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Analysis of PCBs in waste oil by enzyme immunoassay
ELSEVIER
The Scienceof the Total Environment 196 (1997) 57-61
Analysis of PCBs in waste oil by enzyme immunoassay Nadine Lambert”,*, Titan S. Fanb, Jean-FranCois Pilette” “Analytical
Product Division, bBeacon Analytical
Millipore Systems,
S.A., BP 307, 78054 Saint Quentin-en-Yvelines 4 Washington Ave., Scarborough, 04074 Maine,
cedex France USA
Received 29 July 1996; accepted 8 October 1996
Abstract Immunoassays constitute a powerful analytical technique for sensitive, quantitative, fast and economical determination of trace contaminants in environmental monitoring. The enzyme-linked immunosorbent assay (ELISA) has been used extensively to detect numerous medicinal, agricultural and environmental compounds. It is an effective tool for screening samples that will be subsequently analyzed by another method, as well as for rapid, quantitative analysis of a large number of samples. A commercially available kit has been applied successfully to the screening of polychlorinated biphenyls (PCBs) in transformer oil samples. The kit uses a competitive inhibition enzyme immunoassay (EIA) for recognition of the PCB structure. The test specificity is restricted to PCBs, with high sensitivity for Aroclors 1016, 1242, 1248, 1254 and 1260. Kit performance was validated by comparison of EIA and GC results for 35 transformer oil samples. 0 1997 Elsevier Science B.V. Keywords:
Immunoassay; PCBs; Waste oil
1. Introduction
Polychlorinated biphenyls are toxic molecules, which when ingested, bind themselves to human fat tissues and act as possible carcinogens. There is little evidence that PCBs are harmful to humans
unless they are ingested. PCBs exhibit physical properties that promoted their use in electrical equipment such as trans* Corresponding author. Tel.: + 33 1 30127059;fax: + 33 1 30127180.
formers or condensers. When leaking, those equipments contaminate water, soil and food supplies with PCBs. Moreover, the burning of PCBs generates dioxins. According to the US EPA (Environmental Protection Agency) a liquid that has a PCB level less than 50 ppm is not considered PCB contaminated, PCB level of 50 ppm or more constitute contaminations, and concentration over 500 ppm the liquid is ‘pure PCBs’. The high lipophilicity and stability of PCBs resulted in their widespread distribution in the global ecosystem and led to their ban in 1976
004%9697/97/$17.000 1997Elsevier ScienceB.V. All rights reserved. PI1 SOO48-9697(96)05389-2
58
A’. Landwrt
PI ul.
The
Srimw
of the
(EEC) and 1977 (USA). Their hazardous potential needs a careful monitoring system. Jmmunoassays have been used extensively in medical diagnostics for over three decades with an excellent record of reliability, speed of analysis, ease of use and cost-effectiveness. Immunochemical methods have gained universal appeal and are now widely used for the detection of low molecular weight components in environmental analysis [l-3].
2. Material
Sample
Enoironmrnt
196
(1997)
57-61
reach the groove of the strip and immediately the strip is removed from the chamber and allowed to dry. The PCB zone is cut and eluted with 0.4 ml of methanol (for a 5 ppm detection level) or with 1.6 ml of methanol (for a 50 ppm detection level). The eluate (after centrifugation, 1 min at 2000g) is used directly for EIA analysis. However it is noteworthy that other action levels such as 10 and 30 ppm (corresponding to mandated levels in Austria), extracts after TLC are diluted in methanol.
and methods
The EnviroGardTM immunoassay used in this study was originally developed for analysis of PC3 in soil as previously described by Harrison and Menylchuk in 1995 141.As this PCB test kit is designed to operate in aqueous solutions, a sample preparation procedure was developed to adapt it to the analysis of transformer oil. Il.
Total
preparation
21.1. Sflmpk estwction procedure Transformer oil sample (0.5 ml) is added to 1.25 ml of acetonitrile in an extraction vial. The extraction vial is vortexed for 1 min at high speed and then centrifuged for 1 min at 2000 x g.
