EPA Method 1613B: Dioxin & Furan Analysis in Industrial Wastewater Using the Empore™ C18 SPE Disk
- Apr 30
- 11 min read
Updated: May 1
Authors: Guotao Lu
Study summary: A validated EPA Method 1613 Rev. B workflow using the Empore™ C18 SPE Disk extracts 16 toxic 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) from 1 L industrial wastewater samples at pH 2. Across four matrices — POTW final effluent, petroleum refinery API separator effluent, refinery final treatment effluent, and river water — all 16 native compounds fell within the EPA-required 75%–125% acceptance window, with an average native recovery of 109%. Empore™ C18 disks tolerated the demanding low-pH, large-volume conditions without C18 phase loss, enabling clean isotope-dilution HRGC/HRMS quantitation of dioxins and furans in industrial effluents.
TL;DR: EPA Method 1613B requires processing 1 L of wastewater at pH 2 for trace-level (ppt) dioxin and furan analysis — conditions that strip C18 phase from many SPE disks and ruin HRMS chromatograms. The Empore™ C18 SPE Disk holds up to these conditions across four real-world wastewater matrices (POTW, refinery, river water) and delivers all 16 monitored PCDDs/PCDFs within EPA's 75%–125% acceptance window — average native recovery 109%. With an in-situ glass fiber prefilter, full extraction completes in under 20 minutes per sample.
Study at a Glance
Introduction: Dioxin Monitoring in Industrial Wastewater
EPA Method 1613 Revision B was developed by the U.S. Environmental Protection Agency's Office of Science and Technology for isomer-specific determination of the 2,3,7,8-substituted, tetra- through octa-chlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs). Detection is performed by isotope dilution using high-resolution capillary column gas chromatography / high-resolution mass spectrometry (HRGC/HRMS).
EPA Method 1613B specifies extraction directions for several matrices including aqueous, solid, and tissue samples. The CDS Empore™ disk extraction procedure described in this application note is applicable to aqueous matrices, making it well-suited for industrial wastewater monitoring under the Clean Water Act and related regulatory programs.
There are two well-known sample-preparation challenges in this method:
Large sample volume — 1 liter per extraction
Low pH — around pH 2
Why disk integrity matters: EPA specifically emphasizes that the potential damage of C18 phase being stripped from the extraction disk packing at pH 2 is to complicate the chromatographic analysis with high background, which could obscure compounds of interest. Empore™ C18 disks consistently work with large samples and low pH without loss of C18 phase from the silica support in the membrane disks.
In this application note, one-liter aqueous samples from four types of wastewater were spiked with isotopically labeled analogs of CDDs/CDFs:
Publicly Owned Treatment Works (POTW) final effluent
Petroleum refinery American Petroleum Institute (API) separator effluent
Petroleum refinery final / secondary treatment effluent
River water
Samples containing sediment were filtered either off-line or in-situ (using a prefilter placed above the Empore™ disk in the extraction glassware). After acidification to pH 2 and addition of 5 mL methanol, samples were extracted with a C18 disk. Both the prefilter and the C18 disk were then placed in a Soxhlet thimble and extracted for 16–24 hours with toluene. The extract was subjected to standard cleanup and concentration techniques per EPA Method 1613B, and detected by isotope-dilution HRGC/HRMS.
The validation data presented herein were determined on three independent lots of C18 disks. Method detection limits (MDLs) were not determined as part of this validation.
