHERMOMECHANICAL PULPING) EFFLUENTS USING AN ENZYME-LINKED IMMUNOSORBENT ASSAY K. Li, A. Serreqi, C. Breuil and J. N. Saddler* Chair of Forest Products Biotechnology, Department of Wood Science, Faculty of Forestry, University ofBritish Columbia, Vancouver, BC, V6T 1Z4, Canada
ABSTRACT An enzyme-linked immunosorbent assay (ELISA) based on polyclonal antibodies was used to quantify resin acids in the effluents from a chemithermomechanical pulp (CTMP) mill. Using a direct ELISA format, with polyclonal antibodies immobilized on the plate, the assay had a 50% inhibition concentration (150) of 49.7 ppb and a detection limit of 4.5 ppb. Good recoveries were obtained from DHA spiked buffer. CTMP effluents with different levels of resin acids were quantified and the ELISA data were shown to compare favourably with both total abietic type resin acids and total resin acids as determined by gas chromatography (GC). © 1997 IAWQ. Published by Elsevier Science Ltd.
KEYWORDS Chemithermomechanical pulp (CTMP); dehydroabietic acid (DHA); Enzyme-linked immunosorbent assay (ELISA); gas chromatography (GC); polyclonal antibodies; resin acids. INTRODUCTION Resin acids are often considered to be the primary source of fish toxicity of effluents from pulp and paper mills using softwoods as the furnish (Taylor et al., 1988). They are released from wood chips into the pulp mill effluents, no matter which pulping process is used (McLeay & Assoc., 1987). For mechanical pulping processes, effluents usually contain very high levels of chemical oxygen demand (COD) and biochemical oxygen demand (BOD) because little wash water is used, and consequently, resin acid concentrations are also at elevated levels (Liu et al., 1991). The most common resin acids found in Canadian softwood pulp mill effluents are abietic acid, dehydroabietic acid (DHA), pimaric acid, palustric acid, isopimaric acid, neoabietic acid, sandaracopimaric acid and levopimaric acid (Figure 1).
* Corresponding author 93
94
K. LI et al.
Quantification of resin acids in pulp mill effluents is usually achieved by conventional analytical methods such as high performance liquid chromatography (HPLC), gas chromatography (GC) or gas chromatography _ mass spectrometry (GC-MS) (Foster and Zinkel, 1982; NCASI, 1975, 1986; Voss and Rapsomatiotis 1985; Lee et aI., 1990; Richardson et al., 1992). However, due to the lengthy procedures involving extraction, derivatization and chromatographic separation and quantification, these methods are not suitable for routine analysis of large numbers of samples in a short period of time. Therefore, a simple and reliable alternative would be highly desirable.
Abietic
Abietare
..
~
Def¥lroabietic
eOOH
eOOH
Lewpimaric
Neoabietic
Palustric
..... ,~
.... "~ ......
'
"'-
eOOH
eOOH
San:laracopimaric
Isopimaric
"'-eOOH Pimaric
Figure 1. Common resin acids found in Canadian softwood pulp mill effluents.
Imrnunoassays are widely used in the detection and quantification of toxic chemicals, such as pesticide residues and industrial pollutants (Van Emon and Lopez-Avila, 1992; Hock, 1993). Since immunoassays are based on the interaction between an antibody and antigen (Voller et al., 1979), they are target specific and the antibodies are capable of recognizing analytes from a complex sample matrix, thus minimizing the work needed for sample pretreatment. An enzyme-linked immunosorbent assay (ELISA) based on polyclonal antibodies has been developed for dehydroabietic acid (DHA), one of the major resin acids found in pulp mill effluents (Li et aI., 1994). Since the polyclonal antibodies against DHA cross-reacted with the abietic type resin acids (Li et al., 1994), which usually predominate in pulp mill effluents, we tried to use these polyclonal antibodies as a probe to quantify resin acids in the wastestreams from a chemithermomechanical pulp (CTMP) mill. The results were compared with those derived from Gc. MATERIAL AND METHODS All resin acid standards were obtained from Helix Biotech Corporation (Richmond, BC, Canada). Carbonate-bicarbonate buffer capsules, phosphate-citrate buffer with sodium perborate capsules, 0• phenylenediamine (OPD) methyl heneicosanoate (MHS), O-methylpodocarpic acid (OMPA) were purchased from Sigma (St. Louis, MO). N-methyl-N-nitroso-p-toluene sulfonamide (Diazald), and tricosanoic acid (TCA) were purchased from Aldrich Chemical Company, Inc. (Milwaukee, Wis). Ethyl acetate (EtOAc) was obtained from Fisher (HPLC grade). BACTO dehydrated skim milk was purchased from Difco Laboratories (Detroit, MI). ELISA plate was IMMULON® 4 (flat bottom, Catalog No. 01-010• 3855) from Dynatech Laboratories Inc. (Chantilly, VA) and optical density was read on a THERMOmaxTM microplate reader (Molecular Devices Corp., Menlo Park, CA). Individual resin acids were identified and
Quantification of resin acids
95
quantified on a Hewlett Packard HP 5890 series II gas chromatograph (GC) equipped with an HP 7673 auto injector, flame ionization detectors and a dual column system (DB-5 and DB-17 fused silica capillary column, 0.25-mm ID, 30-m long, 0.25 Ilm thickness, from J & W Scientific). Treated and untreated CTMP mill effluents were obtained from a mill located in British Columbia, Canada, using softwoods as the furnish. The polyclonal antibodies were raised from rabbits immunized with DHAM-SUC-KLH, a conjugate prepared by reaction of dehydroabietylamine with succinic anhydride (DHAM-SUC), and subsequently conjugation to keyhole limpet hemocyanin (KLH) (Li et ai., 1994). The enzyme tracer was DHAM-SUC• HRP, prepared by conjugation of DHAM-SUC to horseradish peroxidase.