A mass of 2 g ‘of cerium ammonium nitrate is weighed into an oxidation bottle. Then, 1 ml of acetonitrile, 0.5 ml of iso-octane and 1 ml of H$O, are added to the bottle. The oxidation bottle is vortexed 10 s at high speed before the addition of 1 ml of sample. The bottle is vortexed again 30 s and 8 ml of ‘Quenching Buffer’ is added. The oxidation bottle is vortexed 30 s and allowed to settle for at least 5 min.
The topmost iso-octane layer (from the oxidation bottle) is transferred into a microcentrifuge tube. The iso-octane sample extract is applied on the TLC strip: 50 ,uI for a detection at 5 ppm, or 70 jr1 for a detection at 50 ppm. The TLC strip is developed in hcxane. The solvent front is let to
21.4. Imnutnoassay method: A schematic representation of the kit protocol is shown in Fig. 1. The sample or standard is incubated for 15 min. at room temperature in a tube coated with anti-PCB antibodies. After washing away the unbound material with tap water, a PCB-enzyme conjugate is added to each tube and incubated for 5 min. During this incubation, the anti-PCB antibody captures the PCB-enzyme conjugate at any of the PCB binding sites which have not already been occupied by PCB from the sample or the standard. The tubes are washed again with tap water and a colourless enzyme substrate is added. The tubes are allowed to incubate for an additional 5 min at room temperature before adding dilute sulfuric acid to stop the reaction. Color development is proportional to the amount of enzyme bound, and therefore inversely proportional to the PCB concentration in the sample or standard. The optical density (O.D.) of each tube is read on a portable photometer. Samples are compared to 5 and 50 ppm PCBs calibrators. A sample having greater color than a calibrator contains less PCB than the calibrator concentration, while a sample having less color than a calibrator may contain more PCB than the calibrator concentration. The actual concentration of the calibrator solutions are less than the nominal concentrations in order to guarantee detection of the less strongly recognized Aroclors such as 1242, and to limit the frequency of false negative results. This strategy allows 95% confidence detection of positive samples at any calibration level.
N. Lamhert et al. i The Science of’ the Total Enrironntent 196 (1997) 57-61
Dilution of sample or calibrator is incubated in tube containing immobilized antibodies.
Sample matrix is washed away, only PCBs bound to antibodies,
0
59
PCB Anti-PCB
Antibody
leaving
zncttbati6n 2 : PCB-HRP binds to free anti-PCB on immobilized antibodies.
sites
@Zi
Enzyme E= HRP
Conjugate (Horse Radish
Peroxydase
Colorless substrate and chromogen are converted to blue color in proportion to amount of bound enzyme. Less color mmPCB. Stop solution inactivates the HRP. changes color to yellow, and stabilizes color.
Fig.
1. Schematic
representation
S = Substrate C = Chromogen
of the EnviroGard
3. Results and discussion
TheprimarypurposeoftheEnviroGardPCBassay is to screen out samples having PCB concentrations around certain mandated levels. Results were interpreted relative to four different calibrators; at 5 and 50 pg/g nominal concentration
Enzyme)
TM Competitive
EIA
for PCB
in oil.
(Tablel)andat1Oand30~gg/gnominalconcentration corresponding to the Austrian norm (Table 2). The correlation study consisted of 35 different waste oil samples that were previously analyzed by GC-ECD (Method 8080) and found to have PCB concentrations between 0 and 670 ppm. Results are reported in Tables 1 and 2.
60
N. Lamberr
et al. / The Science
of
the Total
Environment
196 (1997)
57-61
Table 1 Comparison of results obtained by PCB EIA and GC-ECD Sample no.
