Supplemental Material

Supplemental Material Baird, P., D. Robinette, S. Hink. 2017. A Remote Marking Device and Newly Developed Permanent Dyes. Wildlife Society Bulletin Supplement 1. Detailed Methods of the Dye Machine We first reviewed other hands-off dyeing methods (Table S1-1), dismissing for our use all but the remote squirting units of Mosley and Mueller (1975) and Wendlen et al. (1996). We took ideas from both of these papers and modified them to eliminate inaccurate aim, disturbance, and their cumbersomeness. CONSTRUCTION The remote control system consisted of a transmitter, a remote distribution unit, and up to four battery-operated squirt guns. We purchased the transmitter as a complete unit but constructed the other main components exclusively for this project. We used an Airtronics AV2 75MHz transmitter with a switchable frequency chip necessary to operate several units in close proximity without interference. We used components of the transmitter, two servo motors and a receiver unit, in the remote distribution unit. We also needed an antenna kit for the RC unit, 8 AA cell batteries, a 24 AWG solid hook up wire, and a black dual lock fastener. The remote dye applicator, “squirt gun unit” (Figure S1-1) consisted of the motor and pump assembly (2G) removed from a voice-activated toy squirt gun, selected for its compact design and ability to pump solution through a three-foot section of 0.32 cm (1/8th in) diameter hose. We secured the pump and motor housing inside a molded ABS plastic case (1G), via dual lock fasteners (7G). This allowed easy removal for cleaning. We also needed 24 AWG Speaker Wire (25 ft) , and a Hi-Reliability 3.5mm Plug. 1 We drilled a hole through the plastic case to make space for the hose from the pump, and fitted it with a 0.953 cm(3/8 in) rubber grommet (5G). We drilled another 2.54 cm (1 in) diameter hole in the case for the neck and cap of a 4 ounce plastic bottle (3G), fitted the hole with a large 2.54 cm (1 in) grommet (6G), and pushed the bottle neck through the hole. The bottle was held in place snugly with 2 strips of the dual lock fastener. We drilled a hole in the bottle, placed a 0.953 cm (3/8 in) grommet in the hole, and inserted the suction hose through there to the pump. We drilled a final hole in the rear of the unit where we placed a small 3.5 mm jack (4G). We then wired the jack to the motor and pump assembly with 24 gauge wire (8G) so that the squirt gun unit could be activated via a 7.62 m (25 ft) wire from the remote distribution unit. For each gun that was to be activated by the RC unit, we cut a 7.62 m (25 ft) connector with 2.0 gauge wire and fitted it with 2.5 mm plugs on each end. After painting the squirt gun unit flat tan to blend in with the surroundings, we sealed it with silicone sealant to reduce damage from salt and sand. We constructed eight of these units. The central part of the system was the remote distribution unit. Components and accessories for this unit (Figures S1-2, S1-3) were housed in an aluminum case (2R) for good shielding properties, which we painted tan to blend in with the sand. The case contained the switch, antenna, LED lights, the Airtronics receiver (3R), two servo motors with levers (4R), the terminal block (5R), four C batteries (1R) and holder (6R), attached with dual lock fasteners. We placed the lever so that it would be straight up when it was midway between full left and full right and marked the case when the servo levers were in full left and full right position. The levers activated the switches (8R) just prior to their full “on” state. The LED lens mounts (12R) were pushed into holes that we had drilled in the case, and we pushed four super-bright LED's (10R) into these mounts. We placed a flashing red or green 2 LED (11R) into the mount directly above the switch, and mounted the switch (7R) from the inside. We