An Insect-based Detector of Bovine Manure Odors

Transcription

1 Iowa State Uiversity From the SelectedWorks of D. Raj Rama 1997 A Isect-based Detector of Bovie Maure Odors D. Raj Rama Reid R. Gerhardt Available at:

2 AN INSECT-BASED DETECTOR OF BOVINE MANURE ODORS D. R. Rama, R. R. Gerhardt ABSTRACT. We desiged, costructed, ad tested a sigle-stream isect odormeter for the measuremet of bovie maure odors. The biosesig elemet i the odormeter is a group of six gravid face flies (Musca autumalis DeGeer) i a 360 ml chamber. A microphoe, mouted above the chamber, seses souds made by the flies activity. The microphoe sigal is processed by a aalog root-mea-square power-measurig circuit coected to a computer data acquisitio system. The apparatus allows electroic measuremet of isect activity. Iitial experimets demostrate the feasibility of this system for odor detectio (yes/o). Kietic experimets show that a dose-respose curve ca be produced by ijectig a series of icreasigly odorous pulses ito the system. As expected, the repeatability of this techique is highly depedet o cotrol of critical biological variables such as the flies odor exposure history ad reproductive state. If face fly resposes ca be positively correlated with huma odor pael resposes, the this techique might eable rapid, reliable ad iexpesive quatificatio of maure odors. Keywords. Odor measuremet, Biosesor, Maure odors, Odor pael, Isect, Face fly. Nuisace odor from aimal productio operatios is oe of the greatest problems facig agriculture today. This fact is reflected i the growig umber of atioal ad iteratioal cofereces o livestock odor, o the umber of odor-related articles i producer-orieted magazies, ad o the umber of compaies sellig odor cotrol additives ad process techologies. The problem is caused by the covergece of two pheomea: icreased suburba ecroachmet o agricultural lads ad icreased aimal ad hece waste desities o farms. A partial list of techological solutios to the problem icludes gas treatmet by plasma reactors (Zhag et al., 1994), gas treatmet by combied physicochemical ad biological processes i aerobic biofilters (Scholtes et al., 1988; Sweete et al., 1991; Zeisig, 1988), odorat destructio with alumia platium catalysts (Mote, 1991), reduced emissio through ehaced treatmet of the solid ad liquid waste streams (Powers et al., 1995; Westerma ad Zhag, 1995; Wilkie et al., 1995; Zhag ad Westerma, 1995), ad reduced emissio through additives for aimal feeds ad for maure storage ad hadlig areas (Barrigto et al., 1995; Hobbs ad Pai, 1995; Williams ad Schiffma, 1995). However, there is little cosesus o which of these techologies are the most reliable ad cost effective, i part because of the difficulty i assessig treatmet performace. Article was submitted for publicatio i March 1997; reviewed ad approved for publicatio by the Structures & Eviromet Div. of ASAE i August The authors are D. Raj Rama, ASAE Member Egieer, Assistat Professor, Departmet of Agricultural ad Biosystems Egieerig, ad Reid R. Gerhardt, Professor, Departmet of Etomology ad Plat Pathology, The Uiversity of Teessee, Koxville, Te. Correspodig author: D. Raj Rama, Agricultural ad Biosystems Egieerig Dept., The Uiversity of Teessee, PO Box 1071, Koxville, TN ; tel.: (43) ; fax: (43) ; <drama@utk.edu>. Ulike most agricultural water pollutats, whose cocetratios are readily measured, the quatificatio of maure odor is cumbersome (Hobbs et al., 1995; Sorel et al., 1983; Joes et al., 1994). Aalytical techiques are cofouded by the large umber of odorous compouds that ca cause malodor (O Neill ad Phillips, 199; Schaefer, 1977); o sigle techique ca detect all of them, let aloe accout for the differetial sesitivity of the huma ose to these compouds. Electroic oses are curretly much less sesitive tha the huma ose (Hobbs et al., 1995), but huma sesory evaluatio techiques (i.e., odor paels) require sophisticated airhadlig systems ad daily paelist calibratio to achieve eve moderate repeatability (Joes et al., 1994). Furthermore, a forced-choice system is preferred over a sigle-stream system to reduce iheret respose biases i paelists (Koster, 1986, refereced i Joes et al., 1994). Thus, high variability ad cost limit the utility of odor pael measuremets, while isesitivity limits the use of physicochemical sesig methods. Despite the difficulties associated with odor measuremet, agriculture s eed for accurate, reliable, iexpesive odor quatificatio is growig. Quatificatio is eeded for objective performace assessmets, which are ecessary for decidig betwee competig maagemet practices ad techologies. Simplicity ad low-cost are required because the temporal ad spatial variatios i odor productio (across seasos ad states) make o-site odor measuremets critical to the developmet of odor cotrol strategies. To this ed, we have explored the possibility of usig what amouts to a isect odor pael to quatify bovie maure odors. The face fly (Musca autumalis DeGeer) is a Europea ative itroduced to North America i the 1950s. The isect is closely related to the house fly, but is a pest of cattle, ad is depedet o them for its developmet ad survival. The primary habitat of larval flies is cattle maure (Hammer, 1941; Teskey, 1969). They ca develop i the maure of Trasactios of the ASAE VOL. 40(6): America Society of Agricultural Egieers / 97 /

