the efficacy of diatomaceous earth against the common bed bug

MC Bed Bug Management Report, 2006THE EFFICACY OF DIATOMACEOUSEARTH AGAINST THE COMMONBED BUG, CIMEX LECTULARIUSA report for Mount Sylvia DiatomiteStephen L. Doggett, Merilyn J. Geary,David Lilly* & Richard C. RussellDepartment of Medical Entomology, ICPMR, Westmead Hospital, WESTMEAD, NSW2145. *Ecolab Pest Elimination, SILVERWATER, NSW, 2128.Doggett, Russell & Jones 0May 2008

Efficacy Testing of Diatomaceous Earth for Bed BugsThe Efficacy of Diatomaceous Earthagainst the Common Bed Bug,Cimex lectularius.A report for Mount Sylvia DiatomiteStephen L. Doggett, Merilyn J. Geary,David Lilly* & Richard C. RussellDepartment of Medical Entomology,ICPMR and University of Sydney, Westmead Hospital,WESTMEAD, NSW 2145.Ph: 02 9845 7265 Fax: 02 9893 8659Email: Stephen.Doggett@swahs.health.nsw.gov.au* Ecolab Pest Elimination, 2 Wetherill St,SILVERWATER, NSW, 2128.©2007, 2008. This report was produced by The Department of MedicalEntomology on behalf of and for Mount Sylvia Diatomite. All images arecopyright to the Department of Medical Entomology. The Department ofMedical Entomology, ICPMR, accepts no responsibility for the use of thisreport by other parties. The use of brand names and any mention orlisting of commercial products or services in this report does not implyendorsement by the Department of Medical Entomology, ICPMR, ordiscrimination against similar products or services not mentioned.Warning and DisclaimerEvery effort has been made to make this document to be as completeand accurate as possible, but no warranty or fitness is implied. Theinformation provided is on an ‘as is’ basis. The authors shall have neitherliability nor responsibility to any person, organisation or entity withrespect to any loss or damages arising from the information contained inthis document.Department of Medical Entomologyi

Efficacy Testing of Diatomaceous Earth for Bed BugsEXECUTIVE OVERVIEWCurrently the world is experiencing a major resurgence in bed bugsinfestations, largely due to insecticide resistance. Bed bugs are nowresistant to most of the current commercially available insecticides, andthere is an urgent need for new products to be registered to combat thisemerging public health pest.Diatomaceous Earth dust (DED) has many advantages over presentlyregistered insecticides, including indefinite shelf life, long residual action, lowrisk of insecticide resistance developing, and very low mammalian toxicity.Apropos of this, DED was supplied by Mount Sylvia Diatomite to test theefficacy of the product against the Common bed bug, Cimex lectularius.In the laboratory investigations, DED produced a complete kill of adult bedbugs with all dose rates. At the dose rate equivalent to 1g of product/m 2this took 15 days; 2g/m 2 – 10d; 4g/m 2 – 13d; 8g/m 2 – 9d.For the first instar stage, DED again produced a complete kill with all doserates and at a faster rate than adults. At the dose rate equivalent to 1g ofproduct/m 2 this took 9 days; 2g/m 2 – 9d; 4g/m 2 – 3d; 8g/m 2 – 4d.Testing under different humidity levels demonstrated that the product wasslower acting, however a complete kill was again achieved. At the dose rateequivalent to 1g of product/m 2 this took 18 days; 2g/m 2 – 20d; 4g/m 2 –11d; 8g/m 2 – 11d.Testing of the coarser grained Diatomaceous Earth Fines, produced acomplete kill of adult bed bugs with all dose rates, albeit of a longerduration. At the dose rate equivalent to 1g of product/m 2 this took 17days; 2g/m 2 – 12d; 4g/m 2 – 11d; 8g/m 2 – 11d.Testing of DED in a simulated environment (a mesocosm) found theproduct to be efficacious with a complete kill achieved within 9 days.It was found that DED could be readily applied via typical pest controlequipment used for insecticidal dust application.The experiments demonstrated that DED is highly efficacious against bedbugs under a range of conditions, and the dose rate is not critical.Stephen L. DoggettSenior Hospital Scientist &Principal Investigator27/May/2008Department of Medical Entomologyii

