IRON-BASED MICROTRACERS AND THEIR USE IN FEED APPLICATIONS

Nikolay Barashkov, David Eisenberg
Micro Tracers., Inc, 1370 Van Dyke Ave., San Francisco, CA 94124, United States



ABSTRACT
This presentation is devoted to a wide variety of analytical ferromagnetic tracers used to assure the quality of mixed formula animal and poultry feeds. When formulated in vitamin, mineral or medicated premixes, a MicrotracerTMr serves to mark the presence of the premix in the finished feeds as well as to identify the premix and feeds containing it as proprietary. When assayed quantitatively, a MicrotracersTM can be used to document efficacy of mixing as well as adequacy of batch to batch “cleanout” of mixers and other feed manufacturing equipment.
Iron-based tracers coated with food dye(s), as well as  the recent development of several new types of ferromagnetic microtracers will be discussed
INTRODUCTION
The worldwide formula feed industry manufactures more than 400 million tons annually. Manufacturers waste labor, energy and capital when they mix feeds longer than necessary to achieve a complete blend. Excess mixing may also cause degradation of vitamins and medications. If feed is not completely mixed, portions of the feed will contain either too much or too little of the formulated ingredients. This excess variability causes economic losses to users of the feed and may increase the incidence of illegal drug residues.  Periodic routine mixer testing is both economically and ethically justified. The mid-1990s brought an increased interest from regulatory authorities in many countries in assuring medicated feeds are mixed completely and that all micro ingredients are added as formulated.
Failure to consistently add the formulated levels of micro ingredients may be a greater problem in technologically advanced countries, where computerized micro-bin systems are commonly used to add micro ingredients. Feed manufacturers owe a response to the public to ensure their feeds are properly manufactured. This interest coincides with their self-interest to make the best product reasonably possible. This benefits the feed manufacturer's customers and the feed manufacturer himself if he is an integrated producer. By developing and implementing well-designed sampling and analysis plans to document the efficacy of these manufacturing processes the feed manufacturer can validate his mixing and micro ingredient addition operations.
Different substances including protein, microminerals [1], salt [2], and drugs [3] have been suggested as markers to measure feed mixing efficiency. The tracers for livestock feeds, which include chromium mordanted fiber for the particulate phase and cobalt-ethylene diaminetetracetic acid for the liquid phase have been proposed [4].
U.S. Patent Applications [5,6] describes two types of markers (tracers) which are environmentally stable and resistant to food and feed processing. These products, Generally Recognized As Safe ("GRAS"), consisting of a cellulosic fiber fraction can be used to mark all the baked products from a single bakery or manufacturer. The first type of markers, i.e. the fibrous cell fraction could be included in the fiber component for labeling purposes. Another type of markers is made of specific fibrous cell types from non-traditional bakery food ingredients. These markers would make the identification more specific for a particular baked product, but might not be able to be included in all the baked goods from a particular bakery. Sub-cellular markers could also be isolated and included as part of the fiber portion of the label. The presence of a cellular and/or a secondary sub-cellular fraction would offer additional identification probabilities for baked products. According to [5,6], the secondary marker can be in the form of one or more reactants, particularly chromophoric reactants, which are added to the cellulosic markers. These are compounds that will react with specific reagents to produce color many times more intense than that of dyes or other colorants. Copper is an example of a metallic ion that is commonly found in foods and that can be determined to be located in a particular structure rather than distributed throughout the food matrix.
Robinson [7] proposed several types of tracers, suitable for use as markers containing information about the following: a) a crop variety designation related to the agricultural product; b) a specific trait associated with the agricultural product; c) the agricultural product's genetic content. According to these inventions, the information containing in these markers which can be made of a paper, cardboard, plastic, rubber, metal, fiber or other such material or materials, can be human-readable, machine-readable or both. The human-readable markers include alpha-numeric characters, graphical designs or several different color codes. This type of marker can also be machine readable. For example, it can be scanned or imaged by an optical reader device having optical character reading or other such capabilities.