3 other species, but prefer fresh (ca. < h old) cattle maure for ovipositio. Like other isects with coprophagous larvae, the face fly must be able to detect the odor of both cattle ad fresh cattle maure for survival. Cattle may migrate several kilometers while flies mature i maure pats. Therefore, ewly emerged adult flies require highly sesitive chemoreceptio to locate their hosts. Adult female face flies are attracted to cattle early i their gootrophic developmet to obtai protei eeded for egg maturatio from lacramatios ad other body secretios. Whe the eggs are mature, the gravid female is o loger attracted to cattle but is istead attracted to fresh bovie maure, where ovipositio takes place (Miller ad Treece, 1968; Thomas et al., 197). While adult females of all gootrophic developmet stages may be attracted to maure, attractio is greatest whe the females have completed their ovaria developmet (Bay ad Pitts, 1976). We sought to use the maure seekig respose of gravid female face flies as a reliable biosesor for maure odors. Though we realized that europhysiological techiques such as the electroateogram (EAG) could be used to moitor the respose of the chemoreceptive system, we chose to pursue whole isect detectio methods (i.e., behavioral techiques), for the followig reasos: (1) system simplicity was desired, ad a europhysiological approach violated this objective; ad () a fuctioal, itegrated chemotactic system seemed likely to be more sesitive to stimuli tha the idividual receptor cells comprisig that system. For example, i male silkworm (Bombyx mori L.), the EAG threshold for the sex pheromoe bombykol is 50,000 times greater tha the behavioral threshold (Scheider et al., 1967, cited i Metcalf ad Metcalf, 199). The desig goal was a odormeter that would be small eough for coveiet trasportatio to the field, or that would remai i the lab but would require low volume (< 1 L) odor samples for testig. This places a volume costrait o the system of roughly 5 L. The iitial cofiguratio ivolved a 3 L Plexiglas ad scree box ( cm). A pair of.5-cm diameter air ilet ports o 10 cm ceters blew air ito oe of the screeed walls, while the opposite screeed wall acted as a exhaust. Oe port emitted odorous air, the other clea air, ito the box. We expected the flies to accumulate o the odorous port, but they did ot. This was due to the small scale of the box i compariso to the radius of curvature of face flies host seekig flight: there was t eough room for the isects to fly i ad out of the odor plume, as they would i the wild (Bowe, 1991). This is why flight boxes for mosquitoes are typically over a meter i legth (e.g., Hacock ad Foster, 1993). Durig these early experimets, we oticed a icrease i the fly activity upo applyig odorous air to the chamber. This observatio led to the cocept of a device that would simply detect isect activity, rather tha directioal preferece. I such a device, flight ito ad out of a odor plume would be simulated by presetig the odor stimulus i short bursts; the spatial variatio i odor cocetratio, which would ormally be perceived by the isect s flight through a plume, was replaced with temporal variatios i odor cocetratio. To make a aalogy with huma olfactometers, we decided to develop a siglestream olfactometer rather tha a forced choice device. Havig oted that the maure odor acted as a excitat, we explored the possibility of soically sesig the flies activity. This requires three mai compoets, as follow: (1) a microphoe to covert soud ito electrical sigals; () a amplifier ad root-mea-square (rms) power measuremet circuit to determie the magitude (total power) of the microphoe sigal; ad (3) a computer data acquisitio ad cotrol (DAC) system to record the total power sigal over time. A fast DAC board would allow samplig of the raw audio sigal, with rms computatios performed i software. However, this techique would icrease the computig burde ad overall cost, sice a high throughput DAC board costs cosiderably more (ca. 10 ) tha the aalog circuitry. Cost is a issue because the uderlyig goal of this work is a simple ad iexpesive techique that could be widely used by odor cotrol researchers. This system, with the microphoe iserted i the wall of a 500 ml cardboard caister cotaiig 10 face flies, could easily sese fly activity. However, the iverse-square relatioship betwee soud pressure level ad fly-tomicrophoe distace caused bias i the data: a flight ear the microphoe saturated the output amplifier, while a flight o the opposite side of the box was hardly detectable. To couter this effect, we cosidered usig multiple microphoes placed half-way up the chamber walls, or suspedig a sigle microphoe slightly above the fly cotaier (see the Appedix for a detailed exploratio of this issue). For reasos of performace ad simplicity, we chose the latter method. Additioally, the bias itroduced by fly-to-microphoe distace ca be reduced by coutig evets (buzzes) rather tha itegratig the soud eergy sigal. We left this optio ope by separatig the data acquisitio ad processig fuctios. MATERIALS AND METHODS OVERVIEW The odormeter cosists of two pricipal compoets. The first is a air hadlig system (fig. 1) for providig CO -free, deodorized air to the flies, ad allowig the ijectio of odorous pulses ito the clea air stream. The secod is a microphoe ad associated circuitry that Figure 1 Air hadlig system block diagram, illustratig KOH (potassium hydroxide) filter for removig CO, ad sequetial activated carbo (AC) ad high efficiecy particulate air (HEPA) filter for removig other odors ad particulate materials. Teflo block with septum allows ijectio of odorous gases ito clea air stream, ad microphoe seses fly activity TRANSACTIONS OF THE ASAE