soldered the leads from the battery holder (6R) to the input wires of the main switch (7R), and routed the switch output wires to the terminal block (5R), with two pairs of negative and positive terminals. We bent, joined, and soldered together all the negative leads of the front panel LED's and connected them to the negative terminal block. We then soldered each of the positive leads of the LED's to 100 ohm resistors (13R), wiring each resistor to one of the four switches in the unit, and then wired them to the positive terminal block. This switch wiring enabled the LED to be connected to the positive terminal when we depressed the switch. We then wired the jacks (9R) in the same fashion as the LED's, connecting to the negative lead of the LED and also to the positive lead of the LED. In this way, the LED was wired in parallel to the jack and would indicate current flow to the jack. We installed a blinking red or green indicator LED (11R) above the switch, wired in series via the positive lead and a 100- ohm resistor to the terminal block (positive-positive & negative-negative), which indicated when the unit was active. The receiver was also wired directly to the positive and negative terminals, and all wiring connections were enclosed with shrink tubing to prevent any short circuits. We then reinstalled the servo motors and plugged the connectors into the receiver in the case, with the plastic antenna mount-a straw-like antenna support with antenna. All these exited through the drilled hole in the lid, which was now connected to the case. We camouflaged the squirt gun with a bowl, primed and painted flat tan like the other units, with sand added to the freshly painted surface, and from a distance, the bowl looked like a mound of sand. The entire system fit into a fishing tackle box (Accessories Table S1-2). OPERATION 3 Each transmitter control sent a signal to the two-channel receiver in the RC distribution unit, and each receiver channel controlled its own servo motor. Each motor could move a lever left or right, and this activated the left or right switch in the unit, with power supplied to the corresponding LED and jack on the front panel. Plugged into this jack was the 7.62 m (25 ft) wire connected to the jack on the squirt gun. When the squirt gun received power, the pump and motor were turned on, and dye was pumped out of the nozzle as long as the transmitter control was held in the ‘on‘ position. The dye machine is set so that servo A (left control), or B (right control) operated by their respective channels, can move a lever left or right. This movement activates the left or right switch in the unit, with power supplied to the corresponding LED and jack on the front panel. Plugged into this jack is the 7.62 m (25 ft) wire connected to the jack on the squirt gun. When the squirt gun receives the power, the pump and motor are turned on, and dye is pumped out of the nozzle as long as the transmitter control is held in the ‘on‘ position. Up to four squirt gun units can be operated from one RC distribution unit. All equipment needs a pre-check that includes a battery check for the RC distribution unit and the transmitter, and a test of the controls and squirt gun. DYEING ROUTINE We determined each bird’s preferred incubation position, which we needed for proper machine setup and aimed the nozzle at the position the bird would normally face when sitting on the eggs. We made a small indention at the edge of the nest where we placed the nozzle of the dye gun, dug a small trough, and buried the nozzle with the tip sticking up about 2 cm, and angled slightly up, pointed towards the area where the tern had been sitting. We stretched out the hose to its full one m length, and covered it lightly with loose sand or dirt. We plugged the 4 connection wire into the back of the gun and covered the gun with the camouflaged