Beta-agonist residues in broilers: kidney and liver data
Beta-agonist residues in broilers concentrate in kidney and liver, not in the breast meat that dominates the food supply. In a 168-bird trial, clenbuterol reached 117.76 micrograms per kg in kidney at the first withdrawal sample and dropped below the decision limit by day 30. Breast muscle peaked far lower. Neither dose of any compound improved body weight.
Why beta-agonist residues in broilers matter for export testing
Beta-adrenergic agonists are banned as growth promoters across the European Union. Clenbuterol and salbutamol sit on routine screening lists for poultry under national residue-control programs. Any exporter shipping into a market with that framework needs three answers: what the test looks for, where in the carcass it will be found, and how long it takes to clear.
That third question is harder than it looks. In cattle, pigs and sheep these compounds repartition nutrients toward lean tissue and away from fat. Poultry works differently. Feed conversion in a modern broiler already sits close to a biological ceiling, and published work on beta-agonists in chickens has given inconsistent performance results.
The trial below measured what actually happens in edible tissue when three compounds enter through drinking water for seven days and are then withdrawn. It produced two datasets: a growth performance record and a tissue residue record covering kidney, liver and breast muscle across an eight-point withdrawal schedule.
How the broiler study was designed
| Design element | Specification |
|---|---|
| Birds | 168 one-day-old Ross 308 broiler chicks from a commercial hatchery |
| Groups | 7 total, 6 treated plus 1 untreated control, 24 birds per group |
| Replicates | 4 floor pens per group, 6 birds per pen, about 10 birds per square meter |
| Allocation | Complete randomized design, computer-generated, balanced for initial body weight |
| Compounds | Salbutamol, clenbuterol and terbutaline, purity 98 percent or higher |
| Concentrations | 2.5 and 5 mg per liter of drinking water |
| Route | Drinking water, prepared fresh daily, supplied ad libitum |
| Treatment window | 7 consecutive days, days 13 to 19 of age |
| Withdrawal | Untreated water and basal diet from day 19 to day 46 |
| Sampling days | 21, 22, 23, 25, 28, 30, 39 and 46 of age |
| Birds per sample | 3 per treatment group per timepoint, selected without replacement |
| Tissues | Breast muscle, liver and kidney |
| Analytical method | LC-MS/MS with stable isotope labeled internal standards, compliant with Commission Implementing Regulation (EU) 2021/808 |
| Weighing days | 20, 22, 24, 26, 33 and 40 of age, after an 8 hour fast |
The exposure route deserves a flag. Drinking water was chosen to model covert administration, not an authorized feed protocol, because medicated water is one of the practical routes a banned compound takes into a flock. Water intake was never measured per bird. The nominal concentration is therefore a group-level exposure marker, not an individual dose. That distinction matters when the residue numbers are read against dose.
Housing followed standard commercial practice for the strain. Temperature started at 32 to 33 degrees Celsius in week one and fell by 2 to 3 degrees per week to reach 22 to 24 degrees Celsius by day 21. Humidity held between 50 and 60 percent. Ammonia stayed below 10 ppm. Feed ran starter, grower and finisher phases at 22, 20 and 18 percent crude protein. No growth promoters, anabolic additives or other veterinary medicinal products were used outside the experimental compounds.
How the analytical method performed
| Analyte | Matrix | Linear range (micrograms per kg) | R squared | Decision limit, CC alpha (micrograms per kg) | Detection capability, CC beta (micrograms per kg) |
|---|---|---|---|---|---|
| Clenbuterol | Muscle | 0.05 to 1.00 | 0.9992 | 0.11 | 0.13 |
| Clenbuterol | Liver | 0.10 to 1.00 | 0.9987 | 0.12 | 0.15 |
| Clenbuterol | Kidney | 0.05 to 1.00 | 0.9985 | 0.11 | 0.14 |
| Salbutamol | Muscle | 0.25 to 2.00 | 0.9990 | 0.53 | 0.58 |
| Salbutamol | Liver | 0.25 to 2.00 | 0.9982 | 0.55 | 0.62 |
| Salbutamol | Kidney | 0.25 to 2.00 | 0.9980 | 0.54 | 0.60 |
| Terbutaline | Muscle | 0.25 to 2.00 | 0.9988 | 0.52 | 0.57 |
| Terbutaline | Liver | 0.25 to 2.00 | 0.9981 | 0.56 | 0.63 |
| Terbutaline | Kidney | 0.25 to 2.00 | 0.9980 | 0.55 | 0.61 |
Linearity held across every analyte and matrix combination, with coefficients of determination at 0.998 or higher. Sample preparation used solid phase extraction clean-up. Quantification relied on stable isotope labeled internal standards added before extraction.