4 provide a electroic measure of the flies activity level. Acillary compoets iclude the PC-based data acquisitio ad cotrol (DAC) system that cotrols the air pump ad measures, displays, ad records the sigal from the sesig circuitry, ad the soud-deadeig chamber that houses the fly cotaier ad atteuates ambiet oise. AIR-HANDLING SYSTEM The air-hadlig system (fig. 1) provides approximately L mi 1 of CO -free deodorized air to the fly chamber. A rotary vae pump (Fürgut DC1/16f, MRM Iteratioal, Duluth, Ga.) draws ambiet air through 1.5 L of 0.1 M KOH solutio to remove CO. Pheolphthalei idicator (ca. 1 g L 1 ) i the KOH solutio turs clear whe the ph drops below 8., idicatig that the solutio eeds replacemet. The first versio of this scrubber was costructed of PVC, which is rated as compatible for prologed use with strog bases icludig KOH. However, the PVC appeared to react with the KOH over time, visibly yellowig, ad givig off a strog odor that brought the flies to a stadstill. Replacig the PVC scrubber with oe made of borosilicate glass ad Teflo solved the problem. After passig through the pump, the CO -free air is deodorized by a combied activated carbo ad high efficiecy particulate air (HEPA) filter (Survivair, Ic. model , Sata Aa, Calif.). Upo exitig the activated carbo/hepa filter, the air passes through Teflo tubig, to the ijectio block. The ijectio block is machied from a solid piece of Teflo, ad allows isertio of a hypodermic eedle through a replaceable septum ito the air stream. From the ijectio block, the air passes through Teflo tubig to a port i the base. This PVC base is a structural support for the fly cotaier ad for the microphoe. To facilitate loadig ad uloadig flies i a differet lab, the fly cotaier is easily attached ad removed from the base with a pair of wig uts. Rubber o-rigs seal the air passages betwee the base ad the fly cotaier. The fly cotaier itself (fig. ) is a 7.5 cm tall, 7.8 cm iside-diameter, PVC pipe sectio (total volume of 0.36 L) mouted o a 0.6 cm thick piece of PVC. A.5 cm hole i the cylider wall, allows fly loadig ad uloadig. This loadig port is sealed with Parafilm. The top of the Figure Fly cotaier ad base, showig o-rigs ad moutig studs. Plexiglas microphoe mout (ot show) is attached to the base ad suspeds microphoe 1.5 cm above chamber. This etire apparatus is housed iside a isulated box to atteuate ambiet oise. cylider is covered with a 13 μm (0.5 mil) thick piece of clear polyethylee film affixed by a rubber bad. This material was chose for its low mass ad dampig (it is soically lively ad ulikely to atteuate soud sigificatly), ad its air-impermeability. Air eters ad exits the cotaier through a pair of ports i the cotaier bottom, ad the cotaier floor is covered with a alumium scree to prevet flies from eterig the ports. Exhaust from the cotaier flows through a Teflo tube ad is discharged ito the lab. Durig all experimets, the air pump was tured o for 1 mi every 10 mi, yieldig a 1- mi pumpig time ad a 9-mi quiescet period (table 1). Maure origiated from healthy aimals at The Uiversity of Teessee Koxville Experimet Statio Dairy. It was collected while still warm, partitioed ito 0 ml sample cotaiers, ad froze withi oe hour of collectio. Prior to experimets, a froze maure sample is thawed by immersio i warm (ca. 70 C) water for at least oe hour. After this reheatig period, the warm maure is deposited i a L Erlemeyer flask, ad the flask sealed with several layers of Parafilm. Maure odor is the collected from the flask headspace by meas of a 10 ml syrige ad hypodermic eedle. Table 1. Typical experimetal timelie. Odor presetatios bega after a two-hour settlig period, ad each odor presetatio was made by rapidly emptyig a 10 ml syrige full of odorous gas ito the clea air stream, approximately 15 s ito the 60 s pumpig period Time After Experimet Evet Start No. (hh:mm:ss) Evet Commet 1 1:00:00 Flies trasferred from rearig box to test cotaier; cotaier Aspirator exposes flies to high moisture ad CO cocetratios placed i isulated box for storage ad trasport to testig lab (huma breath) 0:10:00 Place test cotaier i apparatus 3 0:00:00 Start experimetal sequece o computer; air pump o Typically 10:30 A.M. 4 0:01:00 Pump off; sese fly activity for 9 mi 5 0:10:00 Pump o (1 mi) 0:10:00- :00:00 Repeat steps 4 ad 5 Settlig time for flies 6 :00:00 Pump o 7 :00:15 Odor ijected 8 :01:00 Pump off; sese fly activity for 9 mi 9 :10:00 Pump o :10:00- Repeat steps 7-9, staggerig ijectios by at least 30 mi Ed VOL. 40(6):

