Hatchery Losses in Flocks of Layer and Broiler Breeders Due to Feed Contamination with Nicarbazin and Narasin: A Case Report.

In the current study, we investigated decreased hatchability and increased embryonic mortality in two farms of layer breeders (flocks A1 and B1) and a farm of broiler breeders (flocks C1 and C2) from Austria, which also presented discoloration of eggshells in 2% of the eggs. After conducting clinical evaluations and the approval that the feed operator was common for flocks A1 and B1, and C1 and C2, it was decided to investigate the feed. Our findings revealed that the feed contained levels of nicarbazin and narasin up to five and 14 times, respectively, above the maximum limits allowed by the European Union for nontarget species. On the other hand, there were no significant abnormalities in vitamin levels, which were also described as the etiology of the noticed abnormalities. Switching to a noncontaminated feed resulted in the clinical signs and production parameters returning to expected ranges. This report emphasizes the significance of considering feed contamination by nicarbazin and narasin as a potential cause of hatchery losses in nontarget species, even in the absence of other clinical signs.

Reporte de caso- Pérdidas en la eclosión de parvadas de reproductoras ponedoras y pollos de engorde debido a la contaminación del alimento con nicarbazina y narasina: Reporte de un caso. En el presente estudio, se investigó la disminución de la incubabilidad y el aumento de la mortalidad embrionaria en dos granjas de reproductoras ponedoras (parvadas A1 y B1) y una granja de reproductoras de pollos de engorde (parvadas C1 y C2) de Austria, que también presentaron decoloración del cascarón en el 2% de los huevos. Luego de realizar evaluaciones clínicas y la aprobación de que el operador de alimento era común para las parvadas A1 y B1, y C1 y C2, se decidió investigar el alimento. Nuestros hallazgos revelaron que el alimento contenía niveles de nicarbazina y narasina de hasta cinco y 14 veces, respectivamente, por encima de los límites máximos permitidos por la Unión Europea para especies no objetivo. Por otro lado, no se observaron anomalías significativas en los niveles de vitaminas, lo que también se describió como la etiología de las anomalías observadas. El cambio a un alimento no contaminado provocó que los signos clínicos y los parámetros de producción regresaran a los rangos esperados. Este informe enfatiza la importancia de considerar la contaminación del alimento por nicarbazina y narasina como una causa potencial de pérdidas en la eclosión de especies no objetivo, incluso en ausencia de otros signos clínicos.

Determination and Identification of Nicarbazin, Measured as 4,4'-Dinitrocarbanilide (DNC), in Chicken Tissues by Liquid Chromatography With Tandem Mass Spectrometry: Final Action 2013.07.

BACKGROUND: AOAC Method 2013.07 was adopted as First Action in 2013. Since then, the method has been used in numerous residue depletion studies with favorable comments from analysts. OBJECTIVE: To analyze data from residue depletion studies to support Final Action status. METHOD: Ten residue depletion studies were conducted during May 2014 through May 2019. For each study, harvested incurred tissues were analyzed for nicarbazin using AOAC Method 2013.07 in 1 of 4 laboratories. Each analytical run included one or more fortified quality control test portions. The data from these known fortified matrix test portions were analyzed for reproducibility and repeatability. RESULTS: For muscle tissues, relative recovery was 90.4% (95% CI 83.8 to 97.5); RSDr was 5.4% (95% CI 3.8 to 9.2); and RSDR was 7.9%. In the liver, values were 94.5% (95% CI 91.1 to 98.0), 5.8% (95% CI 4.1 to 9.9), and 6.8%, respectively. In the kidney, values were 91.5% (95% CI 85.3 to 98.1), 5.2% (95% CI 3.7 to 8.8), and 9.0%, respectively. In skin with adhering fat, values were 94.5% (95% CI 89.2 to 100.1), 8.9% (95% CI 6.3 to 15.1), and 8.9%, respectively. In all cases, repeatability and reproducibility were within acceptable limits. CONCLUSIONS: The data and positive feedback support the transition of AOAC Method 2013.07 from First Action to Final Action. HIGHLIGHTS: Final action status is supported by data collected during routine use of the method rather than a traditional multi-laboratory collaborative study. Data were subjected to statistical analysis using the pC-metamer, and then transformed back to the traditional C-metamer.

