A step-by-step, EU-compliant n=9 sampling SOP for Indonesian tuna exporters. Clear actions on lot definition, carton selection, 9-unit sampling, screening vs confirmation (rapid kits vs HPLC/UPLC), pass/fail criteria, and COA documentation that clears EU import checks in 2026.
If you’ve ever had a container held at EU border because the histamine COA wasn’t “n=9,” you know the pain. The lab may have given you a single number or a composite result. The competent authority asked for nine. Meanwhile, your buyer is calling. We’ve been there with Indonesian tuna since the mid-2010s, and the path to consistent EU releases is simpler than it looks when you run a tight n=9 SOP.
Here’s our 2026 guide, focused only on EU histamine for tuna. No fluff. Just what works.
The EU rule in 60 seconds: limits and pass/fail
For tuna and other high-histidine species placed on the EU market, the EU sampling plan under Regulation (EC) No 2073/2005 uses:
- n = 9 individual unit samples from the same lot
- m = 100 mg/kg
- M = 200 mg/kg
- c = 2
A lot passes when all three are true:
- The average of the 9 results is ≤ 100 mg/kg (≤ m)
- At most 2 results are between 100 and 200 mg/kg
- No result is > 200 mg/kg
Note: Fishery products that undergo enzyme maturation in brine have different limits (m = 200, M = 400). That’s not tuna in our trade stream, so we stick with 100/200.
What does n=9 actually mean?
It means nine separate analytical results on nine separate unit samples from the same lot. Not 9 injections from one homogenate. Not 3 composites of 3 unless you’re only screening. EU border checks and many buyers now ask for the full set of 9 individual values plus the average.
How do I define a lot for EU histamine sampling of frozen tuna?
Define a lot so it’s homogenous in species, product form, production date window, and cold-chain history. We recommend:
- Species and form: e.g., Yellowfin tuna saku, IQF; Skipjack cube, IQF; Bigeye loin, IVP
- Production window: max 48 hours per lot ID
- Same vessel pool or raw-material intake batch
- Same finished-product spec and glazing
- One continuous cold chain from blast freeze to container load
Examples we use in practice:
- 10,000 kg of Yellowfin Saku (Sushi Grade), IQF, produced 10–11 March, from the same intake pool. One lot, one n=9 test.
- 7,500 kg of Skipjack Cube (WGGS / IQF), 3 production shifts across three days. We split this into two lots because day three ran hotter in the gut room. Two n=9 tests.
When in doubt, split the lot. Rescuing a borderline average later is much harder than testing two tighter lots upfront.
How many cartons should I open to collect the 9 units?
Open at least 9 cartons, distributed across the lot. Don’t pull all nine from the same pallet face.
- If you have 10–20 pallets, pick 3 cartons each from the front, middle, and rear pallets. That’s 9.
- If cartons contain multiple loins/pieces, take one unit from each carton.
- For small retail packs, assemble each unit sample by combining sealed packs from one carton. One unit still represents one carton.
If your lot is tiny (for example 9–12 cartons total), still spread selection across the stack to capture variability.
Step-by-step n=9 sampling SOP (what we actually do)
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Prepare. Pre-label 9 sterile bags and a chain-of-custody form. Calibrated thermometer ready. Sanitized knife for cutting internal core.
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Keep it cold. Sample at -18 C or colder. Work fast. Minimize surface thaw. If you see any partial thawing, note it and reject that unit; pick a replacement carton.
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Randomize with intent. Use a simple randomizer or an A-B-C pattern across the container. Never pick just the easy-to-reach cartons.
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Collect unit samples. Most EU labs prefer 100 g per unit. If mass is limited, confirm your lab accepts 50 g per unit minimum.
- Loins/saku: cut from the inner core to 100 g.
- Cubes: take sub-aliquots from deep inside the bag to avoid surface bias.
- Steaks: cut from the center (avoid trim and bloodlines for consistency).
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Seal and label. Include lot ID, carton ID, unit number 1–9, product form, temp at sampling.
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Courier to lab. Frozen gel or dry ice. Include chain-of-custody and test request specifying: Histamine, n=9, HPLC/UPLC-FLD, report all 9 values and the average, EU 2073/2005 criteria.
Practical takeaway: 9 units, 9 cartons, 9 individual numbers. Ship frozen. Ask the lab to print the acceptance criteria on the COA.