Headline result:
All 16 native PCDDs/PCDFs across all 4 wastewater types fell within the EPA Method 1613B acceptance range of 75%–125%
Average native compound recovery = 109%
Only 1 compound below 90%: 12378-PeCDD in POTW effluent (88%)
Only 1 compound above 120%: 123678-HxCDD in river water (123%)
All combined post-extraction filtrates were "not detected" — confirming complete retention by the disk
With in-situ prefilter and settle/decant approach, all extractions completed in under 20 minutes
Why the Empore™ C18 Disk for EPA Method 1613B
EPA Method 1613B places two demanding requirements on the SPE format: 1 L sample volume and pH 2 acidic conditions, with target analytes at parts-per-trillion levels. The Empore™ C18 disk delivers on both:
Resistant to C18 phase loss at pH 2 — particle-loaded PTFE membrane keeps C18-bonded silica securely in place even at low pH, preventing the high chromatographic background that EPA specifically warns against
Handles 1 L large-volume samples — disk format processes large volumes faster than cartridge SPE, with all four wastewater extractions in this study completed in under 20 minutes
Compatible with in-situ glass fiber prefilter — accommodates particle-laden industrial effluents (refinery API separator water, POTW effluent) without clogging
Both 47 mm and 90 mm formats available — 90 mm with prefilter for particle-laden samples; 47 mm for particle-free samples
Validated across 4 real-world wastewater matrices using three independent lots of C18 disks
Materials
SPE Media
Empore™ C18-bonded silica disk — 90 mm (for samples with particulates) or 47 mm (for particle-free samples)
Glass fiber prefilter — Whatman GMF 150, 1 µm pore size (or equivalent), placed on top of the Empore™ disk for particle-laden sample
Glassware & Apparatus
All-glass filtration assembly (for 90 mm or 47 mm disk; manifold acceptable for multiple extractions)
Soxhlet Dean-Stark apparatus for 16–24 hour toluene extraction of disk + prefilter
Glass wool plug
HRGC/HRMS Configuration
Note on instrument parameters: The original application note focused on extraction performance using Empore™ C18 disks. Detailed instrument-specific HRGC/HRMS parameters (capillary column, temperature program, mass resolution settings) follow the directions specified in the official EPA Method 1613 Rev. B publication. Parameters explicitly stated in this validation work are summarized below.
Validated EPA Method 1613B Extraction Procedure
This extraction option is applicable to aqueous samples only. The full workflow for dioxin/furan extraction from wastewater using Empore™ C18 disks is as follows.
1. Sample Preparation
Measure the sample volume. Add isotopically labeled analogs, 5 mL methanol, and adjust pH to 2. Allow the sample to sit so that any sediment can settle as much as is practical.
2. Filtration Apparatus Assembly
Assemble an all-glass filtration assembly using a 90 mm CDS Empore™ C18 extraction disk. For samples with no particulates, a 47 mm disk can be used. Use of a manifold for multiple extractions is acceptable.
For samples with significant particulates: use an in-situ glass micro-fiber prefilter (Whatman GMF 150, 1 µm pore size or equivalent), placed on top of the Empore™ disk prior to placement of the glass reservoir and clamp.
3. Prewash the Apparatus and Disk
Add 15 mL of toluene to the reservoir, washing down the sides of the glass reservoir in the process. Pull a small amount through the disk under vacuum, turn off vacuum, and allow the disk to soak for about one minute. Pull the remaining solvent through and allow the disk to dry. Repeat the wash step using 15 mL of acetone.
4. Condition the Disk
Pre-wet the disk by adding 15 mL methanol to the reservoir. Pull a small amount through, then let it soak for about one minute. Pull most of the remaining methanol through, leaving 3–5 mm on the surface of the disk.
⚠ CRITICAL STEP: The disk must not be allowed to go dry from this point until the sample extraction has been completed. The disk is composed of hydrophobic materials and must be pre-wetted with a water-miscible solvent (methanol) so water can pass through. If the material accidentally dries before sample addition, simply repeat the pre-wetting step.
5. Equilibrate with Reagent Water
Add 50 mL of reagent water to the disk and draw most through, again leaving 3–5 mm of water on the surface. Repeat using a second 50 mL aliquot of reagent water.
6. Sample Extraction
Add the water sample to the reservoir and, under full vacuum, filter as quickly as the vacuum will allow. Drain as much water from the sample bottle as possible. Particulate-free water may pass through the disk in as little as 10 minutes without reducing analyte recoveries. Allow the entire sample to pass through the disk, then dry the disk by maintaining vacuum for about 3 minutes.
For heavily particle-laden samples: allow the sediment to settle, decant as much liquid as practical into the reservoir, allow most of the liquid to filter, then swirl the sediment portion and add it to the reservoir. Before the entire sample has filtered, rinse the sample bottle with reagent water and add to the reservoir to transfer any particulates remaining in the bottle. Drain the sample bottle as completely as possible.
7. Transfer to Soxhlet Thimble
Disassemble the extraction glassware and carefully transfer both the Empore™ disk and the particle-laden GMF prefilter to the thimble of a Soxhlet Dean-Stark apparatus. Place a glass wool plug over the filters. Add about 50 mL toluene to the sample bottle, replace the cap, and agitate well to rinse all remaining residues into the toluene. Transfer the toluene to the Soxhlet apparatus. Repeat the bottle rinse three times. Using 10 mL toluene, rinse the extraction glassware reservoir and transfer to the Soxhlet.