Multiple response optimization of a QuEChERS extraction and HPLC analysis of diclazuril, nicarbazin and lasalocid in chicken liver.

A method for the simultaneous determination of three commonly used coccidiostats in chicken liver was developed, comprising a multi-residue QuEChERS (quick, easy, cheap, effective, rugged and safe) extraction step, and a liquid chromatography-ultra violet-fluorescence (HPLC-UV/FL) analysis. The QuEChERS extraction was optimized using an experimental design approach that includes a screening step to obtain the critical variables, an optimization step using multiple response surface analysis and the calculation of a desirability parameter. The optimized method was validated with fortified samples, reaching an average recovery of 91% and an overall precision of 5.5% (mean of three analytes at three levels). Limits of detection calculated on fortified samples were 20 µg kg-1 for lasalocid, 15 µg kg-1 for nicarbazin and 120 µg kg-1 for diclazuril. These values resulted at least one order of magnitude lower than the maximum allowed residue limit (MRL) of the studied coccidiostats for chicken liver.

Current research, regulation, risk, analytical methods and monitoring results for nicarbazin in chicken meat: A perspective review.

This review presents up-to-date information about current research on nicarbazin, one of the most used anticoccidials in poultry production. The focus is to elucidate regulation concerning nicarbazin, limits for its residues in food, how maximum residue limits in different countries are calculated regarding edible chicken tissues and the possible implications in human health. Analytical methods to extract and quantify this residue, expressed as dinitrocarbanilide (DNC) are presented and discussed, including qualitative screening and quantitative/confirmatory analytical methods. Monitoring results and occurrence of DNC residues in chicken meat are discussed. Additionally, the causes of eventual chicken meat contamination and possible solutions to reduce or eliminate DNC residue in tissues are also presented. The paper concludes with perspectives, the current state of DNC residue analysis and suggestions for future research, especially considering the gap in the study of residue recycling effect due to continuous chicken litter use.

Development and validation of a multiresidue liquid chromatography/tandem mass spectrometry method for 11 coccidiostats in feed.

A confirmatory method for the determination of 11 regulated coccidiostats including the ionophore antibiotics lasalocid, maduramicin, monensin, narasin, salinomycin, and semduramicin and the chemical coccidiostats decoquinate, diclazuril, halofuginone, nicarbazin, and robenidine in animal feed was developed and validated. The procedure was intended for the identification and quantification of the coccidiostats at concentrations relating both to the unintentional carryover as stated in Regulation 574/2011 and to the authorized levels in target feed. The analytes were determined by LC/MS/MS in the positive or negative electrospray ionization mode. The method performance characteristics were estimated in the relevant application field from 0.003 to 200 mg/kg. Validation criteria of linearity, specificity, trueness, precision, LOD, and LOQ along with measurement uncertainty were estimated for all analytes. Absolute and relative matrix effects were also studied. The results proved that the method performance was satisfactory, and it was successfully applied to carryover control by analyzing 165 feed samples collected within regulatory monitoring plans. Finally, since the carryover phenomenon in feed may result in the presence of residues in food products of animal origin, a survey has been carried out on the occurrence of coccidiostats in 167 eggs and animal muscles.

Feed additives diclazuril and nicarbazin in egg and liver samples from Croatian farms.

In total 307 egg and 275 liver samples were examined for nicarbazin and 365 eggs for diclazuril over a 30-month period. Enzyme-linked immunosorbent assay methods used for quantification were validated according to European Commission Decision 2002/657/EC. Non-compliant samples were confirmed by LC-MS/MS. Mean diclazuril concentrations in egg samples were 0.31 µg kg⁻¹, which is below the MRL. In only one egg sample, 2.26 µg kg⁻¹ was determined by enzyme-linked immunosorbent assay, although confirmation by LC-MS/MS gave a value of 1.6 µg kg(-1). Mean nicarbazin levels determined were 1.85 µg kg⁻¹ in egg and 21.1 µg kg⁻¹ in liver samples. Four samples, one egg and three livers, yielded elevated concentrations of nicarbazin, but only in the egg sample the LC-MS/MS method confirmed nicarbazin (106 µg kg⁻¹) above the MRL value.

Determination of narasin and monensin in bovine, swine, and chicken tissues by liquid chromatography with tandem mass spectrometry: first action 2011.24.