1 2 3 4 5 10 19 20 58 62 96 98 102 103 104 III II? 294 295 296 339 357 368 390 397
CC results (ppm)
69 630 39 60 55.4 20 5.4 100 14 6.5 3 I 105 40 52 1.3 1.2 490 410 570 60 44 39 20 traces
Immunoassay results 5 wm
Correlation
50 wm
Correlation
>5 >5 25 >5 >5 25 >5 >5 >5 >5 <5 15 >5 >5 25 <5 <5 >5 >5 15 >5 >5 >5 r5 15
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
>50 >50 <50 >50 <5050 <50 t50 <50 <50 >50 150 >50 150 <50 >50 >50 >50 >50 >50 <50 r50 <50
Yes Yes Yes Yes No Yes Yes Yes Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes Yes Yes No Yes No Yes
GC-ECD results are indicated in parts per million @pm). EIA results are relative to the calibrators included with the kit, at the nominal concentrations 5 and 50 ppm.
One false negative sample was observed; sample 5. 55 ,ug/g by GC-ECD (Table 1) which was very closed to the 50 ppm calibrator. The four other samples showing GC-EIA disagreement (103, 357 and 390 in Table 1 and sample 400 in Table 3) are all apparent false positives. The fact that theses samples contained predominantly Aroclor 1260, which is better recognized than Aroclor 1248 by the test, probably accounts for the apparent false positive results. It is possible to reduce the false positive rate in such situations by using matching Aroclor calibrators with an actual concentration closer to the nominal concentration. The test kit is biased away from false negative results so that the least reactive Aroclors will be detected. The probability of a false positive result depends on the actual level
of contamination and on the Aroclors in the sample. Interpretation of the results based on the data of Table 1 yielded 8.6% false positive and 3% false negative at 50 ppm action level. There were no false-negative and no false-positive results at 30 ppm action level (Table 2). For screening methods directed toward environmental applications, falsepositive results are preferred over false-negatives. Finally agreement between the immunoassay test kit and GC was 89%. 4. Conclusion
The evaluation described above demonstrated that the PCB EIA kit is an effective screening tool for analysis of PCBs in waste oil.
N. Lambert
rt al. ! Thr Science
of‘ the Total
Environment
196 (1997)
57
hl
61
Table 2 Comparison of results obtained by PCB EIA and GC-ECD. Sample no.
GC results (ppm)
__-. I5 25 30 100 200 300 400 29 558 29 406 29 688
670 65 7.8 500 49 25 7.8 410 490 570
Immunoassay results
10 wm
Correlation
30 ppm
Correlation
>I0 > IO > to >I0 >I0 >I0 >I0 >I0 > IO > 10
Yes Yes Yes Yes Yes Yes No Yes Yes Yes
>?O >30 >30 >30 >30 <30 <30 >30 >30 r30
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
GC-ECD results are indicated in parts per million (ppm). EIA results are relative to the calibrators included with the kit, at the nominal concentration of 10 and 30 ppm.
Assay sensitivity in the 5-50 ppm range makes the test results relevant to regulatory decision making. The method is accurate, the false-positive and false-negative results that were observed can be explained by differences in the immunoreactivities of the Aroclors present in the test samples and the Aroclors used as standard in the assay. The use of this kit offers a significant timeand cost-saving alternative to laboratory analytical methods that are currently used to measure PCBs in oil. This test kit will also be useful in the laboratory as a prescreening method to eliminate samples negative for PCBs and to define dilution requirements for
samples that are to be analyzed methods.
by standard
References [l] Sherry, J.P.. 1992. Environmental chemistry: The immunoassav, oation. Crit. Rev. Anal. Chem.. 23: 217-230. . [2] Ferguson, B.S., D.E. Kelsey, T.S. Fan and R.J. Bushway. 1993. Pesticide testing by enzyme immunoassay at trace levels in environmental and agricultural samples. Sci. Total Environ.. 132: 415-428. [3] Skerritt, J.H., 1995. Field Immunoassays for pesticide residues. In: D.A. Kurtz, J.H. Skerritt and L.S. Stankers (Edsj, New Frontiers in Agrochemical immunoassay. AOAC, Arlington, pp. 91-102. [4] Harrison, R.O. and N. Melnychuk. 1995. Rapid analysis of PCBs in soil by enzyme Immunoassay. Intern. J. Environ. Anal. Chem., 59: 1799185.