Competitive direct ELISA: The microtitre plate was coated with diluted primary antibodies (I :8000, 100 Ill/well) in carbonate-bicarbonate buffer (pH 9.6) at 37°C overnight. The plate was washed four times using PBS buffer to remove the unbound material. The remaining binding sites on the plate were blocked with 2% milk in PBS buffer (200 Ill/well) at 37°C for 1 hr. Once the blocking agent was removed and the plate washed, the sample solution (or standard solution) in PBS and the enzyme tracer (I :8000 in PBS with 0.1 % milk) were mixed in equal volume and added to the microtitre plate (100 Ill/well), and the plate was incubated at 37°C for 1.0 hrs. After washing, enzyme substrate OPD (1.0 mg/ml in citrate-phosphate buffer, 100 Ill/well) was added and the reaction was stopped after 15 min using sulfuric acid (2.5 M, 50 Ill/well). The colour intensity was measured at 490 nm using a plate reader. GC analysis: Individual resin acids were quantified using the following procedures. Briefly, untreated CTMP effluent (20.0 ml) was spiked with surrogate (O-MPCA, 50.0 Ill, 1.0 mg/ml in methanol), adjusted to pH 3 with 1M HCl, and extracted twice with an equal volume of ethyl acetate. The organic phases from each extraction were combined and dried over anhydrous MgS0 4 . After filtration, the solvent was then removed under reduced pressure. The extract was dissolved in 1.0 ml of diethyl ether and spiked with an internal standard (50 ilL of MHS in methanol, 1.0 mg/ml) and a methylation standard (50 ilL of TCA in methanol, 1.0 mg/ml). The mixture was then derivatized with diazomethane, which was generated in-situ by reaction of N-methyl-N-nitroso-p-toluene sulfonamide (Diazald) with alcoholic potassium hydroxide and delivered to the sample vial by a stream of nitrogen gas, until a persistent yellow colour was obtained. The ether was evaporated under nitrogen and the methyl esters of fatty and resin acids were redissolved in 1.0 rnL of methanol. Individual resin acids were identified and quantified by Gc. Helium and nitrogen were used as the carrier and make-up gases, respectively. Injector and detector temperatures were 260°C and 290°C, respectively. The oven temperature was programmed at 60°C for 2 min, with an increase of temperature at 35°C/min to 170°C, then 0.6°C/min to 200°C and finally 35°C/min to 280°C when it was kept at this temperature for 10 minutes. Inhibition% 80
Y = 1.07 + 28.85 R = 0.998
* Log X
60
40
20
a
10
100
DHA concentration (ppb) Figure 2. Standard curve for DHA in the direct ELISA.