The single-laboratory validation (SLV) of an LC-MS/MS method for determination and confirmation of two ionophores, narasin and monensin, in animal tissues is described. The data demonstrated linearity of matrix-matched calibration curves using a weighted (1/x) regression and selectivity of the method for narasin and monensin in the presence of lasalocid, salinomycin, maduramycin, nicarbazin, and sulfadiazine. Recoveries varied from 86.2 to 103.5% for narasin and 89.1 to 105.1% for monensin. Intertrial repeatability precision [relative standard deviation of repeatability (RSDr)] varied from 3.9 to 13.8% for narasin and 3.3 to 16.3% for monensin in fortified tissue. Precision of the method was verified in incurred tissues. The LOQ of the method was validated and ranged from 0.45 ng/g in milk, to 4.0 ng/g in chicken fat, but was 0.75 ng/g for most tissues. Two confirmatory ions for each analyte were examined across all matrixes, resulting in estimated false-negative rates of 0.00% (95% confidence interval of 0.00-0.68%) for monensin ions (540 samples) compared to the U.S. and European Union (EU) acceptance criteria. The confirmatory ions for narasin demonstrated 0.00% false-negative rates (95% confidence interval of 0.00-0.58%) when compared to either the U.S. or EU criteria in 630 samples. The method was robust when small changes in method parameters were made and stability of fortified tissues, extracts, and calibration solutions were estimated. The data satisfy the requirements of the AOAC Stakeholder Panel on Veterinary Drug Residue for SLV studies, and the method was adopted Official Methods of Analysis First Action 2011.24 by the AOAC Expert Review Panel on Veterinary Drug Residues.

A novel ion-pairing chromatographic method for the simultaneous determination of both nicarbazin components in feed additives: chemometric tools for improving the optimization and validation.

The development, optimization and validation of an ion-pairing high performance liquid chromatography method for the simultaneous determination of both nicarbazin (NIC) components: 4,4'-dinitrocarbanilide (DNC) and 2-hydroxy-4,6-dimethylpyrimidine (HDP) in bulk materials and feed additives are described. An experimental design was used for the optimization of the chromatographic system. Four variables, including mobile phase composition and oven temperature, were analyzed through a central composite design exploring their contribution to analyte separation. Five responses: peak resolutions, HDP capacity factor, HDP tailing and analysis time, were modelled by using the response surface methodology and were optimized simultaneously by implementing the desirability function. The optimum conditions resulted in a mobile phase consisting of 10.0 mmol L(-1) of 1-heptanesulfonate, 20.0 mmol L(-1) of sodium acetate, pH=3.30 buffer and acetonitrile in a gradient system at a flow rate of 1.00 mL min(-1). Column was an INERSTIL ODS-3 (4.6 mm×150 mm, 5 µm particle size) at 40.0°C. Detection was performed at 300 nm by a diode array detector. The validation results of the method indicated a high selectivity and good precision characteristics, with RSD less than 1.0% for both components, both in intra and inter-assay precision studies. Linearity was proved for a range of 32.0-50.0 µg mL(-1) of NIC in sample solution. The recovery, studied at three different fortification levels, varied from 98.0 to 101.4 for HDP and from 99.1 to 100.2 for DNC. The applicability of the method was demonstrated by determining DNC and HDP content in raw materials and commercial formulations used for coccidiosis prevention. Assays results on real samples showed that considerable differences in molecular ratio DNC:HDP exist among them.

Application of total error approach to assess the performance of a biological method (ELISA) to detect nicarbazin residues in eggs.

Nicarbazin, a coccidiostat, is used as a feed additive in poultry but not in laying hens. Feed contamination may however occur resulting in residues being present in eggs. As a Maximum Residue Limit (MRL) does not exist for nicarbazin residues in eggs a "Differential Action Level" (DAL) of 100 microg/kg has been established by the Veterinary Medicines Directorate (VMD). We have studied a commercial ELISA kit validated to detect and quantify nicarbazin in eggs with a sensitivity of 3 microg/kg. We used the total error approach to assess the performance of and validate the kit at the DAL level. The accuracy profile has been successfully obtained for the ELISA kit. The method cannot however be validated as a semi-quantitative method and we have consequently determined a cut-off based on 5% false negative rate according to European Decision 2002/657 on blank and spiked samples (70 microg/kg). The cut-off value established was 20 microg/kg using the 95th percentile.

Survey of the anticoccidial feed additive nicarbazin (as dinitrocarbanilide residues) in poultry and eggs.