The Scienceof the Total Environment 196 (1997) 57-61
Analysis of PCBs in waste oil by enzyme immunoassay Nadine Lambert”,*, Titan S. Fanb, Jean-FranCois Pilette” “Analytical
Product Division, bBeacon Analytical
Millipore Systems,
S.A., BP 307, 78054 Saint Quentin-en-Yvelines 4 Washington Ave., Scarborough, 04074 Maine,
cedex France USA
Received 29 July 1996; accepted 8 October 1996
Abstract Immunoassays constitute a powerful analytical technique for sensitive, quantitative, fast and economical determination of trace contaminants in environmental monitoring. The enzyme-linked immunosorbent assay (ELISA) has been used extensively to detect numerous medicinal, agricultural and environmental compounds. It is an effective tool for screening samples that will be subsequently analyzed by another method, as well as for rapid, quantitative analysis of a large number of samples. A commercially available kit has been applied successfully to the screening of polychlorinated biphenyls (PCBs) in transformer oil samples. The kit uses a competitive inhibition enzyme immunoassay (EIA) for recognition of the PCB structure. The test specificity is restricted to PCBs, with high sensitivity for Aroclors 1016, 1242, 1248, 1254 and 1260. Kit performance was validated by comparison of EIA and GC results for 35 transformer oil samples. 0 1997 Elsevier Science B.V. Keywords:
Immunoassay; PCBs; Waste oil
1. Introduction
Polychlorinated biphenyls are toxic molecules, which when ingested, bind themselves to human fat tissues and act as possible carcinogens. There is little evidence that PCBs are harmful to humans
unless they are ingested. PCBs exhibit physical properties that promoted their use in electrical equipment such as trans* Corresponding author. Tel.: + 33 1 30127059;fax: + 33 1 30127180.
formers or condensers. When leaking, those equipments contaminate water, soil and food supplies with PCBs. Moreover, the burning of PCBs generates dioxins. According to the US EPA (Environmental Protection Agency) a liquid that has a PCB level less than 50 ppm is not considered PCB contaminated, PCB level of 50 ppm or more constitute contaminations, and concentration over 500 ppm the liquid is ‘pure PCBs’. The high lipophilicity and stability of PCBs resulted in their widespread distribution in the global ecosystem and led to their ban in 1976
004%9697/97/$17.000 1997Elsevier ScienceB.V. All rights reserved. PI1 SOO48-9697(96)05389-2
58
A’. Landwrt
PI ul.
The
Srimw
of the
(EEC) and 1977 (USA). Their hazardous potential needs a careful monitoring system. Jmmunoassays have been used extensively in medical diagnostics for over three decades with an excellent record of reliability, speed of analysis, ease of use and cost-effectiveness. Immunochemical methods have gained universal appeal and are now widely used for the detection of low molecular weight components in environmental analysis [l-3].
2. Material
Sample
Enoironmrnt
196
(1997)
57-61
reach the groove of the strip and immediately the strip is removed from the chamber and allowed to dry. The PCB zone is cut and eluted with 0.4 ml of methanol (for a 5 ppm detection level) or with 1.6 ml of methanol (for a 50 ppm detection level). The eluate (after centrifugation, 1 min at 2000g) is used directly for EIA analysis. However it is noteworthy that other action levels such as 10 and 30 ppm (corresponding to mandated levels in Austria), extracts after TLC are diluted in methanol.
and methods
The EnviroGardTM immunoassay used in this study was originally developed for analysis of PC3 in soil as previously described by Harrison and Menylchuk in 1995 141.As this PCB test kit is designed to operate in aqueous solutions, a sample preparation procedure was developed to adapt it to the analysis of transformer oil. Il.
Total
preparation
21.1. Sflmpk estwction procedure Transformer oil sample (0.5 ml) is added to 1.25 ml of acetonitrile in an extraction vial. The extraction vial is vortexed for 1 min at high speed and then centrifuged for 1 min at 2000 x g.
