11.1 Sampling, Preservation & Chain of Custody
Key Takeaways
- A grab sample captures conditions at one instant while a composite sample represents average conditions over a period, and permits specify which is required.
- Flow-proportional composites weight each aliquot by flow and are required where flow varies significantly.
- Most samples are preserved at 4 degrees Celsius, with acid preservation to pH below 2 for metals and for nutrients requiring sulfuric acid.
- Bacteriological sample bottles contain sodium thiosulfate to neutralize residual chlorine and must not be rinsed before sampling.
- Holding times run from collection to analysis, and an exceeded holding time invalidates the result regardless of how carefully the analysis was performed.
11.1 Sampling, Preservation & Chain of Custody
Laboratory Analysis is a full content area on both the water and wastewater standardized exams, and a surprising share of the questions are about the sample, not the test. A perfectly executed analysis of a badly collected sample produces a precisely wrong number.
1. Grab vs. Composite
| Grab sample | Composite sample | |
|---|---|---|
| What it is | A single sample taken at one point in time | Multiple aliquots combined over a period, commonly 24 hours |
| Represents | Conditions at that instant | Average conditions over the period |
| Required for | Parameters that change rapidly or are lost on standing: pH, temperature, dissolved oxygen, chlorine residual, bacteriological, oil and grease, volatile organics, sulfide, cyanide, settleable solids | BOD, COD, TSS, nutrients, metals — most NPDES effluent parameters |
| Never used for | Averaging a variable stream | Anything that changes with time in the container or is lost to volatilization |
The rule that catches people: you can never composite a bacteriological, dissolved oxygen, chlorine residual, pH, volatile organic, or oil and grease sample. They change in the bottle, or the compositing procedure itself destroys them.
Composite types
| Type | Method | When required |
|---|---|---|
| Time-proportional | Equal aliquot volume at equal time intervals | Acceptable where flow is relatively constant |
| Flow-proportional (flow-weighted) | Aliquot volume or frequency varies with flow | Required where flow varies significantly — the usual NPDES requirement |
Worked example. A permit requires a flow-proportional 24-hour composite with a total volume of 2,000 mL and 24 hourly aliquots. Total daily flow is 1.20 MG; the 8:00 a.m. hourly flow is 0.075 MG.
Equal 83 mL aliquots would under-represent the peak hour and bias the result low.
2. Where to Sample
- Collect from a well-mixed, representative location — not from a quiescent corner, not at a wall, not immediately downstream of a chemical injection point.
- For a channel, sample at the centroid of flow at mid-depth, avoiding both the surface film and the bottom sediment.
- For automatic samplers, keep intake line velocity at least 2 ft/s so solids are drawn in rather than settling out in the tubing, and keep the line as short and steep as practical.
- Purge taps and sample lines adequately before collecting — except where the protocol specifically requires stagnant water, as with first-draw lead and copper samples (Section 5.4).
- Sample before and after the unit process you are evaluating when troubleshooting.
3. Preservation and Holding Times
Preservation slows the biological, chemical, and physical changes that begin the moment the sample leaves the stream.
| Parameter | Container | Preservation | Typical holding time |
|---|---|---|---|
| pH | P or G | None | Analyze immediately (15 min) |
| Temperature | — | None | Immediate |
| Dissolved oxygen (probe) | — | None | Immediate |
| Chlorine residual | P or G | None | Analyze immediately (15 min) |
| Turbidity | P or G | Cool 4 °C, dark | 48 hours |
| BOD / CBOD | P or G | Cool 4 °C | 48 hours |
| COD | P or G | Cool 4 °C, H₂SO₄ to pH < 2 | 28 days |
| TSS / TDS | P or G | Cool 4 °C | 7 days |
| Ammonia, TKN, total phosphorus | P or G | Cool 4 °C, H₂SO₄ to pH < 2 | 28 days |
| Nitrate / nitrite | P or G | Cool 4 °C | 48 hours |
| Metals (total) | P or G | HNO₃ to pH < 2 | 6 months |
| Mercury | P or G | HNO₃ to pH < 2 | 28 days |
| Cyanide | P or G | NaOH to pH > 12, cool | 14 days |
| Oil and grease | Glass only | Cool 4 °C, H₂SO₄ or HCl to pH < 2 | 28 days |
| Bacteriological | Sterile, with sodium thiosulfate | Cool 4 °C | Drinking water 30 hours; wastewater 8 hours |
The four rules that matter most
- Holding time runs from collection to analysis. Exceeding it invalidates the result, and the analysis must be repeated on a fresh sample. There is no waiver for a good reason.