A survey was carried out on the occurrence of dinitrocarbanilide (DNC), the marker residue for nicarbazin, in poultry produced in Ireland during 2002-2004. Liver (n = 736) and breast muscle samples (n = 342) were tested. DNC residues were found in 40 and 26% of liver and breast muscle samples at levels greater than 12.5 and 5 microg kg(-1), respectively. DNC residues were found at >200 microg kg(-1) in 12 and 0% of liver and muscle samples, respectively. Samples of breast muscle (n = 217) imported from 11 countries were also tested for DNC residues. A lower incidence of DNC residues (6%) was found in imported breast muscle. Egg samples (n = 546) were tested and DNC residues were found in nine samples, with levels ranging between 14 and 122 microg kg(-1). Analysis of poultry, carried out as part of official food inspection in the period 2004-2006, indicated a reduction in the number of broiler liver samples containing DNC at >200 microg kg(-1), to approximately 7%. Low levels of DNC residues continue to be found in <2% of egg samples.

Investigation of the causes for the occurrence of residues of the anticoccidial feed additive nicarbazin in commercial poultry.

Investigations were undertaken to identify causes for the occurrence of high levels of the zootechnical feed additive nicarbazin in broiler liver at slaughter. The first investigation on 32 commercial broiler flocks involved sampling and analysis for nicarbazin (as dinitrocarbanilide, DNC) in liver from birds during a 3-10-day period after withdrawal of nicarbazin from their feed and before commercial slaughter. DNC residues in liver samples of broilers scheduled as being withdrawn from nicarbazin for > or =6 days ranged from 20 to >1600 microg kg(-1) (the specified withdrawal period for nicarbazin is 5 days and the Joint Expert Committee on Food Additives (JECFA) maximum residue limit (MRL) is 200 microg kg(-1) liver). Further on-farm investigations on 12 of these flocks, selected on the basis of the feeding system in use and the levels of DNC residues determined in liver, identified issues in feed management contributing to elevated residues in broiler liver. A significant correlation (0.81, p < 0.01, n = 10) between DNC residues in liver samples and in feed samples from the feeding pans was observed. The second investigation on 12 commercial broiler flocks involved sampling and analysis for DNC in liver samples and feed samples from feeding pans and from the feed mill at the three thinnings of birds for commercial slaughter. In the case of one flock, a clear relationship between nicarbazin in feed from the feed mill (10.5 mg kg(-1) DNC), in feed from the feeding pans (6.6 mg kg(-1) DNC) and in liver (583 microg kg(-1) DNC) at first thinning (9 days scheduled withdrawal from nicarbazin) was observed. Such a clear relationship was not observed in other cases, particularly at second and third thinnings, pointing to re-exposure of birds to nicarbazin late in the flock production cycle, probably from the litter. Guidelines outlining best farm practice to eliminate nicarbazin residues in poultry have been published in booklet and poster format for broiler producers and deal with feed system cleaning, feed bin management, feed deliveries, feed usage and records.

Development and validation of a lateral flow device for the detection of nicarbazin contamination in poultry feeds.

Concentrations of the coccidiostat nicarbazin as low as 2 mg/kg in feed can result in violative drug residues arising in poultry liver. A lateral flow device (LFD) was developed for the detection of contaminating concentrations of nicarbazin following solvent extraction of poultry feeds. Test results, as determined by both visual and instrumental measurement, are available within minutes. For 22 feed samples, nicarbazin-free and fortified at 2 mg/kg, the % relative inhibition ranged from 0 to 45% and from 53 to 85%, respectively. Nicarbazin contamination at the critical concentration (2 mg/kg) can be determined in all cases providing the sampling is representative. A wide range of feed samples taken at a mill that incorporated nicarbazin into poultry feed were analyzed. Data generated for these samples by both the LFDs and a mass spectrometric method were compared, and a significant correlation was achieved.

The effect of cooking on veterinary drug residues in food: nicarbazin (dinitrocarbanilide component).