bowl. The loose wire connector stretched out in the direction that the RC distribution unit would be placed. We connected the RC distribution unit to the gun via the wire which was plugged into the selected jack, and placed the unit in an optimal position such that it was 3-8 m away from the target nest, as far away from other nests as possible, and with the front panel in the line of site of the blind. Although Line of site was not necessary for proper function of the unit, it was helpful because a transmitter pre-check, via switching on the RC Unit and testing an unused channel, could be done while viewing the LED's with binoculars. if the unit was not functioning, the light would not go on. We recommend shutting off the transmitter after the test to conserve power. Once the tern settled on the nest and was in its preferred position, we rechecked the placement of the nozzle. If it was not placed correctly, we flushed the tern from the nest and repositioned it. We repeated this up to three times, and if the nozzle was still not positioned correctly, we left. If the positioning was correct, we fired the dye gun by switching on the transmitter and activating the appropriate control for 1-2 sec. We verified the dye shot by the reaction of the tern and by visual observation with binoculars. 5 Supplement 1 Tables and Figures Table S1-1. Marking techniques used in other studies. Reference Technique Problems with technique Cavanaugh et al. Coat live or dummy Mortality of eggs + eggs conspicuous to predators 1992 eggs Donehower and Dye on ground in Dyed other species + difficult to remove from Bird 2005 front of nest habitat Evans and Griffith Dye on bushes and Marked non-target animals and no control over 1973 hay which animals marked or where mark placed Mosley and Squirt apparatus Intrusive, cumbersome, inaccurate, disturbed habitat Mueller 1975 Mossman 1960 Powder in nests Transferred to eggs -conspicuous to predators Paton and Pank Coat live or dummy Dyed eggs with some mortality + eggs conspicuous 1986 eggs to predators Wendlen et al. Squirt apparatus Intrusive, cumbersome, inaccurate, disturbed habitat 1996 Table S1-2. Accessories and other supplies for the entire remote dye system. Supplies Qty Source Stock # Large Plastic Bowl 1 Target N/A Plastic Bird Eggs 50 Art Store N/A Heat Shrink Tubing 1 Mouser 524-12-1080 1 Mouser 5168-4300 Tool Kit Size Solder Pack 1 Mouser 533-5055 Electrical Tape 1 roll Home Depot N/A Paint Primer 1 can Home Depot N/A Paint 1 can Home Depot N/A (assortment) Quick Bond Gel and Accelerator 6 Figure S1-1 Remote dye applicator “squirt gun. “ 1G= Mouser Molded ABS Plastic Case (5.9x3.5x2.2) 2G=ShootNShout Squirt Gun Motor and Pump 3G= 4 oz. Plastic bottle 4G= Mouser Hi-reliability 3.5 mm jack 5G= Mouser Rubber Grommets for hose from pump (ID: 9/32 OD: 3/8) 6G= Rubber Grommets for hole for neck of bottle (ID: 3/4 OD: 1.0) 7G= Mouser Black Dual Lock Fastener (1 FT) 8G= Mouser 24 AWG Solid Hookup Wire from jack to motor & pump Figure S1-2 Top view of remote distribution unit. 1R= C cell battery 2R= Aluminum case 3R=Airtronics receiver 4R=Airtronics servo motor 5R=Terminal block 6R=Battery holder wire lead 7R=Main switch 8R=Miniature snap switch 9R=3.5 mm jack 10R=1 ¾ “ green LED 11R=1 ¾ “ red LED 12R=Clear clip lens mount 13R= ¼ watt carbon film resistors 100 Ohm 7 Figure S1-3. Front view of remote distribution unit. 2R= Aluminum case 7R=Main switch 9R=3.5 mm jack 10R=1 ¾ “ green LED 11R=1 ¾ “ red LED 12R=Clear clip lens mount 14R=Bumpon taped square 8 SUPPLEMENT 1 LITERATURE CITED CAVANAUGH, P., C. GRIFFIN, AND E. HOOPES. 1992. A technique to color-mark incubating gulls. Journal of Field Ornithology, 63: 264-267. DONEHOWER, C. and D. BIRD. 2005. A method for color-marking birds at resting sites, Journal of Field Ornithology 76:204-207. EVANS, J., AND R. E. GRIFFITH Jr.. 1973. A fluorescent tracer and marker for animal studies. Journal of Wildlife Management 37:73–81 MOSELEY, L. J., AND H. C. MUELLER. 