96
K. LI et al.
RESULTS AND DISCUSSION A direct ELISA fonnat was adopted due to the simplicity of the assay protocol and the short assay time required. A typical standard curve for DHA is shown in Figure 2. From the regression equation, the concentrations required for a 50% inhibition (Iso) and detection limit, defined as the amount of DHA which yielded a 20% inhibition (Midgley et ai. 1969), were calculated as 49.7 ppb and 4.5 ppb, respectively. There was a day to day variation due to the small differences in timing, temperature or reagent age, etc. Therefore, a standard curve was included with each assay to ensure an accurate result. Table 1. DHA concentration in CTMP effluent detennined by the direct ELISA using different dilutions 1:40
1:80
1: 160
1:320
1:640
DHA equivalent found (Ppb)*
133.80 ± 12.24
59.73 ±6.08
33.12 ± 3.15
16.74 ± 1.49
8.14 ± 0.78
DHA equivalent in original (ppm)
5.35
4.78
5.30
5.36
5.21
Ave = 5.20 ppm
s = 0.24
CV%=4.6
* Mean value was calculated on eight replicate wells. Prior to applying the direct ELISA to CTMP effluents, negative sample matrices, such as assay buffer, tap water and biotreated bleached kraft mill effluent (BKME), were spiked with DHA and assayed by the direct ELISA. As the recoveries were usually in the range of 85 to 110% (data not shown), we subsequently decided to assess this method of quantifying resin acids in CTMP effluents. As CTMP effluents usually contain high levels of COD and BOD when softwoods are used as the furnish, resin acid concentrations are also typically at elevated levels (Liu et al., 1991). As a result, effluent samples must be diluted in order to bring the concentration into the quantification window. Due to the high dilution factor required, any possible contaminating material present in the effluent did not exert any adverse effect on the quantification of resin acids. It was apparent (Table 1) that a good linearity was maintained over a wide range of dilutions. Table 2. Individual resin acid concentration of the three different CTMP streams as detennined by GC and ELISA No.1 a
No.2 b
No.3 c
PIM 900 SAN 100 ISO 1800 PALILEV 400 DHA 3600 ABA 2700 NED 300 7-0XO-DHA 1100 GC(total resin acids, ppb) 10,800 GC(total abietic type resin acids, ppb) 8,100 ELISA (DHA equivalents, ppb) 7,049 GC% 87.0%
1400 400 2800 3800 10800 5200 1400 300 26,200 21,500 21,465 99.8%
24800 5300 34800 33200 104700 93100 10100 9500 315,400 250,600 251,231 100.2%
a. Sample from aerobic stabilization basin b. Sample from preacidification tank c. Sample from preheater site
Quantification of resin acids
97
The precision obtained with this initial work encouraged us to further assess the accuracy of the assay. The resin acid content in the wastewaters from various stages of CTMP was analyzed by both the direct ELISA and Gc. The DHA equivalents determined by ELISA and the resin acid concentrations as determined by GC of three typical CTMP samples were compared (Table 2). These samples represented waste streams having low, medium and high levels of resin acids, from the aerobic stabilization basin, preacidification tank and preheater site, respectively. Although the ELISA could not provide direct information about the concentration of individual resin acids, the DHA equivalents as determined by ELISA compared favourably with the total concentration of abietic type resin acids as determined by GC. This indicated that the polyclonal antibodies could be used effectively to quantify the abietic group of resin acids (Li et al., 1994). For all cases, the GC data were the average of triplicates (CV from 3-14%) and the ELISA data were the average from three dilutions for each sample (four replicate wells for each dilution, CV from 8-19%). Since it is generally the total concentration of resin acids that will determine effluent toxicity, it would be more desirable to have a value for the total resin acid content rather than for just the content of abietic type resin acids. In order to obtain this information, DHA equivalents as determined by ELISA were correlated with the total resin acid content as determined by Gc. When all of the available data from the ELISA and GC determinations were plotted, good agreement was obtained, with coefficient of correlation of 0.993, significant at P < 0.001 (Figure 3).
ELISA (ppm) 350
ELISA = -0.74 + 0.75 * GC (r = 0.99)
300
•• •
250 200
•
150
•
100
• • •
•
•
••
•
50 0 0
50
100
150 200 250 GC (ppm)
300
350
Figure 3. The correlation between the total resin acids present in CTMP effluents, as determined by GC, and the DHA equivalents determined by direct ELISA.
The information obtained from this correlation allowed us to estimate the total resin acid content based on the ELISA response. Using this regression equation and the assumption that ELISA quantified abietic type resin acids, the abietic type resin acids will account for 66.6 to 74.8% of the total resin acids resulting in values from 5 to 350 ppm. This result is similar to both data in the literature and the values and ratios that we have observed in our own work. For example, analysis of the resin acid concentrations from a bleached CTMP mill wastewater indicated that abietic type resin acids accounted for 75.3% of the total resin acids (McCarthy et al., 1990). Usually, abietic type resin acids predominate in most pulp mill effluents. Data from Ontario Municipal/lndustrial Strategy for Abatement (MISA) programme involving 29 pulp mills indicated that abietic type resin acids typically account for 69.5% of the total resin acids (MIS.A, 1993). Alt.hough the ELISA values were lower than the total resin acids obtained by the chromatographIc method, thIS was not surprising as the polyclonal antibodies primarily detected abietic type resin acids, which have a similar