These compounds are banned in food-producing animals rather than regulated, so there is no maximum residue limit to compare a result against. The regulatory reference point is the decision limit: the concentration at which a laboratory can conclude with a defined statistical confidence that the analyte is present. Two recovery gaps are documented in the method. Terbutaline in muscle recovered at 67.4 to 70.9 percent, and salbutamol in liver at 75.3 to 93.5 percent. Both matrix pairs still met the repeatability and reproducibility criteria in the regulation.
Body weight: no growth promotion at either dose
| Group | Day 20 | Day 22 | Day 24 | Day 26 | Day 33 | Day 40 |
|---|---|---|---|---|---|---|
| Untreated control | 952 plus or minus 51 | 1,088 plus or minus 52 | 1,462 plus or minus 91 | 1,700 plus or minus 73 | 2,727 plus or minus 176 | 3,610 plus or minus 277 |
| Salbutamol 2.5 mg/L | 896 plus or minus 57 | 979 plus or minus 55 | 1,368 plus or minus 115 | 1,547 plus or minus 108 | 2,446 plus or minus 159 | 3,237 plus or minus 260 |
| Salbutamol 5 mg/L | 880 plus or minus 63 | 995 plus or minus 71 | 1,457 plus or minus 97 | 1,610 plus or minus 112 | 2,554 plus or minus 215 | 3,384 plus or minus 326 |
| Clenbuterol 2.5 mg/L | 869 plus or minus 52 | 1,002 plus or minus 80 | 1,369 plus or minus 101 | 1,527 plus or minus 145 | 2,380 plus or minus 476 | 3,106 plus or minus 626 |
| Clenbuterol 5 mg/L | 855 plus or minus 72 | 982 plus or minus 89 | 1,367 plus or minus 116 | 1,560 plus or minus 119 | 2,449 plus or minus 256 | 3,214 plus or minus 501 |
| Terbutaline 2.5 mg/L | 889 plus or minus 67 | 1,002 plus or minus 55 | 1,406 plus or minus 113 | 1,565 plus or minus 163 | 2,553 plus or minus 255 | 3,409 plus or minus 251 |
| Terbutaline 5 mg/L | 879 plus or minus 63 | 1,004 plus or minus 72 | 1,395 plus or minus 132 | 1,590 plus or minus 129 | 2,467 plus or minus 235 | 3,223 plus or minus 482 |
All values are in grams. At day 20, before any bird had been removed for residue work, every treated group weighed significantly less than the untreated control on a one-way ANOVA with Tukey HSD testing at the 0.05 level. The control group stayed heaviest at every later measurement. The 2.5 and 5 mg/L arms of each compound differed inconsistently, with no clear concentration-dependent pattern.
After day 20 the analysis is descriptive only. Birds were being removed on schedule for tissue sampling, so group sizes shrank and no statistics were reported for the later weighings. The direction of the difference never reversed.
Where the residues go: kidney, liver and breast muscle
Quantifiable beta-agonist residues in broilers appeared in all three tissues at the first post-treatment sample on day 21. The distribution was consistent and predictable. Kidney and liver carried the highest concentrations. Breast muscle carried the lowest and cleared fastest.