For the last 30 years the ferromagnetic tracer particles which could be separated from the bulk mix much faster and easier than prior tracers by means of magnetic separators have been successfully used in practice. Number of patents assigned to Micro Tracers, Inc. [8-12] disclose the composition of vehicle which comprises a ground soft iron or other ferromagnetic material. Certified FD&C dyes may be used as identifiable coating materials. Since the tracer particles were to be counted, even major losses of the distinguishing adsorbed layer to the bulk material by abrasion or diffusion could be tolerated without loss of accuracy in the particle count. In animal feed applications, essential nutrients or drugs may themselves be used as identifiable coatings. An advantage of the tracers disclosed in inventions, assigned to Micro Tracers, Inc is that they can be separated from a mix much faster and easier than prior tracers by means of a magnetic probe. These tracers may be separated from a mix by means of a magnetic probe from either a sample of mix, or a moving stream of mix, conveyed during production The vehicle for the ferromagnetic tracers can be magnetized by a magnetic field, but that will lose its magnetism in the absence of such a field. The particulate ferromagnetic material passes a U.S. 40 mesh screen but is retained by a U.S. 325 mesh screen. Finer or coarser ranges may be preferred depending upon the nature of the dry mix to be marked with the tracer.
Coated tracers may be produced by first dry-blending 0.1% to 5% by weight of water soluble FD&C colors with the particulate ferromagnetic vehicle. Water is then added to the dye mixture to dissolve the dye and then mixing is continued. The mix is then dried, and screened. The portion of the dry mix passing U.S. 40 mesh screen but retained by U.S. 325 mesh screen is packaged as the finished product. The screening may be varied depending upon application. Certified FD&C dyes such as Blue #1, Blue#2, Red #2, Red #3, Red #40, Yellow #5, Yellow #6 and others serve as easily identifiable coating materials. Mixtures of dyes differing in diffusion rates may be used to increase the number of different tracers and to produce truly unique markers. For example mixtures of Blue #1 and Yellow #6 produce blue streaks or rings with yellow nuclei by the detection procedure described herein below. Mixtures of Blue #1 and Red #2 produce blue streaks or rings with red nuclei. .
Three types of existing iron-based MicrotracersTM include the following: 1. Microtracer F (Iron Grit 25,000 particles per gram); 2. Microtracer FS (Stainless steel greet 50,000 particles per gram); 3. Microtracer RF (Reduced iron powder > 1,000 000 particles per gram). When formulated in vitamin, mineral or medicated premixes, a microtracer serves to mark the presence of the premix in the finished feeds as well as to identify feed additives and feeds containing such additives as proprietary. When assayed quantitatively, a MicrotracersTM can be used to document efficacy of mixing as well as adequacy of batch to batch “cleanout” of mixers and other feed manufacturing equipment.
Detection of the tracer in samples of finished feed may be easily achieved by means of a magnetic probe. Alternatively, the tracer may be detected in a moving stream of feed by means of a magnetic probe.
Below we will consider two applications of iron-based tracers coated with single color food grade dye which include the validating the mixing process and testing for "Cross Contamination" in Medicated Feeds. Besides traditional tracers, the recent development of several types of new types of microtracers will be discussed.
RESULTS AND DISCUSSION
1. Validating the mixing process.
At least five issues must be considered in validating the mixing process [13,14]: 1. Selection of - one or more tracers; 2• Addition of the tracer to the test feed; 3• Sampling the feed; 4• Analysis the samples; 5• Interpreting the results.
I.1. Selection of the tracer. Whatever analyte is chosen for the mixer test, it will then be used as a tracer for all other ingredients. If it yields results typical of a complete mix, one will assume all other ingredients are mixed completely. This may not always be a correct assumption. Powdered feed additives may become electrostatically charged and stick to the walls of mixers that accumulate as clumps that drop off rarely. If one tests for this ingredient as a tracer,  analytical results will be consistent among samples but always low on average unless one happens to test the rare clump. Such an analyte would not accurately reflect the mixing of other ingredients and would probably be an inappropriate choice for use in evaluating mixer performance.