K. LI et al.
98
carbon skeleton to DHA. However, the good correlation still allowed us to estimate the total reSIn acid content based on the ELISA response. While most of the immunoassays are designed for a single analyte, it has been reported that with careful hapten design, methods can be developed to detect a particular class of compounds (Hock, 1993). Our results support this statement and they show that polyclonal antibodies can be used to quantify abietic type resin acids in the pulp mill effluents. Although identification of individual compounds must depend on other methods, such as GC and GC-MS, quantification and screening of large number of samples could be efficiently carried out by an ELISA based method. Recently, an HPLC based method of quantifying DHA has also shown good correlation in estimating the amount of total resin acids present in mill effluents (Shepard et aI., 1996). However, this procedure still requires the use of expensive equipment and a dedicated technician. With further work, a simple assay kit should allow the quicker, routine analysis of many mill streams using an ELISA based method. CONCLUSION This work describes the use of an immunochemical technique for the quantification of resin acids present in CTMP effluents. Using a direct ELISA format with polyclonal antibodies immobilized on the plate, the assay had a 50% inhibition concentration (150) of 49.7 ppb and a detection limit of 4.5 ppb. This method has been successfully applied to various CTMP effluents and requires little sample pretreatment and operator time. The ELISA provided a good estimation for total abietic type resin acids and correlated well with the total resin acid concentrations determined by Gc. Based on the regression analysis of data from both ELISA and GC, it is predicted that abietic type resin acids in CTMP will usually account for 67 to 75% of the total resin acids. REFERENCES Foster, D. O. and Zinkel, D. F. (1982). Qualitative and quantitative analysis of diterpene resin acids by glass capillary gas-liquid chromatography. J. Chromatog., 248, 89-98 Hock, B. (1993). Enzyme immunoassays for pesticide analysis. Acta Hydrochim. Hydrobiol., 21(2),71-83. Lee, H. B., Peart, T. E. and Carron, 1. (1990). Gas chromatographic and mass spectrometric determination of some resin and fatty acids in pulp mill effluents as their pentafluorobenzyl ester derivatives. 1. Chromatog., 498, 367-379. Li, K., Chester, M., Kutney, J. P., Saddler, 1. N. and Breuil, C. (1994). Production of polyclonal antibodies for the detection of dehydroabietic acid in pulp mill effluents. Analytical Letters, 27(9),1671-1688. Liu, H. W., Lo, S. N. and LavalJee, H. C. (1991). Characteristics of pollutants of source and biological treatment of a CTMP effluent. APPITA, 44, 133-138. McCarthy, P. J., Kennedy, K. J. and Droste, R L. (1990). Role of resin acids in the anaerobic toxicity ofchemithermomechanical pulp wastewater. Wat. Res., 24(11),1401-1405. McLeay and Associates Ltd. (1987). Aquatic Toxicity of Pulp and Paper Mill Effluent: A Review. Prepared for Environment Canada, Fisheries and Oceans Canada, Canadian Pulp and Paper Assoc., and Ontario Ministry of the Environment, Report EPS 4/PF/I, 191pp Midgley, A. R, Niswender, G. D. and Rebar, R W. (1969). Principles for the assessment of reliability of radioimmunoassay methods (precision, accuracy, sensitivity, specificity). Acta Endocrino/. Suppl., 142, 163-179. MISA (1993). Draft Development Document for the Pulp and Paper Scetor Effluent Limits Regulation. Environment Ontario, Queen's Printer for Ontario, ISBN 0-7778-0837-4. NCASI (1975). Improved Procedures for the Gas Chromatographic Analysis of Resin and Fatty Acids in Kraft Mill Effluents, Natl. Counc. Pap. Ind. Air Stream Improv. Tech. Bull. No. 281. NCASI (1986). Procedures for the Analysis of Resin and Fatty Acids in Pulp Mill Effluents, Nat/. Counc. Pap. Ind. Air Stream Improv. Tech. Bull. No. 501. Richardson, D. E., Bremmer, J. B. and O'Grady, B. V. (1992). Quantitative analysis of total resin acids by high performance liquid chromatography of their coumarine ester derivatives. J. Chromatog., 595, 155-162. Shepard, D. B., Sigfusson, D. A. and Sim S. Y. (1996). Effluent management: Determination ofresin acids in pulp mill effluent. Preprints of Papers to be presented at the 1996 Spring Conference, Canadian Pulp & Paper Association, Session 5, Paper 3, May 15-18, 1996, Jasper, Alberta. Taylor, B. R.: Y.eager, K. L., Abernethy, S. G. and Westlake, G. F. (1988). Scientific Criteria Document for Development of ProvIncIal Water Quality Objectives and Guidelines: Resin Acids. Ontario Ministry of the Environment, Water Resources Branch, Toronto, Ontario. ISBN 0-7729-4347-8, Queen's Printer for Ontario.
Quantification of resin acids
99
Van Emon, J. M. and Lopez-Avila, V. (1992). Immunochemical methods for environmental analysis. Anal. Chern., 64, 79A-88A. Voller, A., Bidwell, D. E. and Bartlett, A. (1979). The Enzyme Linked Immunosorbent Assay (ELISA), A guide with abstracts of microplate applications. ISBN 0.906036.01.1 Voss, R. H. and Rapsomatiotis, A. (1985). An improved solvent extraction based procedure for the gas chromatographic analysis of resin and fatty acids in pulp mill effluents. J. Chrornatog., 346, 205-214.