A mass of 2 g ‘of cerium ammonium nitrate is weighed into an oxidation bottle. Then, 1 ml of acetonitrile, 0.5 ml of iso-octane and 1 ml of H$O, are added to the bottle. The oxidation bottle is vortexed 10 s at high speed before the addition of 1 ml of sample. The bottle is vortexed again 30 s and 8 ml of ‘Quenching Buffer’ is added. The oxidation bottle is vortexed 30 s and allowed to settle for at least 5 min.
The topmost iso-octane layer (from the oxidation bottle) is transferred into a microcentrifuge tube. The iso-octane sample extract is applied on the TLC strip: 50 ,uI for a detection at 5 ppm, or 70 jr1 for a detection at 50 ppm. The TLC strip is developed in hcxane. The solvent front is let to
21.4. Imnutnoassay method: A schematic representation of the kit protocol is shown in Fig. 1. The sample or standard is incubated for 15 min. at room temperature in a tube coated with anti-PCB antibodies. After washing away the unbound material with tap water, a PCB-enzyme conjugate is added to each tube and incubated for 5 min. During this incubation, the anti-PCB antibody captures the PCB-enzyme conjugate at any of the PCB binding sites which have not already been occupied by PCB from the sample or the standard. The tubes are washed again with tap water and a colourless enzyme substrate is added. The tubes are allowed to incubate for an additional 5 min at room temperature before adding dilute sulfuric acid to stop the reaction. Color development is proportional to the amount of enzyme bound, and therefore inversely proportional to the PCB concentration in the sample or standard. The optical density (O.D.) of each tube is read on a portable photometer. Samples are compared to 5 and 50 ppm PCBs calibrators. A sample having greater color than a calibrator contains less PCB than the calibrator concentration, while a sample having less color than a calibrator may contain more PCB than the calibrator concentration. The actual concentration of the calibrator solutions are less than the nominal concentrations in order to guarantee detection of the less strongly recognized Aroclors such as 1242, and to limit the frequency of false negative results. This strategy allows 95% confidence detection of positive samples at any calibration level.
N. Lamhert et al. i The Science of’ the Total Enrironntent 196 (1997) 57-61
Dilution of sample or calibrator is incubated in tube containing immobilized antibodies.
Sample matrix is washed away, only PCBs bound to antibodies,
0
59
PCB Anti-PCB
Antibody
leaving
zncttbati6n 2 : PCB-HRP binds to free anti-PCB on immobilized antibodies.
sites
@Zi
Enzyme E= HRP
Conjugate (Horse Radish
Peroxydase
Colorless substrate and chromogen are converted to blue color in proportion to amount of bound enzyme. Less color mmPCB. Stop solution inactivates the HRP. changes color to yellow, and stabilizes color.
Fig.
1. Schematic
representation
S = Substrate C = Chromogen
of the EnviroGard
3. Results and discussion
TheprimarypurposeoftheEnviroGardPCBassay is to screen out samples having PCB concentrations around certain mandated levels. Results were interpreted relative to four different calibrators; at 5 and 50 pg/g nominal concentration
Enzyme)
TM Competitive
EIA
for PCB
in oil.
(Tablel)andat1Oand30~gg/gnominalconcentration corresponding to the Austrian norm (Table 2). The correlation study consisted of 35 different waste oil samples that were previously analyzed by GC-ECD (Method 8080) and found to have PCB concentrations between 0 and 670 ppm. Results are reported in Tables 1 and 2.
60
N. Lamberr
et al. / The Science
of
the Total
Environment
196 (1997)
57-61
Table 1 Comparison of results obtained by PCB EIA and GC-ECD Sample no.