Kidney residue depletion
| Compound and dose | Day 21 | Day 22 | Day 23 | Day 25 | Day 28 | Day 30 | Day 39 | Day 46 |
|---|---|---|---|---|---|---|---|---|
| Clenbuterol 2.5 mg/L | 117.76 | 30.59 | 27.56 | 2.54 | 0.13 | below 0.127 | below 0.127 | below 0.127 |
| Clenbuterol 5 mg/L | 114.29 | 30.00 | 26.18 | 2.44 | below 0.127 | below 0.127 | below 0.127 | below 0.127 |
| Salbutamol 2.5 mg/L | 110.90 | 54.06 | 38.91 | 10.19 | 1.66 | 0.52 | below 0.48 | below 0.48 |
| Salbutamol 5 mg/L | 94.07 | 85.89 | 16.79 | 9.52 | 1.88 | 0.54 | below 0.48 | below 0.48 |
| Terbutaline 2.5 mg/L | 40.57 | 26.13 | 11.10 | 4.15 | 0.77 | 0.24 | below 0.47 | below 0.47 |
| Terbutaline 5 mg/L | 132.79 | 44.57 | 33.26 | 4.58 | below 0.47 | below 0.47 | below 0.47 | below 0.47 |
All values are micrograms per kg. Kidney started high and fell fast. Clenbuterol went from 117.76 micrograms per kg on day 21 to 0.13 by day 28, a fall of roughly 900-fold in a week, and sat below the decision limit from day 30 onward. Salbutamol cleared more slowly and was still readable at 0.52 to 0.54 micrograms per kg on day 30. Terbutaline’s kidney profile is the least orderly of the three, and the two dose arms crossed each other: 132.79 micrograms per kg in the 5 mg/L arm on day 21 against 40.57 in the 2.5 mg/L arm.
Liver residue depletion
| Compound and dose | Day 21 | Day 22 | Day 23 | Day 25 | Day 28 | Day 30 | Day 39 | Day 46 |
|---|---|---|---|---|---|---|---|---|
| Clenbuterol 2.5 mg/L | 120 | 85 | 65 | 28 | 5.5 | 2.0 | below 0.133 | below 0.133 |
| Clenbuterol 5 mg/L | 160 | 110 | 85 | 38 | 7.5 | 3.0 | below 0.133 | below 0.133 |
| Salbutamol 2.5 mg/L | 150 | 105 | 78 | 34 | 6.5 | 1.8 | below 0.45 | below 0.45 |
| Salbutamol 5 mg/L | 190 | 135 | 98 | 42 | 8.0 | 2.4 | below 0.45 | below 0.45 |
| Terbutaline 2.5 mg/L | 90 | 62 | 47 | 17 | 1.4 | 0.5 | below 0.45 | below 0.45 |
| Terbutaline 5 mg/L | 120 | 82 | 63 | 22 | 2.0 | 0.8 | below 0.45 | below 0.45 |
All values are micrograms per kg. Liver held the highest single concentration in the study, 190 micrograms per kg for salbutamol at 5 mg/L on day 21. Depletion was steadier here than in kidney, and the dose arms behaved in the expected order, with 5 mg/L above 2.5 mg/L for all three compounds at every reading. Liver residues still measured 1.4 to 8.0 micrograms per kg on day 28, well above decision limits of 0.12 to 0.56, before clearing by day 39.
Breast muscle residue depletion
| Compound and dose | Day 21 | Day 22 | Day 23 | Day 25 | Day 28 | Day 30 | Day 39 | Day 46 |
|---|---|---|---|---|---|---|---|---|
| Clenbuterol 2.5 mg/L | 30.0 | 16.81 | 26.90 | 5.0 | 0.55 | below 0.07 | below 0.07 | below 0.07 |
| Clenbuterol 5 mg/L | 25.0 | 11.54 | 32.91 | 20.0 | 17.83 | 2.0 | below 0.07 | below 0.07 |
| Salbutamol 2.5 mg/L | 45.0 | 40.22 | 23.66 | 8.0 | 1.16 | below 0.47 | below 0.47 | below 0.47 |
| Salbutamol 5 mg/L | 70.0 | 66.08 | 12.29 | 6.0 | 1.56 | below 0.47 | below 0.47 | below 0.47 |
| Terbutaline 2.5 mg/L | 18.0 | 14.93 | 21.22 | 6.0 | 0.88 | below 0.46 | below 0.46 | below 0.46 |
| Terbutaline 5 mg/L | 25.0 | 15.59 | 36.77 | 7.0 | below 0.46 | below 0.46 | below 0.46 | below 0.46 |
All values are micrograms per kg. Breast muscle started one order of magnitude below liver and two below the highest kidney values, and it cleared faster. The highest reading was 70 micrograms per kg for salbutamol at 5 mg/L on day 21. One striking exception stands out. Clenbuterol at 5 mg/L rose from 11.54 micrograms per kg on day 22 to 32.91 on day 23, still measured 17.83 on day 28, then dropped to 2.0 on day 30. That irregular profile is why muscle residues could not be fitted to a depletion curve at all.