Pergamon
Waf. Sci. Tech. Vol. 35, No. 2-3, pp. 93-99,1997. Copyright © 1997 IAWQ. Published by Elsevier Science Ltd Printed in Great Britain. 0273-1223/97 $17'00 + 0'00
PH: S0273-1223(96)OO919-5
QUANTIFICATION OF RESIN ACIDS IN CTMP (CHEMITHERMOMECHANICAL PULPING) EFFLUENTS USING AN ENZYME-LINKED IMMUNOSORBENT ASSAY K. Li, A. Serreqi, C. Breuil and J. N. Saddler* Chair of Forest Products Biotechnology, Department of Wood Science, Faculty of Forestry, University ofBritish Columbia, Vancouver, BC, V6T 1Z4, Canada
ABSTRACT An enzyme-linked immunosorbent assay (ELISA) based on polyclonal antibodies was used to quantify resin acids in the effluents from a chemithermomechanical pulp (CTMP) mill. Using a direct ELISA format, with polyclonal antibodies immobilized on the plate, the assay had a 50% inhibition concentration (150) of 49.7 ppb and a detection limit of 4.5 ppb. Good recoveries were obtained from DHA spiked buffer. CTMP effluents with different levels of resin acids were quantified and the ELISA data were shown to compare favourably with both total abietic type resin acids and total resin acids as determined by gas chromatography (GC). © 1997 IAWQ. Published by Elsevier Science Ltd.
KEYWORDS Chemithermomechanical pulp (CTMP); dehydroabietic acid (DHA); Enzyme-linked immunosorbent assay (ELISA); gas chromatography (GC); polyclonal antibodies; resin acids. INTRODUCTION Resin acids are often considered to be the primary source of fish toxicity of effluents from pulp and paper mills using softwoods as the furnish (Taylor et al., 1988). They are released from wood chips into the pulp mill effluents, no matter which pulping process is used (McLeay & Assoc., 1987). For mechanical pulping processes, effluents usually contain very high levels of chemical oxygen demand (COD) and biochemical oxygen demand (BOD) because little wash water is used, and consequently, resin acid concentrations are also at elevated levels (Liu et al., 1991). The most common resin acids found in Canadian softwood pulp mill effluents are abietic acid, dehydroabietic acid (DHA), pimaric acid, palustric acid, isopimaric acid, neoabietic acid, sandaracopimaric acid and levopimaric acid (Figure 1).
* Corresponding author 93
94
K. LI et al.
Quantification of resin acids in pulp mill effluents is usually achieved by conventional analytical methods such as high performance liquid chromatography (HPLC), gas chromatography (GC) or gas chromatography _ mass spectrometry (GC-MS) (Foster and Zinkel, 1982; NCASI, 1975, 1986; Voss and Rapsomatiotis 1985; Lee et aI., 1990; Richardson et al., 1992). However, due to the lengthy procedures involving extraction, derivatization and chromatographic separation and quantification, these methods are not suitable for routine analysis of large numbers of samples in a short period of time. Therefore, a simple and reliable alternative would be highly desirable.
Abietic
Abietare
..
~
Def¥lroabietic
eOOH
eOOH
Lewpimaric
Neoabietic
Palustric
..... ,~
.... "~ ......
'
"'-
eOOH
eOOH
San:laracopimaric
Isopimaric
"'-eOOH Pimaric
Figure 1. Common resin acids found in Canadian softwood pulp mill effluents.
Imrnunoassays are widely used in the detection and quantification of toxic chemicals, such as pesticide residues and industrial pollutants (Van Emon and Lopez-Avila, 1992; Hock, 1993). Since immunoassays are based on the interaction between an antibody and antigen (Voller et al., 1979), they are target specific and the antibodies are capable of recognizing analytes from a complex sample matrix, thus minimizing the work needed for sample pretreatment. An enzyme-linked immunosorbent assay (ELISA) based on polyclonal antibodies has been developed for dehydroabietic acid (DHA), one of the major resin acids found in pulp mill effluents (Li et aI., 1994). Since the polyclonal antibodies against DHA cross-reacted with the abietic type resin acids (Li et al., 1994), which usually predominate in pulp mill effluents, we tried to use these polyclonal antibodies as a probe to quantify resin acids in the wastestreams from a chemithermomechanical pulp (CTMP) mill. The results were compared with those derived from Gc. MATERIAL AND METHODS All resin acid standards were obtained from Helix Biotech Corporation (Richmond, BC, Canada). Carbonate-bicarbonate buffer capsules, phosphate-citrate buffer with sodium perborate capsules, 0• phenylenediamine (OPD) methyl heneicosanoate (MHS), O-methylpodocarpic acid (OMPA) were purchased from Sigma (St. Louis, MO). N-methyl-N-nitroso-p-toluene sulfonamide (Diazald), and tricosanoic acid (TCA) were purchased from Aldrich Chemical Company, Inc. (Milwaukee, Wis). Ethyl acetate (EtOAc) was obtained from Fisher (HPLC grade). BACTO dehydrated skim milk was purchased from Difco Laboratories (Detroit, MI). ELISA plate was IMMULON® 4 (flat bottom, Catalog No. 01-010• 3855) from Dynatech Laboratories Inc. (Chantilly, VA) and optical density was read on a THERMOmaxTM microplate reader (Molecular Devices Corp., Menlo Park, CA). Individual resin acids were identified and
Quantification of resin acids
95
quantified on a Hewlett Packard HP 5890 series II gas chromatograph (GC) equipped with an HP 7673 auto injector, flame ionization detectors and a dual column system (DB-5 and DB-17 fused silica capillary column, 0.25-mm ID, 30-m long, 0.25 Ilm thickness, from J & W Scientific). Treated and untreated CTMP mill effluents were obtained from a mill located in British Columbia, Canada, using softwoods as the furnish. The polyclonal antibodies were raised from rabbits immunized with DHAM-SUC-KLH, a conjugate prepared by reaction of dehydroabietylamine with succinic anhydride (DHAM-SUC), and subsequently conjugation to keyhole limpet hemocyanin (KLH) (Li et ai., 1994). The enzyme tracer was DHAM-SUC• HRP, prepared by conjugation of DHAM-SUC to horseradish peroxidase.