The change of concentration of residues of the marker compound for the anti-coccidial drug nicarbazin, N,N'-bis(4-nitrophenyl)urea (dinitrocarbanilide, DNC), was investigated in model oil and aqueous solutions and in chicken muscle and egg. In model aqueous solutions, DNC decreased rapidly in concentration upon heating followed by a much more gradual decomposition. The curves produced when this information was plotted were not typical of exponential decay. In model cooking oil solutions, DNC generally showed a slower decrease in concentration over time when compared with aqueous solutions. DNC residues in egg were stable to microwave cooking and residues in chicken muscle were stable to stewing and microwaving. Other cooking procedures led to a decrease in amount of DNC by 22% to 48% of the total amount of analyte present. Only a small amount (<2%) of residue leached with juices which exuded as the food was cooked.

Efficient HPLC method for the determination of nicarbazin, as dinitrocarbanilide in broiler liver.

A simple, fast and reliable HPLC-UV method has been developed for the determination of dinitrocarbanilide residues in broiler liver. Liver samples (2 g) were extracted with two portions of acetonitrile (10 and 5 ml), defatted with hexane and evaporated to dryness under nitrogen. Extracts were reconstituted in acetonitrile-water (70/30, v/v, 500 microl), loaded onto C18 solid phase (SPE) cartridges and eluted with acetonitrile-water (70/30, v/v, 2.5 ml) into clean test-tubes. Extracts were evaporated to dryness and reconstituted in acetonitrile-water (80/20, v/v, 500 microl). An aliquot of the extract was assayed by high performance liquid chromatography (HPLC) with UV detection at 350 nm. The method was validated according to EU guidelines using liver tissues fortified at levels of 100, 200 and 300 microg/kg, with dinitrocarbanilide. The decision limit (CC(alpha)) and the detection capability (CC(beta)) were calculated from the within laboratory repeatability data to be 228 and 266 microg/kg, respectively. The mean recovery was typically >70% and the limits of quantitation was 12.5 microg/kg (based on the lowest standard on the calibration curve).

Screening for the coccidiostats halofuginone and nicarbazin in egg and chicken muscle: development of an ELISA.

Nicarbazin and halofuginone have been widely used as coccidiostats for the prevention and treatment of coccidiosis in poultry. It has been shown that accidental cross-contamination of feed can lead to residues of these compounds in eggs and/or muscle. This paper describes a direct competitive assay for detecting halofuginone and nicarbazin, developed as qualitative screening assay. In an optimized competitive ELISA, antibodies showed 50% binding inhibition at approximately 0.08 ng ml(-1) for halofuginone and 2.5 ng ml(-1) for dinitrocarbanilide (marker residue for nicarbazin). Extraction from the matrix was carried out with acetonitrile followed by a wash with hexane. The assay's detection capability (CCbeta) for halofuginone was < 0.5 microg kg(-1) in egg and < 1 microg kg(-1) in muscle. For dinitrocarbanilide, the CCbeta was estimated at < 3 microg kg(-1) in egg and < 10 microg kg(-1) in chicken muscle.

An all-in-one dry chemistry immunoassay for the screening of coccidiostat nicarbazin in poultry eggs and liver.

An automated immunoassay for the detection of nicarbazin residues in poultry eggs and liver was developed. The assay was based on a novel all-in-one dry chemistry concept and time-resolved fluorometry. The analyte specific antibody was immobilized into a single microtiter well and covered with an insulation layer, on top of which the label was dried in a small volume. The extracted sample was added automatically to the dry microtiter well, and the result was available within 18 min. Due to the rapidity and simplicity, the quantitative immunoassay could also be used as a high throughput screening method. The analytical limit of detection for the assay was calculated as 0.1 ng mL(-)(1) (n = 12) and the functional limit of detection as 3.2 ng g(-)(1) for egg (n = 6) and 11.3 ng g(-)(1) for liver (n = 6) samples. The sample recovery varied from 97.3 to 115.6%. Typically, the intra-assay variations were less than 10%, and interassay variations ranged between 8.1 and 13.6%.

Liquid chromatographic method for nicarbazin in broiler feeds and premixtures: development, validation, and interlaboratory study.