Apparent depletion half-lives by tissue
| Compound | Matrix | Dose (mg/L) | Rate constant, per day | Apparent half-life (days) | R squared |
|---|---|---|---|---|---|
| Clenbuterol | Liver | 2.5 | 0.462 | 1.50 | 0.992 |
| Clenbuterol | Liver | 5 | 0.450 | 1.54 | 0.992 |
| Salbutamol | Liver | 2.5 | 0.491 | 1.41 | 0.988 |
| Salbutamol | Liver | 5 | 0.488 | 1.42 | 0.990 |
| Terbutaline | Liver | 2.5 | 0.605 | 1.14 | 0.982 |
| Terbutaline | Liver | 5 | 0.587 | 1.18 | 0.984 |
| Clenbuterol | Kidney | 2.5 | 0.961 | 0.72 | 0.987 |
| Clenbuterol | Kidney | 5 | 0.917 | 0.76 | 0.958 |
| Salbutamol | Kidney | 2.5 | 0.596 | 1.16 | 0.998 |
| Salbutamol | Kidney | 5 | 0.573 | 1.21 | 0.973 |
| Terbutaline | Kidney | 2.5 | 0.570 | 1.22 | 0.998 |
| Terbutaline | Kidney | 5 | 0.812 | 0.85 | 0.976 |
Liver half-lives ran 1.14 to 1.54 days. Kidney half-lives ran 0.72 to 1.22 days. The fits were strong at 0.958 to 0.998. Clenbuterol was slowest to clear from liver at 1.50 to 1.54 days and fastest from kidney at 0.72 to 0.76 days. Terbutaline went the other way in liver, clearing in 1.14 to 1.18 days, the fastest liver value of the three.
Call these apparent half-lives, not pharmacokinetic parameters. They come from log-linear regression on group means of three birds, not from individual animal curves. Muscle was never fitted, because its concentration profiles were too irregular.
The counter-intuitive findings
Four results run against the intuition a producer or a laboratory would bring to this data.
The first is that the growth-promoting effect did not appear at all. Beta-agonists are used illegally precisely because they repartition nutrients in ruminants and pigs. In this broiler trial, every treated group weighed less than the control at day 20 and stayed behind. One plausible explanation is a ceiling effect. Decades of selection for growth rate and breast muscle yield have pushed protein accretion in this strain close to what nutrient supply, cardiovascular capacity and skeletal frame can support. Little room is left for an anabolic stimulus to exploit.
The second is that a higher concentration in the water did not reliably produce a higher residue. In several tissue and timepoint combinations the 5 mg/L arm sat at or below the 2.5 mg/L arm. Terbutaline in kidney is the clearest case: 40.57 micrograms per kg in the low-dose group against 132.79 in the high-dose group on day 21, then reversing by day 30. The birds shared drinking water, individual intake was never measured, and nominal concentration is a poor proxy for individual exposure.
The third is where the residue actually sits. Kidney and liver, the organs a consumer rarely eats in quantity, carried 117 to 190 micrograms per kg at the first sample. Breast muscle, the tissue that dominates poultry consumption, peaked at 70 and mostly stayed lower. A testing program built around breast muscle alone is looking at the matrix with the least residue and the fastest clearance.
The fourth is the shape of the breast muscle curve for clenbuterol at 5 mg/L, which climbed between days 22 and 23 instead of falling. Residue behavior in muscle is not a smooth decay.