8. Soxhlet Extraction
Soxhlet-extract for 16–24 hours. The validation data for this study was generated using a 16-hour Soxhlet extraction.
9. Cleanup and Analysis
Concentrate the extract and proceed to cleanup and analysis as per the standard EPA Method 1613B directions. Detect by isotope-dilution HRGC/HRMS.
Results and Discussion
Validation studies were performed on four wastewater types provided by EPA: POTW final effluent, petroleum refinery API separator effluent, refinery final/secondary treatment effluent, and river water. Validation data were generated in a single laboratory, with additional recovery data provided by pulp and paper mills on various matrices from their respective plants. Triplicate (n=3) samples of each matrix were analyzed.
One liter of each wastewater type was also analyzed for background levels. Additionally, the post-extraction filtrate from the triplicate spikes was combined and liquid/liquid extracted to document extraction efficiency. In all cases, the combined filtrates were "not detected" for all analytes — confirming complete retention by the Empore™ C18 disk. Using the in-situ prefilter and settle/decant approach, all sample extractions were completed in under 20 minutes.
Spike Levels
Spike levels ranged from 200 pg/L (2378-TCDD) to 2000 pg/L (OCDD), with most analytes spiked at 1000 pg/L. These levels span the parts-per-trillion range typical of trace dioxin and furan analysis in industrial effluent monitoring.
Overall Recovery Performance
Performance summary across 16 native compounds × 4 wastewater matrices:
All 16 native PCDDs/PCDFs across all 4 wastewater types fell within EPA Method 1613B's 75%–125% acceptance range
Average native compound recovery = 109%
14 of 16 compounds (in all 4 matrices) fell within the tighter 90%–120% range
Only 1 below 90%: 12378-PeCDD in POTW effluent (88%)
Only 1 above 120%: 123678-HxCDD in river water (123%)
Compounds flagged "B" indicate the analyte was also present in the laboratory blank
As expected for isotope-dilution HRGC/HRMS workflows, recoveries for ¹³C₁₂-labeled internal standards were lower than for native analytes. Internal standard recoveries primarily document method behavior; quantitative accuracy depends on the native/IS recovery ratio, which automatically corrects for procedural losses during extraction, cleanup, and concentration.
Table 1 — Recovery Data Across Four Wastewater Matrices
Recovery values are reported as %R (%RSD) with n = 3 replicates per matrix.B = analyte present in blank sample.2,3,7,8-TCDF was not determined in this validation.
Conclusion
A simple and effective method for extracting polychlorinated dioxins and furans from large-volume (1 L) wastewater samples using CDS Empore™ C18 47 mm SPE disks has been validated per EPA Method 1613 Rev. B. The 16 tetra- through octa-chlorinated 2,3,7,8-substituted PCDDs and PCDFs targeted by the method were extracted from four representative aqueous matrices and quantified by isotope-dilution HRGC/HRMS at concentrations from 0.2 ppb to 2.0 ppb:
POTW final effluent
Petroleum refinery API separator effluent
Petroleum refinery final / secondary treatment effluent
River water
With the Empore™ disk extraction procedure:
Recovery exceeded 90% for most native compounds
Only 1 compound (12378-PeCDD in POTW effluent) showed 88% recovery
Only 1 compound (123678-HxCDD in river water) showed 123% recovery
All 16 monitored compounds across all 4 wastewater types fell within the EPA acceptance range of 75%–125%
Average native compound recovery = 109%
Excellent analyte recovery and very clean chromatograms can be obtained using CDS Empore™ C18 disks. For laboratories screening wastewater according to EPA Method 1613 Rev. B — or monitoring chlorinated dibenzo-p-dioxins and dibenzofurans in industrial effluents and surface water — CDS Empore™ C18 disks deliver rapid, economical, and reliable sample preparation.
Frequently Asked Questions
What is EPA Method 1613B used for?
EPA Method 1613 Revision B is the U.S. EPA's standard method for isomer-specific determination of 2,3,7,8-substituted, tetra- through octa-chlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) in aqueous, solid, and tissue matrices. Detection is by isotope dilution using high-resolution capillary column gas chromatography / high-resolution mass spectrometry (HRGC/HRMS). The CDS Empore disk extraction procedure described here is applicable to aqueous matrices, including industrial wastewater monitoring.