1975. A device for color-marking nesting birds. Bird-Banding 46: 341–342. MOSSMAN, A. S. 1960. A color-marking technique. Journal of Wildlife Management 24:104. MURRAY, D.L. and M.R. FULLER. 2000. A critical review of the effects of marking on the biology of vertebrates. Pages 15-64 in F.T. Boitani, editor. Research techniques in animal ecology: controversies and consequences. Columbia University Press, New York, NY, USA. PATON, P. AND L. PANK, 1986. A technique to mark incubating birds. Journal of Field Ornithology 57: 232-233. WENDELN, H., R. NAGEL, and P. BECKER. 1996. A technique to spray dyes on birds. Journal of Field Ornithology 67: 442-446. 9 Supplement 2. Summaries of dyes used in other studies. Dye Species Application method Faded after__ wks (longevity) Visible from >30 m? (visibility) Negative properties Reference 1. Rhodamine B 2. Thief detection powder Rhodamine B in 35% isopropyl + oil/silica gel. Or Malachite Green in 99% propylene glycol. Both in silica gel or petroleum jelly Blacklegged Kittiwakes 1.Squirted from forestry gun 2. In nest material 1. After 1 2. After 2 1.no 2. yes Baird and Gould 1983 Herring Gulls Dye on dummy eggs (adding carrier of silica gel or petroleum jelly caused 100% mortality live eggs) Most: 1-2 days, no longer than 5 weeks at 100m & 8x scope. RB 35% isopropyl “greater retention.” MG & PA greater retention w silica gel than w petroleum jelly but none had MG after 2 wk. No distance noted. Greatest visibility with silica gel. “Some” visible to 350 m using 15x scope after 3 wk but none with petroleum jelly 1. Carcinogenic; spreads & fades when exposed to water 2. Color transfers to egg Rhodamine B carcinogenic; transfer of color to live eggs w silica gel. Embryo death. Hatch success control 70.8%; of dyed live eggs 4.116.8%; low w transferred dye “significant reductions in hatch success and modified behavior” Belant and Seamans 1993 10 Dyes: Auramine (Color Index 493) Malachite Green Crystals (Color Index 495) Victoria Blue B. Cone. (Color Index 559) Resoline B Cone. 200% Ruffed grouse 1. Rhodamine B + H2O & petroleum jelly paste 2. Malachite Green & 70% isopropyl alcohol 1. Herring Dye on dummy eggs & Greater blackbacked Gulls low concentratio n 2. Laughing Gulls high concentratio n Laughing Dye on dummy eggs Gulls high concentratio n 1 Rhodamine B 2 Malachite Green & petroleum jelly Printer's ink & xylene or carbon tetrachloride put in hen eggs, hole sealed w cotton wadding soaked in acetone & glue and thrown at logs near birds to splash them. For dyes (all basic), birds trapped and back & tail feathers saturated with dyes 28 days Faded rapidly till 42 days “readily identifiable at a distance” 1. 42 d 2. Not seen. Difficult to detect Eggs with ink not Bendell and accurate. Auramine: Fowle 1950 acute toxicity (oral, dermal, inhalation) + mutagenicity (germ cells) & carcinogenicity and Reproductive toxicity categories 1A,1B,2 . Victoria Blue B: Acute toxicity, serious eye irritation, Harmful if swallowed. Resolin B: skin irritation, serious eye irritation, may cause respiratory irritation Carcinogenic Cavanagh et al. 1992 Low levels of adult mortality (0.05-0.27%) 0.46% nest w dyed eggs destroyed Cavanagh et al. 1992 11 Batik dye: Deep purple Procion MX Fiber reactive 70% isopropyl + petroleum jelly 3% Rhodamine B Herring and Great-black backed Gulls Paste put on groundbirds walked through & got on feathers One bird 7 days; one bird 31 days. Dye faded completely on the other 4 birds Black-tailed jackrabbits Caged/enclosed rabbitsafter 1 wk, 3 marked (seen “up close”); one sprayed rabbit-4 mo. 2. Five months (we did not consider rabbits who were fed dyed food) Nyanzol LR (red-brown), Nyanzol D (black), aniline red Beechy ground squirrel Sprayed on bushes or hay in enclosure-27 rabbits there for for3 wks. Also sprayed on hay in field. Covered 20% of bodies. 