What this means for residue testing programs
Residue surveillance for beta-agonist residues in broilers has to choose a matrix, a timepoint and a threshold. This dataset informs all three.
On matrix, kidney and liver give the longest detection window and the highest concentrations. Breast muscle is the consumer-facing tissue and has to be tested for food safety reasons, but it will return negative earlier than the organs.
On timepoint, the gap between day 28 and day 39 is decisive. Liver residues on day 28 ranged from 1.4 to 8.0 micrograms per kg, far above the decision limits. By day 39 no analyte exceeded its limit in any tissue, and day 46 confirmed it. A sampling schedule that misses the day 21 to day 30 window will read clean even in a flock that was dosed.
On threshold, decision limits differ by compound by a factor of about five, from 0.11 micrograms per kg for clenbuterol in muscle to 0.56 for terbutaline in liver. Reporting a result without naming the compound and matrix the limit applies to makes the number uninterpretable.
Practical recommendations
- Test kidney or liver when you want the longest window of detection. They held the highest concentrations at every early timepoint.
- Treat the first 10 days after exposure stops as the informative window. Most readable residue sits in days 21 to 30 of age in this design.
- Do not assume a cleaner result from a lower nominal dose. The 5 mg/L arms were sometimes lower than the 2.5 mg/L arms here.
- Report the compound-specific decision limit alongside every result. Clenbuterol and salbutamol differ by roughly fivefold in the same matrix.
- Expect breast muscle to be the weak matrix for surveillance. It starts lowest and clears fastest.
- Check confirmation criteria rather than concentration alone if the regulatory question is whether a banned compound was used at all.
- Treat day 39 onward as clean in this dataset, not as a universal withdrawal rule. Seven-day exposure starting at day 13 is a narrow slice of real-world misuse patterns.
- Keep sample handling disciplined. Samples here were snap-frozen in liquid nitrogen, held at minus 20 degrees Celsius, and analyzed within 30 days.
Limitations
The largest limitation is statistical. The beta-agonist residues in broilers reported here are group means of three birds per treatment per timepoint, and the individual per-bird results were not retained. No standard deviations, no confidence intervals and no assessment of biological variation between birds appear anywhere in the residue dataset. Every residue figure is a point estimate, and the dose reversals that show up in the data cannot be separated from sampling noise.
Three method validation elements are also absent, and the study says so directly. Freeze-thaw stability, processed-sample stability, and carry-over plus dilution integrity were not evaluated. The authors note that these gaps do not affect confirmation of identity, which rested on retention time plus one quantifier and one qualifier MRM transition. They do matter when comparing these concentrations with results from fully validated official-control methods.
Exposure was group-level rather than individual. Medicated water was shared, intake was not measured per bird, and the exact dose any animal received is unknown. The birds were a mixed-sex population and sex was not included as an experimental factor. The treatment window was seven days starting at day 13 of age, which models one specific exposure scenario rather than the range of conditions found in the field. Body weight is the only performance endpoint reported, so nothing can be said about carcass composition or feed efficiency from this dataset. Muscle residue data were never fitted to a depletion curve, because the profiles were too irregular. Growth performance was analyzed statistically only at day 20, because scheduled tissue sampling progressively reduced group sizes afterwards.
Bottom line
Beta-agonist residues in broilers behave predictably in kidney and liver and unpredictably in breast muscle. Clenbuterol at 5 mg/L in drinking water left 160 micrograms per kg in liver and 114.29 in kidney at the first withdrawal sample, while breast muscle never exceeded 70. Kidney and liver cleared to below their decision limits by day 30. Every tissue was clean by day 39 and still clean at day 46. None of the three compounds improved body weight, and a higher concentration in the water did not reliably produce a higher residue. The residue numbers are single point estimates from three birds per group, so treat the pattern as robust and the exact values as indicative.
FAQ
How long does clenbuterol stay in broiler tissue?
In this trial, clenbuterol at 2.5 mg/L left 117.76 micrograms per kg in kidney on day 21 and 120 in liver, and both tissues were below their decision limits by day 30. Breast muscle cleared more slowly in the 5 mg/L arm, still reading 17.83 micrograms per kg on day 28. Every tissue was below its decision limit by day 39.