5 MICROPHONE AND ASSOCIATED CIRCUITRY A electrostatic microphoe is biased at 5 V ad coected to the sigal amplificatio ad rms coversio circuit. The microphoe sigal is amplified 300, the fed to a rms detectio itegrated circuit (SSM-110 True RMS-to- DC Coverter, Precisio Moolithic Icorporated, Sata Clara, Calif.), cofigured to reject sigals below 140 Hz, ad to average the iput sigal for 15 ms. Curret output from the PMI is coverted to voltage by aother amplifier, ad the output voltage is moitored by a 16-bit aalog-to-digital (A/D) coverter (CIO-DAS 160/16, ComputerBoards, Ic., Masfield, Mass.), i a 486-based PC. ANCILLARY COMPONENTS A computer program (Microsoft QuickBASIC, ver. 4.5) collects data from the rms circuit, ad cotrols the airflow through the fly cotaier, via the DAC ad a optically isolated relay. Because of ucertaity regardig the patters of activity accompayig odor presetatio, the raw rms output voltages are read ad stored, without pre-processig. Processig prior to storage would reduce the storage requiremets, but would also reduce our ability to aalyze uforesee effects. To somewhat reduce the data storage requiremets, time ad voltage data are oly stored to disk if the rms sigal exceeds a threshold level (software adjustable, but typically 0.1 V). Figure 3 illustrates a flowchart of the data acquisitio program, showig sigal averagig, timestampig, threshold cosideratios, ad data storage. The high amplificatio of the microphoe sigal makes it susceptible to iterferece from exteral soud sources, such as vehicles outside the buildig, footsteps i the hallway, ad doors closig. Both active ad passive oise reductio methods were cosidered, ad a passive method chose for simplicity. However, a active method, usig a secod microphoe mouted slightly farther from the cotaier top, coupled with a differece amplifier, could ehace oise rejectio ad reduce the system size. The passive method relies o a cm box with walls made of a three-layer sadwich of dese, light, ad dese materials (1.6 cm plywood, 4.8 cm fiberglass isulatio, Figure 3 Simplified flowchart of data collectio program. To allow flexibility i processig, o o-lie data processig is used. 1.6 cm ceilig tile). The fly cotaier base attaches to the bottom surface of the box. The rest of the box ca be lifted off the bottom piece, ad attached securely by meas of two cam-type latches. Because light is ecessary for face fly activity (Teskey, 1969), ad because eve small (1 W) icadescet lamps would rapidly heat the air i the isulated box, a 0.6 cm diameter Plexiglas rod with polished eds is iserted through a hole i the box top to serve as a light pipe. Durig experimets a fluorescet lamp is placed 1 cm from the outer ed of the pipe. FLIES: GROWTH, PRETREATMENT, AND HANDLING Face flies have bee maitaied i coloy for over five years, ad were origially obtaied from a existig coloy at USDA s Beltsville isectory. Adults are kept at 1 to 4 C with 4 h light i cm alumium-framed scree cages. They are provided graulated table sugar, o-fat powdered milk, ad water, ad lib. Female flies used i each experimet are betwee 9 ad 11 days old, ad have ot bee allowed access to cow maure, which is ormally preseted to stimulate ovipositio. These steps are take to esure that the files are gravid ad thus attracted by bovie maure odor. Flies are froze ad dissected after each experimet to esure that they are gravid. Three hours prior to each experimet, six apparetly gravid female flies are aspirated from the rearig cage ad placed i a clea, dry fly cotaier (fig. ). The loadig port is subsequetly sealed with Parafilm. A swatch of moist cotto (ca. 1 cm ) is also placed i the cotaier to prevet desiccatio. The loaded fly cotaier is the placed i a isulated luch box for temporary storage ad trasportatio to the experimetal apparatus. DATA ANALYSIS METHODS The sheer quatity of raw voltage versus time data (ca. 1 MB per experimet) makes extractio of summary parameters ecessary. Sice each oe-mi pumpig evet is followed by a ie-mi quiescet (pump-off) period (table 1), it is reasoable to geerate summary parameters for cosecutive 9-mi periods. The three parameters calculated are the umber of evets (or distict buzzes) takig place per time period, the itegral of (or total) soud eergy per time period, ad the duratio (or total time above threshold) per time period. This aalysis, or data compressio, is accomplished by a secod computer program. The algorithm for computig the umber of evets is to cout the umber of peaks i the voltage waveform, the itegral is computed by summig the voltage sigal over each time period (possible sice the time betwee voltage readigs is equal), ad the duratio is computed by simply coutig the umber of data poits (sice each data poit correspoded to a overthreshold evet). Data files cotaiig 10-mi summary parameters are loaded ito a spreadsheet program for further processig ad aalysis. This icluded percet-ormalizig each summary parameter value to its highest value (i.e., the highest value is always set to 100, ad the rest scaled accordigly), ad mior reformattig. Graphs of ormalized summary parameters versus time were made for the prelimiary experimets to detect maure odor, while graphs of ormalized summary parameters versus odor volume were made for kietic experimets. 170 TRANSACTIONS OF THE ASAE