Can I composite the 9 samples for EU compliance?
Short answer: no. Compositing can hide a >200 mg/kg outlier. That’s a fail in EU logic even if the average looks fine. We only use 3×3 composites for fast internal screening when volumes are heavy, then move to 9 individual units for the official COA.
Rapid kits vs HPLC/UPLC confirmation
Rapid kits are useful for in-plant screening. They can save you from loading a risky lot.
- Good for screening: enzymatic/ELISA or lateral-flow kits with decision points around 50–100 mg/kg.
- Watch-outs: matrix effects on high-fat tuna, kit drift near cut-off, and operator technique. We verify questionable results with instrument methods.
For pre-shipment COAs that will face EU customs, use an ISO/IEC 17025-accredited lab running HPLC/UPLC with fluorometric detection. Ask the lab to list the method validation, LOQ, and accreditation scope on the certificate. We’ve seen more EU importers, especially in late 2025, request the lab’s accreditation number and LOQ in the paperwork.
If you need a working list of reliable Indonesian labs and a one-page sampling template, Contact us on whatsapp. We’ll share what we use.
What internal histamine limit should we target?
We run conservative internal actions so the lot comfortably passes the EU n=9 criteria.
- Target average: ≤ 50 mg/kg
- Unit action limit: 80 mg/kg triggers resampling and root-cause check
- Unit reject: ≥ 100 mg/kg segregate, investigate, and consider splitting the lot
Why so tight? Because cold-chain reality and raw-material variability happen. If two units drift into 120–160 mg/kg, your average must still stay ≤ 100 mg/kg. A 50 mg/kg internal target keeps you safe.
How to document a COA that clears EU checks in 2026
Ask your lab to include:
- Product and species. Example: Yellowfin tuna saku, Thunnus albacares, IQF
- Lot ID, production date(s), sample receipt temperature/condition
- Method: HPLC/UPLC-FLD, LOQ, lab ISO/IEC 17025 accreditation number
- Results: all 9 individual values and the arithmetic average
- Acceptance statement cross-referencing EU 2073/2005 criteria (n=9; m=100; M=200; c=2; mean ≤ m)
We attach the COA to the packing list and health certificate set. Some EU importers now ask for raw chromatograms on request. Be ready to provide them quickly.
Common mistakes we still see (and how to avoid them)
- Compositing for the final COA. Don’t. Keep composites for screening only.
- Pulling all 9 units from one pallet face. Spread across the lot. We map carton IDs to a pallet diagram before sampling.
- Warm sampling rooms. Histamine doesn’t go down at 8 C. Keep the product frozen during sampling and shipping to the lab.
- Single-number COAs. Border inspectors increasingly reject these. Insist on 9 values and the mean.
- Late testing. Test pre-shipment, ideally 48–72 hours before loading. If you wait for the vessel cutoff, you lose your buffer.
When this guidance applies (and when it doesn’t)
Use this SOP for raw and frozen tuna products destined for the EU market. That includes Yellowfin Steak, Yellowfin Cube (IQF), saku, loins, and similar. If you’re dealing with enzyme-matured products in brine, you’re in the higher 200/400 mg/kg bracket with the same n=9 framework. Different species or finished goods like brined anchovies need tailored criteria, so confirm the exact category before you sample.
What if one unit is >200 mg/kg in the n=9 test?
That’s a fail. You can’t blend to dilute in the EU. Segregate and divert the lot outside the EU or destroy. Investigate quickly:
- Vessel handling and time-temperature exposure
- Gutting and ice-on time
- Thawing during cutting or glazing
- Extended pre-freeze holding at plant
We trend histamine by vessel, supplier, and seasonality. It’s amazing how fast root causes become obvious when you chart results monthly.
A final word from the cutting room floor
What’s interesting is how predictable good lots become when you’re strict on lot definition and cold-chain discipline. We’ve found that the most reliable EU pass rates come from two habits: 1) never mixing days into one lot when the plant was running hot, and 2) keeping the 50 mg/kg target in everyone’s head, from QC to loading.
If you want our editable SOP, carton selection map, and a one-page COA checklist we use for Yellowfin Saku (Sushi Grade) and Skipjack Cube (WGGS / IQF), Contact us on whatsapp. And if you’re reviewing specifications for a new EU program, you can also View our products.