A reversed-phase liquid chromatography method for nicarbazin in broiler feeds and premixtures was developed, validated, and interlaboratory studied. The extraction solvent was an acetonitrile-methanol (1 + 1) mixture. For feedingstuffs, water was also added. The 4,4'-dinitrocarbanilide moiety of nicarbazin was detected at a wavelength of 350 nm. Recovery was > or =87%. At 20 mg/kg, the repeatability was 0.7% and the within-laboratory reproducibility was 2.7%. The limit of determination was <20 mg/kg. Other feed additives did not interfere in the assay that proved to be applicable to broiler feeds from different European Union countries. In an interlaboratory study, 4 positive broiler feeds, 1 blank pig feed, and 1 broiler premixture were analyzed by 19 laboratories using the method developed in this study. The relative standard deviation for repeatability (RSDr) of the feedingstuffs (20-240 mg/kg) varied between 2.6 and 10.2%. The HORRAT ranged between 0.70 and 1.22. Recoveries were 91-108%. Three laboratories detected small signals in the blind blank samples, ranging from 0.4 to 2 mg/kg. For the premixture, acceptable results for reproducibility could only be obtained after the sample weight and volume of extraction had been doubled. To avoid excessive dilution of the extracts, the range of the calibration curve had also been doubled. With this modified method, the RSDr was 5.7% and the HORRAT was 1.95 (10 laboratories).

[Simple determination of residual anticoccidial drugs (diclazuril and nicarbazin) in chicken tissues by HPLC].

A simple and rapid determination of anticoccidial drug residues, diclazuril (DCZ) and nicarbazin (NCZ), in chicken tissues has been developed. DCZ and NCZ were extracted with acetonitrile from chicken liver, muscle, and fat. The extract was rinsed with n-hexane saturated with acetonitrile and then evaporated. The residue was dissolved in 1.4 mL of acetonitrile-methanol (1:1), then 1.0 mL of n-hexane saturated with acetonitrile-methanol (1:1) was added, and the mixture was partitioned by the addition of 0.6 mL of water. DCZ and NCZ in the aqueous layers were determined by HPLC on an Xterra RP-18 column with acetonitrile-0.5% ammonium acetate containing 0.01 mol/L tetra-n-butylammonium hydrogen sulfate (43:57) as the mobile phase. The mean recoveries (n = 5) of DCZ and NCZ spiked in chicken tissues at the maximum residue levels were 92.0-95.6% (CV 2.4-3.0%) and 87.3-89.4% (CV 1.7-2.8%), respectively. The detection limits of DCZ and NCZ were 0.01 and 0.004 microgram/g, respectively.

Contamination of animal feedingstuffs with nicarbazin: investigations in a feed mill.

Some mechanisms of nicarbazin contamination were investigated in a feed mill. Three sequential 3-tonne batches of nicarbazin-free feed were produced directly after a batch of nicarbazin-containing feed (125 mg kg(-1)). Sampling of the nicarbazin-free feed took place at two points before pelleting and at one point post-pelleting. The study was repeated on two further occasions, i.e. three separate nicarbazin-containing feeds and 27 tonnes of 'flushing' feeds were manufactured and sampled in total. Pre-pelleting, the highest nicarbazin concentrations (3.4+/- 0.26 mg kg(-1)) were observed in the first tonne milled after the nicarbazin containing ration. Thereafter, concentrations steadily declined in successive batches. Post-pelleting samples contained much higher concentrations of the drug. After 8 tonnes had passed through, the concentrations (7.2+/- 1.29 mg kg(-1)) were between 10 and 20 times greater than the corresponding concentrations detected post-mixing. These concentrations are sufficient to cause violative residues in eggs and broiler liver. The practice of returning post-press sieved material to the pre-press bins was identified as the cause of the problem. Re-routing of sieved material along with better segregation of nicarbazin-containing and nicarbazin-free feedingstuffs markedly reduced the incidence of feed contamination with this compound.

Determination of 4,4'-dinitrocarbanilide (DNC), a component of Nicarbazin, in Canada goose (Branta canadensis) eggshells using high-performance liquid chromatography.

A method was developed using high-performance liquid chromatography to assay 4,4'-dinitrocarbanilide (DNC), the active ingredient in Nicarbazin, in eggshells collected from Canada geese fed a formulated feed fortified with Nicarbazin at doses of 0, 125, 250, and 500 microg/g. The method was developed using chicken eggshells fortified with DNC. The method was used to quantify DNC in both the shell-associated membranes and the calcified shell extracellular matrix. These values were compared to those obtained for a composite sample consisting of both the membranes and the calcified shell extracellular matrix. The validated method was used to quantify DNC in eggshells from geese fed fortified feed to ascertain the effect of Nicarbazin feed concentration on shell DNC concentration. DNC levels in the eggshells were highly correlated with feed dose.