6 PRELIMINARY EXPERIMENTS TO DETECT MANURE ODOR As metioed earlier, our goal is to preset the odor i a short burst so the isects will have a sesory experiece similar to that occurrig durig a flight through a filametous odor plume. Thus, durig all experimets, the syrige is emptied ito the air stream as rapidly as possible (ca. 1 s). The ijectio is made approximately 15 s ito a 60 s pumpig evet. The tubig volume betwee the ijectio block ad cotaier is approximately 0 ml, givig a 0.6 s retetio time i the tube. The theoretical gas retetio time i the fly cotaier is 11 s, meaig that four flushigs of the cotaier ca occur durig the 45 s after a ijectio. As with may complex systems, face flies exhibit hysteresis (or coditioig). That is, fly resposes are depedet o prior odor exposure. A maifestatio of this is the period of high activity observed approximately oe hour immediately after flies are trasferred ito the apparatus. (The flies are exposed to ambiet odors durig the trasfer.) For this reaso, a miimum h waitig period is used prior to odor ijectios. A typical experimetal timelie (table 1) illustrates the critical evets that occur durig each experimet. Durig odor-detectio experimets, 10 ml of odorous gas is ijected every 30 mi, while durig doserespose experimets, the volume of odorous gas varies from 1 to 10 ml every 30 mi. DOSE-RESPONSE EXPERIMENTS A fuctioal odormeter should have a dyamic rage over which icreases i cocetratio result i mootoically icreasig detector respose. After prelimiary experimets showed that the presece of odor could be detected, we performed dose-respose experimets to see if the system could differetiate betwee differet odor cocetratios. The odor source was the same as used previously, but rather tha withdrawig 10 ml of gas from the headspace of the maure cotaier, we ow withdrew 0, 1, 5 or 10 ml, ad the withdrew gas from the headspace of a empty cotaier, to get a total syrige volume of 10 ml prior to ijectio. The gas volume was kept at 10 ml to miimize the variatios i mixig that might have occurred had the total ijected volume decreased. Three separate kietic tests were ru with each set of flies, each takig approximately h to complete. Figure 4 Graph of 3 s of raw data, with seve peaks i fly activity apparet. The threshold voltage to trigger data storage is typically 0.1 V. The first data recorded is therefore at a somewhat higher voltage (0.1 V i this case). Note the quiescet period several secods before ad after the burst of activity; this was a typical patter of activity. Approximately 100 measuremets are made per 1 s iterval, leadig to large (ca. 1 MB) raw data files. to odor over the course of six hours, later dose-respose experimets show that the fly resposes chaged sigificatly betwee the first odor sequece (1:00-1:00 P.M.) ad the last oe (3:30-4:30 P.M.). DOSE-RESPONSE EXPERIMENTS I six dose-respose experimets coducted i the 4 to 3 C temperature rage, a clear relatioship (e.g., data poits i fig. 6) is observed, betwee the duratio of fly activity ad the volume of odorous air ijected ito the fly cotaier (V odor ). The respose decreases over the course of the day, with oly two of the six experimets respodig well o the secod kietic test, ad oe showig the effect durig the third test. The itegral of activity behaves similarly, while the umber of evets is geerally less correlated with V odor ; we will, therefore, limit our discussio to the duratio values. RESULTS AND DISCUSSION PRELIMINARY EXPERIMENTS The fly activity sesig system works well. Figure 4 shows approximately 1 s of raw data collected i a typical experimet. The quiescet voltage from the rms circuit is 0.05 V, typically, ad the 1 s trace i figure 4 reflects a time durig which multiple flights appear to have occurred (hece the multiple peaks i activity). Figure 5 shows 6 h of compressed evet-umber data, alog with dark bars at 1:40, 1:40, :40, ad 3:40 P.M., to idicate the time of odor ijectios. A arbitrary threshold value of 50 ormalized evets per mi is also idicated o the graph, as a dashed horizotal lie. The importace of allowig sufficiet settlig time betwee odor presetatios is highlighted by the secod peak i activity that occurs at 4:00 P.M., 0 mi after the ijectio of odor. Although figure 5 idicates that flies seem to respod well Figure 5 Example of compressed data graph, showig umber of evets per mi (ormalized so that 100 maximum umber recorded durig the day). Bars are 10 mi wide, accoutig for a 1 mi pumpo period ad 9 mi pump-off period. Shaded bars represet itervals where a 10 ml odor ijectio took place durig the pumpo period. The relatioship betwee odor ijectio ad electroically measured fly activity is clearly show. VOL. 40(6):

7 A typical respose curve relatig the ormalized duratio to V odor is show i figure 6. The saturatig ature of the respose is evidet; it is observed durig the first kietic test durig all six experimets. Several two parameter models ca be used to describe this type of respose, icludig a iverted decayig expoetial, but we have chose to use a Michaelis-Mete (i.e., rectagular hyperbola) type equatio because of the mechaistic iterpretatio that ca be give to both parameters. Specifically, give the followig Michaelis- Mete type equatio: D D max V odor k m +V odor (1) where D is the duratio, D max is iterpreted as the maximum duratio of fly activity per time period, ad k m (ml) is a half-velocity volume (sice D 0.5D max at V odor k m ). The half-velocity volume depeds upo the sesitivity of the populatio to the applied odor. Usig a umerical least-squares approach to fid the kietic parameters D max ad k m, yields data show i table. I this case, we use the raw, as opposed to the ormalized, duratio values; this idicates the actual time of activity