At least the following criteria should be considered in selecting the tracer:  A. The tracer should be contributed from only one source; B.  The tracer should be a microingredient; C. There should be an analytical procedure to determine the tracer of known or determinable accuracy and precision; D.  The analytical procedure should be inexpensive; E.  The analytical procedure should be quick: ideally one that may be performed "on-the spot"; F.  One should be able to interpret results objectively.
Vitamin and drugs assays may be used to validate completeness of mix, but they are expensive, are often subject to considerable analytical error and often require weeks before analytical results are reported.  Salt is often used as a tracer to evaluate mixing by determining either sodium or chlorides in feeds. Salt, however, has significant deficiencies as a tracer.  It is added at five to 20 kilos per metric tonne and is there by hardly a microingredient. It may not be reasonable to assume mixing of a medication added at three parts per million is correct based upon acceptable results for salt added at 20,000 parts per million. Further, sodium and chloride may be contributed to feeds by other ingredients, yielding background "noise" confusing interpretation of results.
Minerals and amino acids are also widely used and these largely meet the criteria outlined in this paper and generally yield meaningful information. The cost of analysis for minerals such as manganese and zinc by atomic absorption spectrophotometry is often low, possibly $20/sample if preformed by a commercial laboratory or less if performed by the feed manufacturer. Reproducibility of results on a given sample is often good with a coefficient of variation of five to eight per cent possible. Some minerals are contributed to feeds in significant quantities from only one source: the mineral premix. Amino acid analyses by HPLC may be reproducible with a coefficient of variation of five per cent or less, although they may be more costly to perform than mineral analyses. MicrotracersTM   F are also used widely to evaluate mixing. These non-nutrient particulate tracers are designed to satisfy the criteria outlined in this paper. The analytical error in their determination may be two to three per cent and they may be performed "'on-the spot" by technicians with comparatively little training.
I.2. Addition of the tracer to the test feed. This should be via a premix possibly made by mixing the tracer by hand with other common feed ingredient. The amount of premix added to the test feed should be similar to the lowest addition ingredient normally formulated in the feed. If medicated premixes added at 500 grams/ tonne then the tracer premix might be added at this level. The location of tracer addition is usually where other microingredients are added.  Test results will then validate existing procedure, the tracer may also be added at other locations to determine if the location of microingredient addition has a significant effect on the time required to achieve a complete mix. In several tests, it took 30 second or less to achieve a complete mix when a tracer was added into the center of the mixer is compared with when it added at the end of the mixer.
1.3. Sampling the feed. In validating mixer performance "grab" samples should ideally be taken from within the mixer. The samples should be carefully identified and all test parameters should be carefully recorded. Samples should be adequate in size to permit repeat analysis samples evidencing unusual results. How many samples should be taken? Taking one sample is an infinite improvement over taking none. We usually take ten samples from each of five consecutive batches and believe this adequate to detect major deviations from complete mixing that could lead to significant economic losses or regulatory compliance problems.
1.4. Analysing the samples. One should take a given weight of sub-sample from each sample for analysis without homogenizing the sample. This makes the "level of scrutiny" of the test the weight of the sub-sample analyzed rather than the weight of the sample taken. It makes the test more severe, increasing the likelihood of a filling conclusion but also improving the likelihood a favorable result is correct. If mixing is good the worst of conditions, then it should be good under less severe ones.  The ideal "level of scrutiny" would be batches of feed consumed by target animals poultry or fish at one feeding.. The ideal "level of scrutiny" for the tracer would be one added at the lowest level of any  microingredient formulated in the feed, though this issue is complex and effected by intermediate mixing procedures utilized to achieve adequate dispersion. For example, critical microingredients may be dissolved in a liquid and sprayed onto a dry carrier to facilitate achieving adequate dispersion. Samples should then be analyzed using appropriate methodology, proper instrumentation and skilled personnel.