2.Four rabbits also caught & sprayed w 2% Rhodamine B – coverage 10% of body-& held till dye dried. One seen 7 days later w dye on 66% of body. One lasted 4 months (killed by coyote). We do not report on dyed food fed to rabbits. Animals captured and dyed with dye + H202 Red Median= 30d Red brown Median=30 d Black Median = 60 d 70 m binoculars 135 m spotting scope No control of placement Donehower of dye. No consistency of and Bird dyeing. Batik dye may be 2005 toxic Rhodamine B reported to be carcinogenic. All rabbits absorbed dye through skin- colored them internally. Predators that consumed rabbits became dyed 2-3 d postconsumption Red “most brilliant Commercial fur dye; & faded quickly”/ “hazardous ingredient, Black ”most explosive” consistent & durable”/Red brown “difficult to fix” Evans and Griffith 1973 Evans and Hodenried 1943 12 “Bird marking colour” (Geigy chemical, Bern) Ink-jet printer ink diluted in a 50-50 isopropyl alcohol-water mixture Picric acid Swan spp. Forster 1973 Great blue heron & great egret Spraymore Paintball landmines with inkjet 2 and 44 days Water-soluble Fox 2010 Redshanks Put dye on breast “and underparts” After 4 ½ months Explosive if dry Painted on birds and let dry After 2 months Furness and Galbraith 1980 M. Hammaris pers. comm. Kadlec and Drury 1969 Picric acid “Green color” Herring gulls Rhodamine B, picric acid Snowshoe hares Picric acid, Rhodamine B Snowshoe hares Caught with snaptraps, muskrat traps w padded jaws; drugging with tribromethanol; & clap-net trapping at nests. Sponged on and held till dry. Caught in live traps or 4 months and held-no application method stated Did not state Picric acid 5 months; Rhodamine B faded immediately Yes- “dark and visible” Explosive if dry Treatment did cause some withdrawal and mortality (about 10 per cent) Shock disease from being held in captivity=acute hypoglycemia: treated with dextrose & heat Rhodamine B carcinogenic Picric acid explosive if dry Keith et al. 1968 Keith et al. 1968 13 “Commercial wool dye” Blackcapped Chickadees Dabbed with pen or sponge and let dry Stamp pad ink Blackcapped Chickadees Blackcapped Chickadees Blackcapped Chickadees Dabbed with pen or sponge and let dry Vegetable dyes with detergent 1 Drimark markers in “highly volatile organic solvent” 2 vegetable dyes & detergent 1 Rhodamine B Malachite green 3Picric acid “3 chemicals” 1,2 Powdered aniline dyes soluble in water & alcohol-Bright blue/red/green/ yellow Sage grouse 2 Lesser snow geese, 3 Ross’s geese White geese Dabbed with pen or sponge and let dry Only stained if detergent added, but “ran & blurred” and did not adhere 6-8 weeks but ran “all over”-stained the dyer—“messy” “after a few weeks” Dabbed with sponge Orange faded to tan in in cap. Dried instantly. 4-8 wks but “not easy to see” Green “visible in field but not easily”: 6- 8 weeks, Red 8-10 wks and “easily seen” Painted dye on wings; 11 mo(2 birds),2 mo(3 held in warm building birds),3 mo(2 birds), 2 3 overnight mo(2 birds),33 mo(1-8 birds) Rolling birds in pans of dye & held till dye dried Dye (50-50 water + 2 months in the lab- no grain alcohol ) buried information from field tanks charged w application compressed air leading to sprinklers activated by trip wire. No Kennard 1961 Kennard 1961 Kennard 1961 Orange, Green not visible. Only red visible- no distance noted Solvent not appropriate. Kennard Vegetable dyes rinsed off 1961 2-3 wk Rhodamine B carcinogenic Picric acid explosive if dry Kozlik 1956 Kozlik et al. 1959 No accuracy; often missed birds Moffitt 1942 14 Dye + 50% Grouse alcohol Malachite green, Least terns Rhodamine B Dye-soaked sponge in nest Squirted birds from bottle Thief Detection Powder Paste on sponge in nest Rhodamine B in oil-based silica gel Pigment stains in carbon tetrachloride Leather dye w ethanol solvent red/yellow/light blue/aqua green. Glaucouswinged gulls Cattle egrets Dye on eggs 2 months 2 weeks maximum 2-6 mo California gulls Wood storks Garden sprayer 2-3 months Rhodamine B in alcohol/water Wandering Albatrosses 1. In tub of dye: ¼- ⅓ gal/bird & dye brushed on. 2. Bird held & entire dorsal & ventral sides sprayed. Dye dried. Purple Batik dye in 70% isopropyl + petroleum jelly. Ringnecked Pheasant skins and Dipped feathers into solution and dried. Sprayed on wings. Held birds till dry. 1. 