i a particular experimet. Preseted this way, the duratio is essetially the percetage of time spet above threshold soud level; i.e., a D max of 6 correspods to measurable fly activity 6% of the time. Of ote i table are the high r values observed for all but oe ru, the factor of 3 variatio i D max for the first rus, ad the factor of 9 variatio i the k m values. The factor of three variatio i D max probably reflects differeces i idividual fly activity rates, which may be geetically ad evirometally cotrolled. Regardless of the source of this variability, it does ot detract greatly from the potetial utility of the odormeter, sice the suggested protocol for testig ukow odors would deped o saturatig the fly respose, ot o achievig a particular level of respose. Havig said that, the variatio i k m does detract greatly from the potetial utility of such a system, because k m is essetially a measure of the sesitivity of the flies to low odor cocetratios. Figure 6 Example of ormalized fly duratio kietics (data from d ru o 6/13). Data are represeted by ; solid curve represets fitted lie. Parameter estimatio yields a D max of 45 ormalized uits, ad a k m value of 1.1 ml. Table. Summary kietic parameters for six experimets (ote that some experimets have results from two kietic rus listed) Date Ru No. T ( C) D max a k m b r c 6 Jue Jue Jue Jue Jue July July July July 1996* 1* 9* 6.4* 1.3* 0.98* a D max reflects the maximum time of fly activity (as percetage of possible). b k m is the half-velocity odor volume (ml). c r is the correlatio-coefficiet for the o-liear curve fit. * Zero volume data poit was discarded because of a suspected syrige cotamiatio. However, it is highly likely that the odorous gas preseted to the flies chaged from experimet to experimet, due to imprecise temperature cotrol i the flask. Therefore, variability i k m may be attributed ot oly to iheret variatio i fly sesitivity, but also to variatios i odor source: The flies may be more cosistet i their resposes tha is reflected i the k m values. The testig scheme proposed at the ed of this sectio attempts to address this problem through stadardizatio of fly resposes. Two poits should be oted i the sigle ru with a low r value. First, the egative r value is a result of the oliear curve fit. If the sum of squared errors betwee the fitted curve ad the data is desigated SSE, ad the sum of total errors betwee the fitted curve ad the average observatio is desigated SSTO, the r may be defied as follows: r 1 SSE SSTO Give such a defiitio, r idicates the degree to which the fitted curve accouts for variatio i the depedet variable. I the liear case, it is impossible for SSE to exceed SSTO, ad r is bouded by 0 ad 1. However, i the o-liear case, SSE ca exceed SSTO, ad r ca take o egative values. Secodly, we suspect that syrige cotamiatio may have caused the high respose at zero odor, ad have therefore also listed the parameter estimates ad r value for the same data set, aalyzed without the zero data poit (table, last row). Notice that droppig the zero data poit does ot chage the D max or k m estimate. This is because the kietic expressio is costraied to pass through the origi, ad the zero data poit does ot affect the parameter estimatio, whereas it sigificatly affects the SSE computatio, ad therefore the r value. I four other experimets, virtually o correlatio is observed betwee fly activity ad V odor, but these experimets suffer from differet errors, each of which illustrates a specific limitatio of the isect odormeter. Two failed experimets were coducted at a ambiet temperature of 18 to 19 C, which was apparetly too cold for sigificat fly activity to be obtaied (ot oly is there little correlatio betwee ijected odor volume ad fly () 1704 TRANSACTIONS OF THE ASAE

8 activity, but there is little overall activity at ay time, as compared with experimets coducted i the 4-3 C rage). Cosiderig that Teskey (1969) reports a miimum temperature for activity as 15 C, it is ot surprisig that low activity is observed at 18 to 19 C. Aother failure occurred whe we did ot place the moist cotto swab i the fly cotaier; i this case, the measured activity levels are high, but ot correlated with odor presetatio, perhaps idicatig cotiuous water seekig rather tha sesitivity to maure odors. I the third case, we substituted fresh, roomtemperature maure for the froze, reheated maure ormally used to produce odors. The lower temperature of the fresh sample evidetly made a large differece i the rate of odorat emissio (o doubt varyig across compouds), ad fly resposes were scattered. Thus, temperature cotrol, water availability, ad cosistecy of odor source are all critical to reliable operatio of this device. I light of the successful demostratio of a graded, electroically measurable respose to heterogeeous odor, it is importat to cosider how such a device could actually be used to ivestigate the stregth of ukow odors. If the air hadlig system is improved so that temperature ad relative humidity are strictly cotrolled, the the first step i measurig a ukow odor would be to provide the flies with a series of cocetratios of a stadard, siglecompoet odorat, thus establishig their relative sesitivity. This mimics the ASTM butaol referece method (ASTM Method E , 1975), which uses butaol as a stadardizig aget i huma