Competitive direct ELISA: The microtitre plate was coated with diluted primary antibodies (I :8000, 100 Ill/well) in carbonate-bicarbonate buffer (pH 9.6) at 37°C overnight. The plate was washed four times using PBS buffer to remove the unbound material. The remaining binding sites on the plate were blocked with 2% milk in PBS buffer (200 Ill/well) at 37°C for 1 hr. Once the blocking agent was removed and the plate washed, the sample solution (or standard solution) in PBS and the enzyme tracer (I :8000 in PBS with 0.1 % milk) were mixed in equal volume and added to the microtitre plate (100 Ill/well), and the plate was incubated at 37°C for 1.0 hrs. After washing, enzyme substrate OPD (1.0 mg/ml in citrate-phosphate buffer, 100 Ill/well) was added and the reaction was stopped after 15 min using sulfuric acid (2.5 M, 50 Ill/well). The colour intensity was measured at 490 nm using a plate reader. GC analysis: Individual resin acids were quantified using the following procedures. Briefly, untreated CTMP effluent (20.0 ml) was spiked with surrogate (O-MPCA, 50.0 Ill, 1.0 mg/ml in methanol), adjusted to pH 3 with 1M HCl, and extracted twice with an equal volume of ethyl acetate. The organic phases from each extraction were combined and dried over anhydrous MgS0 4 . After filtration, the solvent was then removed under reduced pressure. The extract was dissolved in 1.0 ml of diethyl ether and spiked with an internal standard (50 ilL of MHS in methanol, 1.0 mg/ml) and a methylation standard (50 ilL of TCA in methanol, 1.0 mg/ml). The mixture was then derivatized with diazomethane, which was generated in-situ by reaction of N-methyl-N-nitroso-p-toluene sulfonamide (Diazald) with alcoholic potassium hydroxide and delivered to the sample vial by a stream of nitrogen gas, until a persistent yellow colour was obtained. The ether was evaporated under nitrogen and the methyl esters of fatty and resin acids were redissolved in 1.0 rnL of methanol. Individual resin acids were identified and quantified by Gc. Helium and nitrogen were used as the carrier and make-up gases, respectively. Injector and detector temperatures were 260°C and 290°C, respectively. The oven temperature was programmed at 60°C for 2 min, with an increase of temperature at 35°C/min to 170°C, then 0.6°C/min to 200°C and finally 35°C/min to 280°C when it was kept at this temperature for 10 minutes. Inhibition% 80
Y = 1.07 + 28.85 R = 0.998
* Log X
60
40
20
a
10
100
DHA concentration (ppb) Figure 2. Standard curve for DHA in the direct ELISA.
