Proper water sample preservation and accurate COD (Chemical Oxygen Demand) determination are essential for reliable water quality assessment, wastewater treatment control, and regulatory compliance. This technical guide covers internationally recognized preservation protocols, container selection, holding times, and the principal COD testing methods aligned with ISO, ASTM, and GB standards. Whether you operate a treatment facility, an environmental monitoring program, or an accredited third-party laboratory, these best practices will help protect data integrity from the point of sampling to the final report.
Technical Overview
Chemical Oxygen Demand (COD) is a aggregate measure of the mass of oxygen required to chemically oxidize the organic and oxidizable inorganic matter in a water sample, using a strong oxidizing agent under strongly acidic and heated conditions. Results are expressed as milligrams of oxygen per liter (mg/L O2), and the parameter serves as a core indicator of organic pollution in discharge permits, receiving-water monitoring, and treatment process optimization.
A COD result, however, is only as reliable as the sample delivered to the laboratory. Collected water is a dynamic system: microbial degradation, volatilization, redox reactions, and adsorption to container walls can measurably alter COD values within hours. For this reason, sample preservation is an inseparable part of COD methodology. Key reference documents include ISO 5667-3 for the preservation and handling of water samples, U.S. EPA 40 CFR Part 136 for approved preservation and holding times, and the determination standards ISO 6060, ASTM D1252, HJ 828 (which superseded GB 11914 in the Chinese regulatory framework), HJ/T 399 for the rapid digestion spectrophotometric method, and ISO 8467 together with GB 11892 for the permanganate index.
Testing Samples
Surface Water and Groundwater
Rivers, lakes, reservoirs, and groundwater typically exhibit low to moderate COD levels. Samples should be collected in pre-cleaned borosilicate glass or chemically resistant polyethylene containers of at least 250 mL, rinsed with the sample before filling where appropriate. Because ambient temperatures accelerate biological activity, samples must be cooled to approximately 4 °C immediately and delivered to the laboratory as quickly as possible, ideally within 24 hours if unpreserved.
Municipal Wastewater and Sewage
Raw sewage carries a high and heterogeneous organic load with significant suspended solids. Composite samples collected over 24 hours are common for compliance monitoring. Containers should be filled to minimize headspace, and the sample must be acidified to pH below 2 with concentrated sulfuric acid to inhibit microbial degradation, then held at 4 °C in the dark. Thorough homogenization before subsampling is critical, because COD is a total parameter that includes particulate organic matter.
Industrial Wastewater and Trade Effluent
Industrial discharges may contain chlorides, sulfides, oils, heavy metals, or refractory organics that influence both preservation strategy and method selection. High chloride concentrations in particular can bias dichromate COD results unless properly masked. Field personnel should verify expected matrices in advance, record the presence of visible phases or floating layers, and ensure that preserved aliquots are well mixed before analysis.
Treated Effluent and Reclaimed Water
Biologically treated effluent and recycled water generally show low COD values where measurement precision matters greatly. Low-range spectrophotometric procedures, careful blank control, and contamination-free containers are recommended. Preservation requirements are identical to those for raw wastewater, although sample stability is somewhat better due to the lower biodegradable fraction.
Testing Items
The core testing items associated with this topic include the following, each with specific preservation requirements:
- CODCr, the dichromate chemical oxygen demand, determined by titrimetric or spectrophotometric techniques.
- Permanganate index (CODMn), typically applied to surface water, drinking water sources, and other low-pollution matrices.
- Supporting parameters frequently tested alongside COD, including BOD5, TOC, TSS, ammonia nitrogen, total phosphorus, and pH, which together characterize the pollution profile of the sample.
- Preservation verification items, such as confirming sample pH below 2 upon laboratory receipt and checking cooler temperature records as part of chain-of-custody documentation.
Testing Methods
Sample Collection and Preservation Protocol
Regardless of the determination technique, the following preservation sequence is applied to COD samples in accordance with ISO 5667-3 and EPA holding-time requirements:
- Select a clean borosilicate glass or high-density polyethylene container of adequate volume, typically 250 to 500 mL.
- Fill the container with the homogenized sample, avoiding cross-contamination from sampling equipment.
- Add concentrated sulfuric acid to lower the pH below 2, generally about 2 mL of acid per liter of sample, to suppress microbial activity and stabilize the oxidizable fraction.
- Refrigerate at approximately 4 °C and protect from light; freezing is not recommended unless validated for the specific matrix.
- Analyze acidified, refrigerated samples within 28 days per EPA 40 CFR Part 136, and as soon as practicable under ISO guidance; unpreserved samples should be analyzed within 24 hours.
- Never use nitric acid, which is itself an oxidant, or hydrochloric acid, which introduces chloride interference.
- Maintain complete chain-of-custody records, field blanks, and duplicate samples for quality assurance.