odor paels. Havig established a half-velocity volume for the stadard gas [k m (sg)], the ukow odor could be applied i icreasig volumes, util the fly respose saturates. The half-velocity volume of the ukow odor [k m (u)] could be computed, ad the ratio k m (sg)/k m (u) used as a measure of odor stregth. Experimets usig precisely cotrolled challege gases could the be coducted to demostrate the istrumets repeatability. Successful passage of this stage would lead to the fial test: experimets that correlate the results from the isect odormeter with those of a huma pael. Such experimets could be coducted with a variety of heterogeeous odors. For example, prelimiary experimets with the curret system have idicated that face flies ca detect swie maure odors, but ot chicke maure odors. Oe strategy for expadig or alterig the system sesitivity would be to use differet isects i the chamber (e.g., the house fly for geeral odors). The proposed system o loger appears small ad simple eough for field work. It is, however, still potetially simpler ad cheaper tha huma odor paels, ad seems worthy of further study. CONCLUSIONS A whole-orgaism biosesor has successfully bee used to detect bovie maure odors. The device electroically measures face fly resposes to odor presetatio, ad ca distiguish betwee low ad high cocetratios of bovie maure odor. Biological variability i the device suggests that a stadardizatio techique be used to ormalize resposes over differet fly populatios, ad the device s usefuless will oly be established whe its respose is correlated with huma odor pael resposes. ACKNOWLEDGMENTS. Support for this work was provided by the Teessee Agricultural Experimet Statio, uder project TN81. I additio, the authors gratefully ackowledge the assistace of the followig idividuals: Paul Elliott, Walker Garer, ad Craig Wagoer, for fabricatig the mechaical parts of all versios of the apparatus; Richard Roy, for providig reagets ad lab supplies; David Russell, for helpig solve oise problems i the circuitry; Dave Paulse ad Eric Marslad, for maitaiig the fly populatio, collectig maure samples ad loadig flies ito the chambers; Drs. Joh B. Wilkerso, Roald E. Yoder, ad C. Rolad Mote for suggestios for improvig the mauscript. REFERENCES ASTM Stadard practices for referecig suprathreshold odor itesity. Desigatio E (Reapproved 1981). Philadelphia, Pa.: Am. Soc. Testig Materials. Barrigto, S. F., K. El Moueddeb ad S. Hegirum Biological additives ad biofilters. I Proc. It. Livestock Odor Cof., Ames, Iowa: Iowa State Uiversity College of Agriculture. Bay, D. E. ad C. W. Pitts Olfactory resposes of face flies, Masca autumalis, to bovie faeces. Aals Et. Soc. Am. 69(5): Bowe, M. F The sesory physiology of host-seekig behavior i mosquitoes. A. Rev. Etomol. 36: Hacock, R. G. ad W. A. Foster Effects of preblood-meal sugar o sugar seekig ad upwid flight by gravid ad parous Aedes aegypti (Diptera: Culicidae). J. Med. Etomol. 30(): Hobbs, P. J., T. H. Misselbrook ad B. F. Pai Assessmet of odours from livestock wastes by a photoioizatio detector, a electroic ose, olfactometry ad gas chromatography-mass spectrometry. J. Agric. Eg. Res. 60: Hobbs, P. J. ad B. F. Pai Odor reductio i fresh pig slurry by dietary maipulatio of protei. I Proc. It. Livestock Odor Cof., Ames, Iowa: Iowa State Uiversity College of Agriculture. Joes, M., P. J. Watts ad R. J. Smith A mobile dyamic olfactometer for feedlot odor studies. Applied Egieerig i Agriculture 10(3): Koster, E. P Limitatios imposed o olfactometric measuremets by the huma factor. I Odour Prevetio ad Cotrol of Orgaic Sludge ad Livestock Farmig, eds. Nielse, V. C., J. H. Voorburg ad P. L Hermite, Lodo, U.K.: Elsevier Applied Sciece Publishers. Lord, H. W., W. S. Gatley ad H. A. Evese Noise Cotrol for Egieers. Malabar, Fla.: Robert E. Krieger Publishig Co., Ic. Metcalf, R. L. ad E. R. Metcalf Chemical ecology of plat kairomoes. I Plat Kairomoes i Isect Ecology ad Cotrol, New York, N.Y.: Routledge, Chapma & Hall, Ic. Miller, T. A. ad R. E. Treece Some relatioships of face fly feedig, ovaria developmet, ad icidece o dairy cattle. J. Eco. Etomol. 61(1): Mote, C. R Evaluatio of a catalytic air-treatmet system for ammoia cotrol i broiler houses. Applied Egieerig i Agriculture 7(): O Neill, D. H. ad V. R. Phillips A review of the cotrol of odour uisace from livestock buildigs: Part 3, Properties of the odorous substaces which have bee idetified i livestock wastes or i the air aroud them. J. Agric. Eg. Res. 53(1):3-50. VOL. 40(6):

9 Powers, W. J., A. C. Wilkie ad H. H. Va Hor Effect of aaerobic digestio ad commercial additives o odors from liquid dairy maure. I Proc. It. Livestock Odor Cof., Ames, Iowa: Iowa State Uiversity College of Agriculture. Schaefer, J Samplig, characterizatio ad aalysis of malodours. Agriculture ad Eviromet 3(/3): Scheider, D., B. C. Block, J. Boeckh ad E. Prieser Die reactio der Maliche Seidespier auf Bombykol ud seie Electroateograme ud Verhalte (The reactio of the male Silk Moth to bombykol ad its electroateogram ad behavior). Z. Vgl. Physiol. 