96
K. LI et al.
RESULTS AND DISCUSSION A direct ELISA fonnat was adopted due to the simplicity of the assay protocol and the short assay time required. A typical standard curve for DHA is shown in Figure 2. From the regression equation, the concentrations required for a 50% inhibition (Iso) and detection limit, defined as the amount of DHA which yielded a 20% inhibition (Midgley et ai. 1969), were calculated as 49.7 ppb and 4.5 ppb, respectively. There was a day to day variation due to the small differences in timing, temperature or reagent age, etc. Therefore, a standard curve was included with each assay to ensure an accurate result. Table 1. DHA concentration in CTMP effluent detennined by the direct ELISA using different dilutions 1:40
1:80
1: 160
1:320
1:640
DHA equivalent found (Ppb)*
133.80 ± 12.24
59.73 ±6.08
33.12 ± 3.15
16.74 ± 1.49
8.14 ± 0.78
DHA equivalent in original (ppm)
5.35
4.78
5.30
5.36
5.21
Ave = 5.20 ppm
s = 0.24
CV%=4.6
* Mean value was calculated on eight replicate wells. Prior to applying the direct ELISA to CTMP effluents, negative sample matrices, such as assay buffer, tap water and biotreated bleached kraft mill effluent (BKME), were spiked with DHA and assayed by the direct ELISA. As the recoveries were usually in the range of 85 to 110% (data not shown), we subsequently decided to assess this method of quantifying resin acids in CTMP effluents. As CTMP effluents usually contain high levels of COD and BOD when softwoods are used as the furnish, resin acid concentrations are also typically at elevated levels (Liu et al., 1991). As a result, effluent samples must be diluted in order to bring the concentration into the quantification window. Due to the high dilution factor required, any possible contaminating material present in the effluent did not exert any adverse effect on the quantification of resin acids. It was apparent (Table 1) that a good linearity was maintained over a wide range of dilutions. Table 2. Individual resin acid concentration of the three different CTMP streams as detennined by GC and ELISA No.1 a
No.2 b
No.3 c
PIM 900 SAN 100 ISO 1800 PALILEV 400 DHA 3600 ABA 2700 NED 300 7-0XO-DHA 1100 GC(total resin acids, ppb) 10,800 GC(total abietic type resin acids, ppb) 8,100 ELISA (DHA equivalents, ppb) 7,049 GC% 87.0%
1400 400 2800 3800 10800 5200 1400 300 26,200 21,500 21,465 99.8%
24800 5300 34800 33200 104700 93100 10100 9500 315,400 250,600 251,231 100.2%
a. Sample from aerobic stabilization basin b. Sample from preacidification tank c. Sample from preheater site
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97
The precision obtained with this initial work encouraged us to further assess the accuracy of the assay. The resin acid content in the wastewaters from various stages of CTMP was analyzed by both the direct ELISA and Gc. The DHA equivalents determined by ELISA and the resin acid concentrations as determined by GC of three typical CTMP samples were compared (Table 2). These samples represented waste streams having low, medium and high levels of resin acids, from the aerobic stabilization basin, preacidification tank and preheater site, respectively. Although the ELISA could not provide direct information about the concentration of individual resin acids, the DHA equivalents as determined by ELISA compared favourably with the total concentration of abietic type resin acids as determined by GC. This indicated that the polyclonal antibodies could be used effectively to quantify the abietic group of resin acids (Li et al., 1994). For all cases, the GC data were the average of triplicates (CV from 3-14%) and the ELISA data were the average from three dilutions for each sample (four replicate wells for each dilution, CV from 8-19%). Since it is generally the total concentration of resin acids that will determine effluent toxicity, it would be more desirable to have a value for the total resin acid content rather than for just the content of abietic type resin acids. In order to obtain this information, DHA equivalents as determined by ELISA were correlated with the total resin acid content as determined by Gc. When all of the available data from the ELISA and GC determinations were plotted, good agreement was obtained, with coefficient of correlation of 0.993, significant at P < 0.001 (Figure 3).
ELISA (ppm) 350
ELISA = -0.74 + 0.75 * GC (r = 0.99)
300
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250 200
•
150
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100
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•
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50 0 0
50
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150 200 250 GC (ppm)
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350
Figure 3. The correlation between the total resin acids present in CTMP effluents, as determined by GC, and the DHA equivalents determined by direct ELISA.
The information obtained from this correlation allowed us to estimate the total resin acid content based on the ELISA response. Using this regression equation and the assumption that ELISA quantified abietic type resin acids, the abietic type resin acids will account for 66.6 to 74.8% of the total resin acids resulting in values from 5 to 350 ppm. This result is similar to both data in the literature and the values and ratios that we have observed in our own work. For example, analysis of the resin acid concentrations from a bleached CTMP mill wastewater indicated that abietic type resin acids accounted for 75.3% of the total resin acids (McCarthy et al., 1990). Usually, abietic type resin acids predominate in most pulp mill effluents. Data from Ontario Municipal/lndustrial Strategy for Abatement (MISA) programme involving 29 pulp mills indicated that abietic type resin acids typically account for 69.5% of the total resin acids (MIS.A, 1993). Alt.hough the ELISA values were lower than the total resin acids obtained by the chromatographIc method, thIS was not surprising as the polyclonal antibodies primarily detected abietic type resin acids, which have a similar