2-3 mo bright, 1 yr “conspicuous patches” “Dye retention unique to Wandering Albatrosses-most species do not retain dye as long.” “Faded in all cases” Dye on eggs-not known if toxic Monaghan et al. 1989 Mosley and Mueller 1975 Mossman 1960 Rhodamine B carcinogenic Paton and Pank 1986 Solvent toxic Price 1931 diethylene glycol monoethyl ether/ methanol/isopropanol/ flammable; respiratory damage Birds dyed in tub too water-logged to fly Rodgers 1986 Batik toxic Wadkins 1948 Rhod. B carcinogenic < 200 m Tickell 1968 15 Malachite green, live birds Rhodamine B extra, brilliant green in 33% alcohol Picric acid Common terns Silver nitrate Common terns Squirted from bottle Explosive in dry form Squirted from bottle Skin & eye irritant Wendeln et al. 1996 Wendeln et al. 1996 16 SUPPLEMENT 2. LITERATURE CITED BAIRD, P., and P. J. GOULD, editors. 1983. The breeding biology and feeding ecology of marine birds in The Gulf of Alaska (RU341). U.S. Dept. of Commerce, NOAA OCSEAP Final Reports 45 (1986): 121-505. BELANT, J. and T. SEAMANS, 1993. Evaluation of dyes and techniques to colormark incubating herring gulls. Journal of Field Ornithology 64: 440451. BENDELL, J. F. S., AND C.D. FOWLE. 1950. Some methods for trapping and marking Ruffed Grouse. Journal of Wildlife Management 14: 480-482. CAVANAUGH, P., C. GRIFFIN, AND E. HOOPES. 1992. A technique to color-mark incubating gulls. Journal of Field Ornithology, 63: 264-267. DONEHOWER, C. and D. BIRD. 2005. A method for color-marking birds at resting sites, Journal of Field Ornithology 76:204-207. EVANS, J., AND R. E. GRIFFITH Jr.. 1973. A fluorescent tracer and marker for animal studies. Journal of Wildlife Management 37:73–81 EVANS, F.C. and R. HOLDENRIED, 1943. A population study of the beechy ground squirrel in central California. Journal of Mammalogy 24:231-260. FORSTER, R. 1973. Swan Marking with colour. The Ring 77: 99-100. HAMARIS, Michael, BRD, USGS, 2010, personal communication. KADLEC, J. A. and W. H. DRURY. 1968. Structure of the New England Herring Gull population. Ecology 49:644–676 KEITH, L. B., E. C. MESLOWAN, AND O. J. RONGSTAD. 1968. Techniques for snowshoe hare population studies. Journal Wildlife Management 32: 801-812. 17 KENNARD, J. H., 1961. Dyes for color-marking. Bird-Banding 32: 228-229. KOZLIK, F., A.W. MILLER, AND W. C. RIENECKER, 1959. Color-marking white geese for determining migration routes. California Fish and Game Report 45: 69-82. KOZLIK , F. 1956. Color marking of white geese. Western Bird Banding Assoc. Newsletter, 31: 7-8 MOFFITT, J. 1942. Apparatus for marking wild animals with colored dyes. Journal of Wildlife Management. 6: 312-318. MONAGHAN, P., J. D. UTTLEY, M. D. BURNS, C. THAINE and J. BLACKWOOD, 1989. The Relationship Between Food Supply, Reproductive Effort and Breeding Success inArctic Terns Sterna paradisaea. Journal of Animal Ecology 58: 261-274. MOSELEY, L. J., AND H. C. MUELLER. 1975. A device for color-marking nesting birds. Bird-Banding 46: 341–342. MOSSMAN, A. S. 1960. A color-marking technique. Journal of Wildlife Management 24:104. MURRAY, D.L. and M.R. FULLER. 