- Bacteriological bottles contain sodium thiosulfate to neutralize any chlorine residual, which would otherwise continue killing organisms in transit and bias the result low. Never rinse the bottle — rinsing removes the thiosulfate. Fill to the marked line, leaving headspace for mixing.
- Oil and grease requires a glass container, because oil adheres to plastic. Collect it as a grab and analyze the entire container — you cannot pour off a portion.
- Never overfill a container that has preservative already in it, and never add preservative to a bacteriological bottle.
4. Sterile Technique for Bacteriological Samples
- Select a cold-water tap without a leaking aerator or swivel; remove hoses and screens; flush 2–5 minutes.
- Do not touch the inside of the cap or the bottle neck, and do not set the cap down.
- Fill to the 100 mL line, leaving headspace.
- Record the free and total chlorine residual, time, date, location, and collector.
- Cool to ≤ 10 °C and deliver within the holding time — 30 hours for drinking water compliance samples, 8 hours for wastewater under 40 CFR Part 136.
- Never sample from a drinking fountain, hose bibb, or a tap with a threaded swivel or leaking valve.
5. Chain of Custody
A sample is in custody when it is in your physical possession, in your view, secured by you, or in a secured area restricted to authorized personnel.
What a chain of custody record must show
| Element | Why |
|---|---|
| Unique sample identification | Ties the container to the record |
| Collection date and time | Starts the holding time clock |
| Sample location and type (grab/composite) | Establishes representativeness |
| Collector's name and signature | Accountability |
| Parameters requested and preservation used | Tells the lab what to do |
| Every transfer, with signature, date, and time of both the person relinquishing and the person receiving | Establishes unbroken custody |
| Condition on receipt, including temperature | Verifies preservation held |
Samples are transported in sealed, iced coolers, often with custody seals across the lid so tampering would be visible. A temperature blank is frequently included so the lab can document the receipt temperature without opening a real sample.
Why this matters legally: monitoring data submitted to SC DES supports enforcement, and permit compliance data can end up in court. A break in the chain of custody — an unsigned transfer, a sample left unattended in an unlocked truck, a cooler received at 15 °C — can make an otherwise valid result indefensible. Falsifying any part of it is grounds for license revocation under Chapter 51 and carries independent criminal exposure (Section 1.1).
6. Field Quality Control Samples
| QC sample | What it detects |
|---|---|
| Field blank | Contamination from containers, preservative, or field handling |
| Trip blank | Contamination during transport, especially for volatile organics |
| Equipment / rinsate blank | Inadequate decontamination of reusable sampling equipment |
| Field duplicate | Sampling and analytical precision combined |
| Field matrix spike | Matrix interference and recovery |
A positive field blank means the contamination came from your process, not the water — investigate before reporting any associated result.
Which parameter must always be collected as a grab sample and can never be composited?
Why do bacteriological sample bottles contain sodium thiosulfate, and what must the sampler avoid?
A 24-hour flow-proportional composite of 2,400 mL total is collected in 24 hourly aliquots. Daily flow is 3.0 MG and the flow during one hour was 0.20 MG. What aliquot volume should that hour contribute?
A cooler of NPDES compliance samples arrives at the laboratory at 16 degrees Celsius with an unsigned transfer line on the chain of custody. What is the consequence?