Dichromate Reflux Method (ISO 6060, ASTM D1252, HJ 828)
The reference technique for CODCr introduces a known excess of potassium dichromate in a boiling mixture of concentrated sulfuric acid, with silver sulfate as a catalyst to ensure oxidation of straight-chain organics. Mercuric sulfate is added to complex chloride ions, which would otherwise consume dichromate and bias results low. The sample is digested under open reflux for approximately two hours or in sealed digestion tubes at about 165 °C, after which the remaining dichromate is back-titrated with standard ferrous ammonium sulfate using a ferroin indicator. The endpoint color change from blue-green to reddish brown allows calculation of oxygen demand, with a typical working range of roughly 10 to 700 mg/L O2 depending on reagent strength.
Rapid Digestion Spectrophotometric Method (HJ/T 399)
This high-throughput variant uses pre-dosed digestion reagents in sealed tubes heated at approximately 165 °C for about 20 minutes. The decrease of orange hexavalent chromium or the increase of green trivalent chromium is measured spectrophotometrically, commonly at around 440 nm for high-range determinations or near 600 nm for low-range determinations. The method is well suited to laboratories processing large sample volumes, provided that calibration standards, reagent blanks, and interference checks are rigorously controlled.
Permanganate Index Method (ISO 8467, GB 11892)
For relatively clean waters, the permanganate index offers a gentler oxidation using potassium permanganate in a hot acidic medium, with a defined digestion period in a boiling water bath. Excess permanganate is back-titrated with sodium oxalate. Because permanganate oxidizes a smaller fraction of organic matter than dichromate, permanganate index values are consistently lower than CODCr values, and the two parameters must never be compared interchangeably.
Interference Control and Quality Assurance
Chloride above approximately 100 to 300 mg/L is the principal interference in dichromate methods and must be masked with mercuric sulfate at the mass ratio specified by the standard, or removed by dilution. Nitrite interference can be eliminated with sulfamic acid. Volatile organics may be lost during open reflux if the condenser is not properly seated. Quality control measures include reagent blanks, calibration verification standards, matrix spikes, duplicate analyses, and control charts. Digestion wastes containing mercury and hexavalent chromium must be segregated and disposed of as hazardous chemical waste in compliance with applicable regulations.
Applications
Combined preservation and COD testing programs support a wide range of regulatory, operational, and scientific objectives:
- Compliance monitoring of municipal and industrial discharge against statutory COD concentration limits.
- Process control at wastewater treatment plants, where COD loading guides aeration demand and biological treatment efficiency.
- Receiving-water and environmental impact assessments for rivers, lakes, and coastal zones.
- Verification of treatment performance during commissioning, upgrades, and regulatory audits.
- Industrial water management, including cooling tower, boiler feed, and reclaimed water quality control.
- Research and method validation studies in accredited third-party laboratories and academic institutions.
FAQ
What is the maximum holding time for COD samples?
Samples acidified to pH below 2 with sulfuric acid and stored at approximately 4 °C may be held for up to 28 days under EPA 40 CFR Part 136, although ISO 5667-3 recommends analysis as soon as practicable. Unpreserved samples should be analyzed within 24 hours because microbial activity rapidly changes the organic content.
Why is sulfuric acid used instead of other acids for preservation?
Sulfuric acid is a non-volatile, non-oxidizing acid under these conditions that reliably suppresses microbial activity. Nitric acid would chemically oxidize the organic fraction and inflate results, while hydrochloric acid would add chloride, a major positive interference in the dichromate determination.
Can plastic containers be used for COD samples?
Yes. Both borosilicate glass and chemically resistant plastics such as HDPE or polypropylene are accepted by major standards for COD. Glass is preferred where trace organic contamination from container materials is a concern. Containers must be cleaned, free of detergents, and dedicated to water sampling.
How is chloride interference in COD testing managed?
Chloride ions are oxidized by dichromate and cause low-biased results. The standard approach is to add mercuric sulfate to complex chloride before digestion, effective for chloride levels up to about 1,000 mg/L depending on the method. For higher salinity samples, dilution or matrix-appropriate alternative procedures should be evaluated.
What is the difference between COD and BOD5?
COD measures the total chemically oxidizable matter in a few hours, while BOD5 measures only the biodegradable fraction consumed by microorganisms over five days. COD values are therefore almost always higher than BOD5 values, and the ratio between them provides useful insight into wastewater biodegradability.
Why do dichromate COD and permanganate index results differ?
Potassium dichromate is a much stronger oxidizing agent than potassium permanganate and oxidizes nearly all organic compounds, whereas permanganate leaves many refractory substances untouched. Consequently, CODCr exceeds the permanganate index for the same sample, and each parameter serves distinct matrices and regulatory contexts.