Efficacy Testing of Diatomaceous Earth for Bed BugsTABLE OF CONTENTSEXECUTIVE OVERVIEW............................................................................... iiThe efficacy testing of Diatomaceous Earth Dust to the Common BedBug, Cimex lectularius, i. Laboratory Investigations................................. 1Background ............................................................................................ 1Methods.................................................................................................. 2Test Animal ........................................................................................... 2Insecticide ............................................................................................. 2Experimental Procedure .......................................................................... 2Results ................................................................................................... 3Discussion .............................................................................................. 4Conclusion.............................................................................................. 5References ............................................................................................. 5The efficacy testing of Diatomaceous Earth Dust to the Common BedBug, Cimex lectularius, ii. Mesocosm Investigations............................... 36Background .......................................................................................... 36Methods................................................................................................ 37Test Animal ......................................................................................... 37Insecticide ........................................................................................... 37Mesocosm Construction ........................................................................ 37Bulb Hand Duster Calibration................................................................. 37Experimental Procedure ........................................................................ 37Results & Discussion............................................................................ 38Bulb Hand Duster Calibration................................................................. 38Application Dose Rate........................................................................... 38Efficacy Results.................................................................................... 39Conclusion............................................................................................ 39References ........................................................................................... 39Department of Medical Entomologyiii

Efficacy Testing of Diatomaceous Earth for Bed BugsThe efficacy testing of Diatomaceous Earth Dust tothe Common Bed Bug, Cimex lectularius.i. Laboratory InvestigationsBackgroundWorldwide since the beginning of this century, there has been a dramatic increasein the number of bed bug infestations, both of the Common (Cimex lectularius) andTropical (Cimex hemipterus) species (Doggett et al. 2003, 2004, 2005). During thisperiod, the number of infestations has increased by some 4,500% across Australia(Doggett & Russell 2007). Numerous suggestions have been offered to account forthe return of this public health menace, however it is thought that insecticideresistance has been the main contributing cause. Resistance has now beenreported overseas with the synthetic pyrethroids and the carbamates (Boase et al.2006, Romero et al. 2007). Many Australian pest managers have reported productfailure (Doggett & Russell 2007) and the Department of Medical Entomology has astrain of bed bugs that were collected off permethrin dust, suggesting thatresistance is a major issue in this country as well.Unfortunately, most of the insecticides registered in Australia belong to thosegroups that have demonstrated resistance. This has meant that control is a majorchallenge and treatment failures have been all too common. Thus there is anurgent need for new insecticides to become available for the control of bed bugs.One candidate insecticide that could be of benefit in bed bug control isDiatomaceous Earth dust (DED). This insecticidal dust has a number of advantages.Being silica based it has an extremely long shelf; one study found that a silicabased product was still efficacious after 406 days of storage (Tarshis 1962). DEDhas an extremely long residual action and tends to be nullified only by the built upof dirt, household dust and other debris. DED’s mode of action limits the possibilityof resistance developing as it has a physical rather than a chemical/metabolicaction (Quarles & Winn 1996), in fact DED is not even listed by the InternationalResistance Action Committee (http://www.irac-online.org/). DED leaves nochemical residue (Quarles & Winn 1996) and has extremely low mammalian toxicityand is often recommended by environmental groups. As DED has a long residualaction, there is a strong potential for this dust to be employed as a prophylacticinsecticide.Currently, DED is registered for bed bug control in the US and has long beenemployed in the management of this insect. However, despite being registeredoverseas, efficacy data has not been published to date. The aim of thisinvestigation is to test the susceptibility of the Common bed bug, Cimex lectularius,Department of Medical Entomology 1