1.5. Interpreting results.  This should be done by comparing the coefficient of variation found from the test data with the coefficient of variation inherent in the method. The method coefficient of variation is what one would expect from repeat analysis of the same sample. Using a maximum permitted coefficient of variation of 10 per cent is arbitrary and capricious unless it can be related to the variability of the analysis. If a coefficient of variation of two per cent can be achieved from a method, this could be used as the goal for judging a mix complete. If a coefficient of variation of 15 per cent was the best that could be achieved front a method, this could be used as a goal for judging a mix complete. Such standards would be unrealistically high however, as 50 per cent of all tests of a complete mix would evidence more variation than the goal. A more realistic goal would be to consider results acceptable if they would occur by chance from a complete mix in at least one per cent of all tests of such a mix. This may mean considering data as evidencing a compete mix when it yields a coefficient of variation is 50 per cent or more greater than the method coefficient of variation. In evaluating particulate tracer counts, one may utilize Poisson statistics to determine if mixing is complete. If one makes no allowance for analytical error, if a mix is complete it should yield test results with I standard deviation equal to the square root of the average count. An average count of 100 should yield a standard deviation of 10 and a coefficient of -variation of 10 per cent. An average count of nine would yield an expected standard deviation of three and an expected coefficient of variation of 33 per cent.
2. Testing for "Cross Contamination" in Medicated Feeds.
Medicated animal, poultry and aquatic feeds are often manufactured at feedmills making feeds for many species or if not at least many formulations for one species. Contamination of medications to non-target feeds is inevitable but quantifiable and controllable. Medicated feed assays, unfortunately, often do not offer a viable control mechanism because: they are expensive, they cannot be performed immediately and they are often inaccurate at contamination levels (i.e. 1 % or less of formulated levels) [15].
Microtracers F offer a vehicle for locating and quantifying "cross contamination" of medicated feeds. Instead of running expensive drug assays, one uses one or more Microtracers F as indicators for the coded medication. The tracer is first mixed into the medicated feed premix, then the premix is added to the feed and mixed. Many samples are taken either only at truck loading or at many locations, depending upon the scope of the study. It is critical that feeds not formulated with the microtracer follow the identical route at the feedmill as the "target" batch containing the microtracer. Samples yielding significant levels of the tracer may then be analyzed for the medication to confirm  the preliminary results provided by the tracer.
2.1. Addition of the Tracer.  The microtracer is generally added to the medicated premix to yield 50 grams of tracer per metric tonne of final feed. Since the tracer has an expected count of 25,000 particles/gram, this means 50 X 25,000 or 1,250,000 colored iron particles are added to a one metric ton batch with 1,250 particles expected per kilogram of feed or 125 per 100 grams, assuming the feed is completely mixed, the tracer is stable and it meets its specified count.
2.2. Sampling.  One should take a minimum of four and preferably ten or more "grab" samples from the target batch formulated with the Microtracer. Samples of the target batch should weigh at least 200 grams and ideally be taken at several points in the feed production system. If one analyzes 80 gram "grab" sub-samples of each target sample, one will expect to find 100 tracer particles per sample. One should then take samples from one or more following batches not formulated with the medication or the tracer. These samples should weigh at least 1-kilo each. If one analyzes 800 grams of a feed supposed to contain no tracer and finds 100 tracer particles, then if total tracer recovery is 100% of theory one may estimate 10% of the total tracer (and thereby the drug) was found as contamination in that sample. By analyzing very large samples of feeds supposed to contain no tracer, the "sensitivity" (ability of the tracer to allow accurate detection of very low level contamination) may be improved by a factor of 10 or more. Individual batches of feed must be isolated and their flow controlled. All samples must be carefully identified and detailed records of all aspects of the test should be made.