What recovery rates does the Empore C18 Disk achieve for EPA 1613B?
In a validated 1 L wastewater workflow with n=3 replicates per matrix, all 16 monitored 2,3,7,8-substituted PCDDs and PCDFs across all 4 wastewater types fell within the EPA Method 1613B acceptance range of 75%–125%. The average native compound recovery was 109%. Only one compound (12378-PeCDD in POTW effluent) showed 88% recovery, and only one (123678-HxCDD in river water) showed 123% recovery — every other native compound recovery fell within the tighter 90%–120% window.
Why does EPA Method 1613B require pH 2 sample acidification?
Acidification to pH 2 protonates polar interferents and improves the partitioning of neutral PCDDs and PCDFs onto the hydrophobic C18 phase. EPA specifically warns that disk phase materials must remain stable at pH 2 — any C18 stripped from the silica support during extraction would generate background contamination that obscures trace dioxin peaks in the HRMS chromatogram. CDS Empore™ C18 disks consistently work at pH 2 and large 1 L sample volumes without loss of C18 phase.
Should I use a 47 mm or 90 mm Empore C18 disk for EPA 1613B?
Use a 90 mm disk when samples contain significant particulates (most industrial wastewaters and natural surface waters) — the larger surface area accommodates a glass-fiber prefilter on top and prevents flow restriction. Use a 47 mm disk when samples are particle-free. The 47 mm format was used in this validation across all four wastewater types in conjunction with a Whatman GMF 150 (1 µm) prefilter and a settle/decant approach.
Why is Soxhlet extraction (16–24 hours) required after disk extraction?
Dioxins and furans bind very tightly to C18 phase and to particulates retained on the prefilter. A simple solvent elution would not quantitatively recover these highly hydrophobic, planar molecules. Soxhlet extraction with toluene over 16–24 hours provides the prolonged refluxing solvent contact needed for complete desorption. The validation in this study used 16 hours and achieved an average 109% native recovery.
Why are isotopically labeled (¹³C₁₂) standards added before extraction?
EPA Method 1613B uses isotope-dilution HRGC/HRMS for quantitation. The ¹³C₁₂-labeled analogs are added to the sample before extraction so they experience the same losses as the native target analytes during SPE, Soxhlet extraction, cleanup, and concentration. Quantitation is then based on the ratio of native to labeled signal, which automatically corrects for procedural losses — making the absolute recovery of the internal standard less critical than the recovery ratio.
How does the in-situ prefilter approach handle particle-laden industrial wastewater?
Many industrial effluents (e.g., petroleum refinery API separator water) contain suspended solids that would clog a bare C18 disk. Placing a Whatman GMF 150 (1 µm) glass-fiber prefilter directly on top of the Empore™ disk in the same filtration assembly captures particulates while still allowing aqueous-phase analytes to load onto the C18. Both filter and disk are then transferred together to the Soxhlet thimble for extraction. With this approach, all four wastewater extractions in this study completed in under 20 minutes.
Can this Empore C18 disk procedure be used for solid or tissue matrices?
The CDS Empore disk extraction procedure described here is applicable to aqueous matrices only within the scope of EPA Method 1613B. Validation has been performed on POTW effluent, refinery API separator effluent, refinery secondary treatment effluent, and river water; pulp and paper mills have reported additional recovery data on plant-specific aqueous matrices. For solid or tissue matrices, refer to the alternative extraction procedures described in the parent EPA Method 1613B document.
References
U.S. Environmental Protection Agency. Method 1613: Tetra- through Octa-Chlorinated Dioxins and Furans by Isotope Dilution HRGC/HRMS (Revision B). Environmental Monitoring Systems Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Cincinnati, OH 45268.
U.S. EPA Office of Water. Clean Water Act, the Resource Conservation and Recovery Act, the Comprehensive Environmental Response, Compensation and Liability Act, and the Safe Drinking Water Act. Issued October 1994. Water Resource Center; Mail Code RC-4100; 401 M Street SW; Washington, D.C. 20460.
Lu, G. CDS Analytical Application Note #222: Determination of Chlorinated Dioxins and Furans in Four Types of Wastewater by EPA Method 1613 Rev. B. CDS Analytical, LLC.
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