54: Scholtes, R., J. V. Klarebeek ad M. A. Bruis Cotrol of ammoia emissios with biofilters ad bioscrubbers. I Volatile Emissios from Livestock Farmig ad Sewage Operatios, eds. V. C. Nielse, J. H. Voorburg ad P. L Hermite, Lodo, U.K.: Elsevier Applied Sciece Publishers. Sorel, J. E., R. O. Gautt, J. M. Sweete, D. L. Reddell ad A. R. McFarlad Desig of a 1-butaol scale dyamic olfactometer for ambiet odor measuremets. Trasactios of the ASAE 6(4): , 116. Sweete, J. M., R. E. Childers Jr., J. S. Cochra ad R. Bowler Odor cotrol from poultry maure compostig plat usig a soil filter. Applied Egieerig i Agriculture 7(4): Teskey, H. J O the behavior ad ecology of the face fly, Musca autumalis (Diptera: Muscidae). Caad. Etomol. 101(6): Thomas, G. D., B. Puffler ad C. E. Morga Further studies of field parsitism of the face fly by the ematode Heterotylechus autumalis i cetral Missouri with otes o the goatrophic cycles of the face fly. Eviro. Etomol. 1(6): Westerma, P. W. ad R. Zhag Aerobic treatmet of aimal waste for odor cotrol. I Proc. It. Livestock Odor Cof., 18-. Ames, Iowa: Iowa State Uiversity College of Agriculture. Wilkie, A. C., H. H. Va Hor, W. J. Powers ad K. J. Riedesel Aaerobic treatmet techology a itegrated approach to cotrollig maure odors. I Proc. It. Livestock Odor Cof., 3-7. Ames, Iowa: Iowa State Uiversity College of Agriculture. Williams, C. M. ad S. S. Schiffma Livestock odor cotrol additives: Effectiveess ad laboratory-scale protocol for evaluatio. I Proc. It. Livestock Odor Cof., Ames, Iowa: Iowa State Uiversity College of Agriculture. Zeisig, H. D Experieces with the use of biofilters to remove odours from piggeries ad he houses. I Volatile Emissios from Livestock Farmig ad Sewage Operatios, eds. V. C. Nielse, J. H. Voorburg ad P. L Hermite, Lodo, U.K.: Elsevier Applied Sciece Publishers. Zhag, R., D. S. Budy ad Y. Yi Removal of ammoia ad odors from cotamiated air i aimal houses by a ferroelelctric plasma reactor. ASAE Paper No St. Joseph, Mich.: ASAE. Zhag, R. ad P. W. Westerma Solid-liquid separatio for aimal waste. I Proc. It. Livestock Odor Cof., Ames, Iowa: Iowa State Uiversity College of Agriculture. APPENDIX: DERIVATION OF SOUND INTENSITY RATIOS The iverse-square relatioship betwee soud pressure level ad fly-to-microphoe distace ca be quatified i the followig maer: Let P s represet the total soud power emitted by a fly, ad assume that the soud is radiated uiformly i all directios. The itesity (S) of soud at a distace r from the fly ca the be computed as follows (Lord et al., 1987): The ratio of maximum soud itesity (S max ) to miimum soud itesity (S mi ) measured by the system is represeted as S max /S mi. This ratio describes the sesitivity of the system to fly positio; i a ideal system, S max /S mi 1, for all real systems S max /S mi > 1. For a sigle microphoe, this ratio ca be expressed as follows: where r max ad r mi are the maximum ad miimum fly-tomicrophoe distaces, respectively. We estimate a umerical value for S max /S mi by assumig r mi is equal to approximately oe wigspa, or 0.5 cm, ad that the chamber has a height ad diameter of 7.5 cm. I the case of a sigle microphoe mouted half-way up the side of the chamber, r max occurs whe the fly is o the top or bottom of the cotaier, o the opposite side of the microphoe. Geometry gives this distace as 8.4 cm, so that S max /S mi 80. Usig multiple microphoes placed half-way up the chamber walls could reduce S max /S mi. For such a system to work, the sigal from each microphoe eeds to be processed by a separate rms circuit prior to summig, because of phase cacellatio that would occur if the raw microphoe sigals were simply added (Lord et al., 1987). For multiple microphoes, the overall S max /S mi value ca be calculated by takig the followig steps: First compute S max for the etire system, usig the followig equatio: S max where represets the umber of microphoes, ad r i(mi) represets the distace betwee microphoe i ad the fly, whe the fly is directly ext to oe of the microphoes. Note that i this case, the fly is very close to oe microphoe, ad fairly far from the other microphoe(s). Next, compute S mi for the etire system, usig the followig equatio: S mi S max S mi S i max P s 4π S i mi P s 4π S P s 4πr P s 4π r mi P s 4π r max where r i(max) represets the distace betwee microphoe i ad the fly, whe the fly is equidistat from all r max r mi P s 4π ri max 1 r i max P s 4π ri mi 1 r i mi (A1) (A) (A3) (A4) 1706 TRANSACTIONS OF THE ASAE

10 microphoes, ad as far away as possible from them (i.e., o the top or bottom surface of the cylider). Combiig equatios A3 ad A4 yields the followig expressio for the overall S max /S mi : S max S mi 1 r i mi 1 r i max (A5) Usig this expressio, the S max /S mi value is calculated as 57 for, ad 38 for 3. As expected, icreasig the umber of microphoes ca dramatically reduce S max /S mi. The performace of a sigle microphoe suspeded slightly above the fly cotaier ca be aalyzed i a similar fashio. The followig equatio applies for a sigle microphoe mouted axially at a distace h mic above the fly cotaier: S max h mic S mi h mic (A6) The r max coditio occurs whe the fly is at the bottom of the cotaier ad agaist the cotaier wall; the r mi coditio occurs whe the fly is directly below the microphoe. The 0.5 term added to h mic reflects the effect of the fly s wigspa. Equatio A6 predicts that a 1.5 cm h mic value results i S max /S mi 4. A sigle microphoe system with 1.5 cm h mic value gives a 37% better S max /S mi value tha a three-microphoe system, ad is also much simpler to implemet. VOL. 40(6):