The basic flow of feed at a feedmill includes the following: 1. feed ingredients after being ground to mash with a hammer mill (if necessary) are gravity fed into a mixer and mixed; 2. the mixed feed is then dumped into a surge bin. 3. the feed is then discharged from the surge bin using a screw conveyer; 4. the feed is then elevated in the feedmill using a bucket elevator system; 5. the feed is then gravity dumped into a holding bin over a pellet mill; 6. the feed is then pelleted and cooled; 7. the feed is then elevated again using a bucket elevator or pneumatic system; 8. the feed is then distributed to holding bins; 9. the feed is then loaded into trucks.; 10. the feed is then carried to farms where it is discharged into bins; 11. the feed is then distributed from the on-farm bins to the animals, poultry or fish for consumption.
Medications are usually added as a microingredient into the mixer, sometimes by hand but often utilizing computer controlled micro-bin systems. "Cross Contamination" of the medication may occur in the micro-bin system even before the medication reaches the mixer. "Cross contamination" to prior batches may also occur, as when mixer discharge gates leak and an earlier batch remains in the surge bin. As an initial investigation, one may take samples of the "non-target" following feed only at truck loadout taking care to be certain the feed takes the identical route as the medicated feed with Microtracer. If one finds very little or no tracer from the loadout samples, this indicates there may be very little "cross contamination" occurring anywhere in the system. If one finds Microtracers in the "non-target"  following feed, however, much more extensive testing is required to diagnose and then have a basis for correcting the problem.
Portions of the medication may be left as "contamination" to non-target batches at all production points. To reliably diagnose the potential for "cross contamination" of medications to non-target batches of feed, it is critical to take multiple "grab" samples at multiple locations of both the target batch (with medication) as well as one or more following batches. It must be re-emphasized that it is critical the following batches take the identical route through the feedmill as the initial medicated batch formulated with Microtracer.

References: 1. Murthy K.S., Das, C.T. Evaluation of mixer efficiency test., Indian J. Anim Nutr. 7 (1990) 159-164. 2. Calderon V.: Sodium chloride - based markers and their use for evaluation of feed mixing efficiency Proc. Western Section, Amer. Soc. Of Animal Science, 2000. v.51, 10-16. 3. Zinn R.A. A guide to feed mixing. Research Updates and Reports Desert Research and Extension Center, Thesis, UC Davis, 1999. 4. Shelford J.A. Additive for livestock feeds, US Pat. Appl. 20040076659, 2004. 5. Bates L.S. Methods of marking  pharmaceutical products, US Pat. Appl. 20060018832, 2006. 6. Bates L.S. Methods of marking products using natural materials having genetically controlled micromorphological structures as markers, US Pat. Appl. 20060019006, 2006. 7. Robinson M.C. System, apparatus and method for marking and tracking bulk flowable material,  US Pat. 6,796,504, 2004. 8. Eisenberg S. Iron-based tracers, US Pat.3,469,990, 1969. 9. Eisenberg S. Tracers , US Pat.4,029,820, 1977. 10. Eisenberg S. Tracer-containing composition, US Pat.4,152,271, 1979. 11. Eisenberg S. Protected iron tracer composition and method of making, US Pat. 4,188,408, 1980. 12. Eisenberg S. Microingredient containing tracer , US Pat.4,654,165, 1987. 13. Eisenberg D.  The use of MicrotracersTM F (colored uniformly sized iron particles) in coding  the presence of coccidiostats in poultry feeds, Zootechnica International, December (1998), 46-51. 14. Eisenberg D. MicrotracersTM F and their uses in assuring the quality of mixed formula feeds, Advances in Feed Technology, 7 (1992), 78-84. 15. Eisenberg D. Validating cross-contamination control, Feed International, September  (2006) 1-4.nufacturers, Las Vegas, 2007.