1 2 3 4 5 10 19 20 58 62 96 98 102 103 104 III II? 294 295 296 339 357 368 390 397
CC results (ppm)
69 630 39 60 55.4 20 5.4 100 14 6.5 3 I 105 40 52 1.3 1.2 490 410 570 60 44 39 20 traces
Immunoassay results 5 wm
Correlation
50 wm
Correlation
>5 >5 25 >5 >5 25 >5 >5 >5 >5 <5 15 >5 >5 25 <5 <5 >5 >5 15 >5 >5 >5 r5 15
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
>50 >50 <50 >50 <50
Yes Yes Yes Yes No Yes Yes Yes Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes Yes Yes No Yes No Yes
GC-ECD results are indicated in parts per million @pm). EIA results are relative to the calibrators included with the kit, at the nominal concentrations 5 and 50 ppm.
One false negative sample was observed; sample 5. 55 ,ug/g by GC-ECD (Table 1) which was very closed to the 50 ppm calibrator. The four other samples showing GC-EIA disagreement (103, 357 and 390 in Table 1 and sample 400 in Table 3) are all apparent false positives. The fact that theses samples contained predominantly Aroclor 1260, which is better recognized than Aroclor 1248 by the test, probably accounts for the apparent false positive results. It is possible to reduce the false positive rate in such situations by using matching Aroclor calibrators with an actual concentration closer to the nominal concentration. The test kit is biased away from false negative results so that the least reactive Aroclors will be detected. The probability of a false positive result depends on the actual level
of contamination and on the Aroclors in the sample. Interpretation of the results based on the data of Table 1 yielded 8.6% false positive and 3% false negative at 50 ppm action level. There were no false-negative and no false-positive results at 30 ppm action level (Table 2). For screening methods directed toward environmental applications, falsepositive results are preferred over false-negatives. Finally agreement between the immunoassay test kit and GC was 89%. 4. Conclusion
The evaluation described above demonstrated that the PCB EIA kit is an effective screening tool for analysis of PCBs in waste oil.
N. Lambert
rt al. ! Thr Science
of‘ the Total
Environment
196 (1997)
57
hl
61
Table 2 Comparison of results obtained by PCB EIA and GC-ECD. Sample no.
GC results (ppm)
__-. I5 25 30 100 200 300 400 29 558 29 406 29 688
670 65 7.8 500 49 25 7.8 410 490 570
Immunoassay results
10 wm
Correlation
30 ppm
Correlation
>I0 > IO > to >I0 >I0 >I0 >I0 >I0 > IO > 10
Yes Yes Yes Yes Yes Yes No Yes Yes Yes
>?O >30 >30 >30 >30 <30 <30 >30 >30 r30
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
GC-ECD results are indicated in parts per million (ppm). EIA results are relative to the calibrators included with the kit, at the nominal concentration of 10 and 30 ppm.
Assay sensitivity in the 5-50 ppm range makes the test results relevant to regulatory decision making. The method is accurate, the false-positive and false-negative results that were observed can be explained by differences in the immunoreactivities of the Aroclors present in the test samples and the Aroclors used as standard in the assay. The use of this kit offers a significant timeand cost-saving alternative to laboratory analytical methods that are currently used to measure PCBs in oil. This test kit will also be useful in the laboratory as a prescreening method to eliminate samples negative for PCBs and to define dilution requirements for
samples that are to be analyzed methods.
by standard
References [l] Sherry, J.P.. 1992. Environmental chemistry: The immunoassav, oation. Crit. Rev. Anal. Chem.. 23: 217-230. . [2] Ferguson, B.S., D.E. Kelsey, T.S. Fan and R.J. Bushway. 1993. Pesticide testing by enzyme immunoassay at trace levels in environmental and agricultural samples. Sci. Total Environ.. 132: 415-428. [3] Skerritt, J.H., 1995. Field Immunoassays for pesticide residues. In: D.A. Kurtz, J.H. Skerritt and L.S. Stankers (Edsj, New Frontiers in Agrochemical immunoassay. AOAC, Arlington, pp. 91-102. [4] Harrison, R.O. and N. Melnychuk. 1995. Rapid analysis of PCBs in soil by enzyme Immunoassay. Intern. J. Environ. Anal. Chem., 59: 1799185.