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carbon skeleton to DHA. However, the good correlation still allowed us to estimate the total reSIn acid content based on the ELISA response. While most of the immunoassays are designed for a single analyte, it has been reported that with careful hapten design, methods can be developed to detect a particular class of compounds (Hock, 1993). Our results support this statement and they show that polyclonal antibodies can be used to quantify abietic type resin acids in the pulp mill effluents. Although identification of individual compounds must depend on other methods, such as GC and GC-MS, quantification and screening of large number of samples could be efficiently carried out by an ELISA based method. Recently, an HPLC based method of quantifying DHA has also shown good correlation in estimating the amount of total resin acids present in mill effluents (Shepard et aI., 1996). However, this procedure still requires the use of expensive equipment and a dedicated technician. With further work, a simple assay kit should allow the quicker, routine analysis of many mill streams using an ELISA based method. CONCLUSION This work describes the use of an immunochemical technique for the quantification of resin acids present in CTMP effluents. Using a direct ELISA format with polyclonal antibodies immobilized on the plate, the assay had a 50% inhibition concentration (150) of 49.7 ppb and a detection limit of 4.5 ppb. This method has been successfully applied to various CTMP effluents and requires little sample pretreatment and operator time. The ELISA provided a good estimation for total abietic type resin acids and correlated well with the total resin acid concentrations determined by Gc. Based on the regression analysis of data from both ELISA and GC, it is predicted that abietic type resin acids in CTMP will usually account for 67 to 75% of the total resin acids. REFERENCES Foster, D. O. and Zinkel, D. F. (1982). Qualitative and quantitative analysis of diterpene resin acids by glass capillary gas-liquid chromatography. J. Chromatog., 248, 89-98 Hock, B. (1993). Enzyme immunoassays for pesticide analysis. Acta Hydrochim. Hydrobiol., 21(2),71-83. Lee, H. B., Peart, T. E. and Carron, 1. (1990). Gas chromatographic and mass spectrometric determination of some resin and fatty acids in pulp mill effluents as their pentafluorobenzyl ester derivatives. 1. Chromatog., 498, 367-379. Li, K., Chester, M., Kutney, J. P., Saddler, 1. N. and Breuil, C. (1994). Production of polyclonal antibodies for the detection of dehydroabietic acid in pulp mill effluents. Analytical Letters, 27(9),1671-1688. Liu, H. W., Lo, S. N. and LavalJee, H. C. (1991). Characteristics of pollutants of source and biological treatment of a CTMP effluent. APPITA, 44, 133-138. McCarthy, P. J., Kennedy, K. J. and Droste, R L. (1990). Role of resin acids in the anaerobic toxicity ofchemithermomechanical pulp wastewater. Wat. Res., 24(11),1401-1405. McLeay and Associates Ltd. (1987). Aquatic Toxicity of Pulp and Paper Mill Effluent: A Review. Prepared for Environment Canada, Fisheries and Oceans Canada, Canadian Pulp and Paper Assoc., and Ontario Ministry of the Environment, Report EPS 4/PF/I, 191pp Midgley, A. R, Niswender, G. D. and Rebar, R W. (1969). Principles for the assessment of reliability of radioimmunoassay methods (precision, accuracy, sensitivity, specificity). Acta Endocrino/. Suppl., 142, 163-179. MISA (1993). Draft Development Document for the Pulp and Paper Scetor Effluent Limits Regulation. Environment Ontario, Queen's Printer for Ontario, ISBN 0-7778-0837-4. NCASI (1975). Improved Procedures for the Gas Chromatographic Analysis of Resin and Fatty Acids in Kraft Mill Effluents, Natl. Counc. Pap. Ind. Air Stream Improv. Tech. Bull. No. 281. NCASI (1986). Procedures for the Analysis of Resin and Fatty Acids in Pulp Mill Effluents, Nat/. Counc. Pap. Ind. Air Stream Improv. Tech. Bull. No. 501. Richardson, D. E., Bremmer, J. B. and O'Grady, B. V. (1992). Quantitative analysis of total resin acids by high performance liquid chromatography of their coumarine ester derivatives. J. Chromatog., 595, 155-162. Shepard, D. B., Sigfusson, D. A. and Sim S. Y. (1996). Effluent management: Determination ofresin acids in pulp mill effluent. Preprints of Papers to be presented at the 1996 Spring Conference, Canadian Pulp & Paper Association, Session 5, Paper 3, May 15-18, 1996, Jasper, Alberta. Taylor, B. R.: Y.eager, K. L., Abernethy, S. G. and Westlake, G. F. (1988). Scientific Criteria Document for Development of ProvIncIal Water Quality Objectives and Guidelines: Resin Acids. Ontario Ministry of the Environment, Water Resources Branch, Toronto, Ontario. ISBN 0-7729-4347-8, Queen's Printer for Ontario.
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Van Emon, J. M. and Lopez-Avila, V. (1992). Immunochemical methods for environmental analysis. Anal. Chern., 64, 79A-88A. Voller, A., Bidwell, D. E. and Bartlett, A. (1979). The Enzyme Linked Immunosorbent Assay (ELISA), A guide with abstracts of microplate applications. ISBN 0.906036.01.1 Voss, R. H. and Rapsomatiotis, A. (1985). An improved solvent extraction based procedure for the gas chromatographic analysis of resin and fatty acids in pulp mill effluents. J. Chrornatog., 346, 205-214.