Which tissue carries the highest beta-agonist residues in broilers?
Kidney and liver. Clenbuterol hit 117.76 micrograms per kg in kidney and 160 in liver on the first withdrawal sample, while breast muscle peaked at 70. Liver held the single highest value in the study, 190 micrograms per kg for salbutamol at 5 mg/L. Breast muscle consistently carried the lowest concentrations and cleared the fastest.
Why did beta-agonists not increase broiler body weight?
All treated groups weighed significantly less than the control at day 20 and stayed behind afterwards. The likely reason is a ceiling effect. Modern Ross 308 broilers have been selected so intensively for growth rate and breast muscle yield that protein accretion already sits near the physiological maximum, leaving little room for a nutrient-repartitioning stimulus to add anything.
What is the decision limit for clenbuterol in chicken muscle?
0.11 micrograms per kg in this study, with a detection capability of 0.13. The decision limit is the concentration at which a laboratory can conclude with defined confidence that the analyte is present. Limits differ by compound and matrix, ranging from 0.11 micrograms per kg for clenbuterol in muscle to 0.56 for terbutaline in liver.
How were the residues measured in this study?
By LC-MS/MS using stable isotope labeled internal standards added before extraction, with solid phase extraction clean-up, following Commission Implementing Regulation (EU) 2021/808. Coefficients of determination were 0.998 or higher in all nine analyte and matrix combinations. Two recovery gaps are reported: terbutaline in muscle at 67.4 to 70.9 percent and salbutamol in liver at 75.3 to 93.5 percent.
Can a higher dose in drinking water give lower residues?
Yes, and it did here. In several tissue and timepoint combinations the 5 mg/L groups showed residues at or below the 2.5 mg/L groups. Terbutaline in kidney is the clearest example: 40.57 micrograms per kg at 2.5 mg/L against 132.79 at 5 mg/L on day 21. Birds shared medicated water, so individual intake was never measured and nominal concentration is not a reliable dose marker.
How long is the withdrawal period for beta-agonists in poultry?
This study cannot supply a withdrawal rule, because the exposure it tested was a single seven-day window starting at day 13 of age. What it does show is the clearance shape: liver residues remained well above the decision limits on day 28, and no analyte exceeded its limit in any tissue on day 39 or day 46.
What are beta-agonist residues in broilers tested for?
For evidence that a banned growth promoter was used. Clenbuterol and salbutamol sit on routine screening lists for poultry under national residue-control programs, and beta-adrenergic agonists are prohibited as growth promoters across the European Union. Because these compounds are banned rather than regulated, there is no maximum residue limit and the decision limit is the regulatory reference point.
Is salbutamol allowed in poultry production?
Not as a growth promoter. It is prohibited in food-producing animals across the European Union under the same framework that bans the other beta-agonists. It remains a human prescription medicine for asthma, which is exactly why residues in meat are a public health as well as a trade issue: they can also confound legitimate therapeutic use and doping controls.
Why is breast muscle the weak matrix for beta-agonist testing?
Because it starts lowest and clears fastest. Breast muscle peaked at 70 micrograms per kg in this study against 190 in liver, and most of it was gone by day 30. Kidney and liver hold higher concentrations for longer, so a surveillance program built only on breast muscle will miss exposures that organ testing would still catch.
What does beta-agonist residue data not tell you?
It does not distinguish absorption, distribution, metabolism and excretion. Tissue concentrations cannot say how much compound an animal absorbed or how fast it was metabolized. Two further gaps apply specifically here: no standard deviations are reported because individual bird results were not retained, and freeze-thaw stability, processed-sample stability and dilution integrity were never assessed.
Are beta-agonist residues in broilers a food safety risk?
The risk framing depends on the matrix and the timing. Clenbuterol and salbutamol residues reached three-figure micrograms per kg in kidney and liver within days of exposure stopping, which is why those organs matter for surveillance. Breast muscle, the tissue most people eat, carried the lowest concentrations and the shortest persistence in this trial.