2000. A critical review of the effects of marking on the biology of vertebrates. Pages 15-64 in F.T. Boitani, editor. Research techniques in animal ecology: controversies and consequences. Columbia University Press, New York, NY, USA. PATON, P. AND L. PANK, 1986. A technique to mark incubating birds. Journal of Field Ornithology 57: 232-233. PRICE, J. B. 1931. An experiment on staining California gulls. Condor, 33: 3. RODGERS, J.A., 1986. A field technique for color-dyeing nesting wading birds without capture. Wildlife Society Bulletin 14: 399-400 18 TICKELL, W.L.N. 1968. Color-dyeing albatrosses. Bird-Banding 39: 36-40. WADKINS, L. 1948. Dyeing birds for identification. Journal of Wildlife Management 12: 388391. WENDELN, H., R. NAGEL, and P. BECKER. 1996. A technique to spray dyes on birds. Journal of Field Ornithology 67: 442-446. 19 SUPPLEMENT 3. BIOCHEMISTRY OF DYES The superiority and permanence of acid or metal phthalocyanine dyes compared to basic dyes lies in the molecular structure of the dye itself, not of the wetting compound. Basic dyes are topical; acid dyes and metal phthalocyanine dyes form hydrogen bonds with the amino acid groups on the feather or fur keratin proteins and they are thus incorporated into the feathers or fur themselves. The two also attract each other by van der Waals forces (Burch 2008, 2010). Acid dyes typically have a colorless cation such as sodium, and a colored anion. Amino acids on feathers or fur have a positive charge so that the negatively charged anion of dye binds to them. Amino acids bind to the colorless anion in basic dyes, and the colored cation rinses off, unless stuck to the feather or fur by a temporary adherent. Felt-tipped markers have also been used to apply dye to feathers. Adherent of dye from felttipped markers works slightly differently (Sukhna and Reichmann 2003). Markers have a suspension of basic dyes in a solvent like alcohol (water-soluble), toluene or benzene (not water soluble), as well as a resin. It is the resin that then sticks to the surface of the feather, along with the dye and solvent. The resin adheres better than does the silica gel of many basic dyes, but since the positive dye itself is still not incorporated into the feather, the colors eventually fade after a few weeks when the resin flakes off (P. Burch 2008). Other markers, e.g. Drimark (Kennard 1961), have a positively-charged metal colorant, an aluminum pigment (less than 12 microns), grounded in a positive acrylic binder. The color adheres to the feathers via butyl acetate, a fluoro chemical surfactant, a liquid acrylic resin solution, and an unsaturated polycarboxylic acid, in an ammoniacal base for adjusting the pH, a preservative, (Germaben), and polyglycol, which serves as a humectant. (http://www.freepatentsonline.com/6561713.html). 20 Since the metal colored part is positive, it also cannot bind to the feathers the way that acid dyes do. With advice from Paul Burch (2008), we found that dyes with longer hydrocarbon chains were better than those with shorter ones, and that dyes with smaller molecules can enter the feather faster but leave more readily than do larger ones. The longer chains also improve the hydrogen bonding and the van der Waals forces between the dye and the protein. Thus a big benefit of acid dyes is due to hydrogen bonding between the dye and the feathers’ amino acids. The dyes are not just topical; they become part of the feather. They are unable to be ingested or preened off and thus are particularly suited for use in wild animals as harmless dyes. In contrast, basic dyes are ingestible, because they are only physically and topically adhered to the top of feathers or fur via resins or gels. They are thus potentially more toxic than are acid or phthalocyanine dyes because they can be ingested. SUPPLEMENT 3. LITERATURE CITED BURCH, PAULA, 2008 personal communication BURCH, P., 2010http://www.pburch.net/dyeing.html (7 February 2010). KENNARD, J. H., 1961. Dyes for color-marking. Bird-Banding 32: 228-229. SUKHNA, C. and C. REICHMANN 2003. http://www.freepatentsonline.com/6561713.html 21