Efficacy Testing of Diatomaceous Earth for Bed Bugsto DED in order to provide efficacy data towards having DED registered for thecontrol of bed bugs (Cimex spp.) in Australia.MethodsThe methods are loosely based on Fletcher & Axtell (1993).Test AnimalThe bed bugs used are the Common bed bug, Cimex lectularius, from a colonyestablished by the Department of Medical Entomology during 2004. The coloniesheld by the Department are the only bed bug colonies in Australia. The founderspecimens were sourced from various infestations within the Sydney metropolitanarea and have not been profiled for insecticide susceptibility, however are knownfrom in-house efficacy testing to have a degree of insecticide resistance to both thesynthetic pyrethroids and the carbamates. The colony is maintained within 1Lcontainers, provided with a blood meal once per week via anesthetised rats, andheld at 25 o C, with 80%RH. Active unengorged bed bug adults were removed andused for the tests. Sex and age of the bed bugs were not determined.InsecticideThe DED was supplied by Dr Ian Neering of Mount Sylvia Diatomite. No batchnumber or date of manufacture was provided.Experimental ProcedureThe bed bugs were exposed to residues of the DED on filter paper within Petridishes (Figures 1 & 2). In each experiment, a variety of doses were used toestablish optimal dose rates. The DED was applied at the dose rates equivalent to1, 2, 4 and 8g/m 2 , which covers the dose for which the product is currentlyregistered against other insect pests. The surface area of the filter paper wascalculated and the amount of DED required to treat the area at these ratesdetermined. The dust was applied to the filter paper and spread evenly over thesurface via a small paint brush. For each dose rate of DED, ten bed bugs whereexposed per Petri dish, with a total of four replicates (i.e. 40 bugs in total exposedper treatment). The controls consisted of four replicates of ten bugs on filter paperin Petri dishes not exposed to the DED (i.e. a further 40 bed bugs). Mortality wasdetermined daily from 24 hours onwards by counting bed bugs that do not moveafter the dish is tapped. The experiments were conducted at 22±1 o C, in adarkened environment, relative humidity of 55±5%.For each dose, the daily results were statistically compared with the control via oneway ANOVA.Experiment 1 tested the efficacy of DED against adult bed bugs.Department of Medical Entomology 2

Efficacy Testing of Diatomaceous Earth for Bed BugsExperiment 2 tested the efficacy of DED against first instar bed bugs, using amodified test chamber (Figure 2).Experiment 3 tested the efficacy of DED against adult bed bugs in high humidity(approx 70%RH).Experiment 4 tested the efficacy of Diatomaceous Earth ‘fines’ (DEF) against adultbed bugs. Only two replicates were used for this experiment.ResultsA summary of Experiment 1 results are provided in Table 1 and graphed in Figure3. Raw results are provided in Table 5 and complete statistical analysis in Table 9.In summary, complete adult mortality was achieved with the high dose (8g/m 2 ) byDay 9 and by Day 15 with the lowest (1gm 2 ). Mortality was significantly differentfrom the controls within 1 day with the high dose and by Day 3, with all the dosesof DED.A summary of Experiment 2 results are provided in Table 2 and graphed in Figure4. Raw results are provided in Table 6 and complete statistical analysis in Table 10.Mortality was more rapid with the first instar nymphs compared with the adults;100% death was achieved with the high dose by Day 4 and complete mortalitywith all the doses by Day 9. Even within one day, mortality with all the treatmentswas significantly different from the controls.A summary of Experiment 3 results are provided in Table 3 and graphed in Figure5. Raw results are provided in Table 7 and complete statistical analysis in Table 11.The high humidity slowed down the death rate and it was not until Day 11 when100% mortality was achieved with the high dose, and Day 20 that all bed bugs haddied for each treatment. Despite this, mortality was significantly different from thecontrols with all treatments by Day 3 and for the two highest doses by Day 1.Experiment 4 summary results are provided in Table 4 and graphed in Figure 6.Raw results are provided in Table 8 and complete statistical analysis in Table 12.Complete adult mortality was achieved by Day 11 in the high dose and by Day 17with all the doses. Mortality was significantly different from the controls with alltreatments by Day 9 and for the highest dose by Day 4.Department of Medical Entomology 3

Efficacy Testing of Diatomaceous Earth for Bed BugsDiscussionThe experiments demonstrate that DED is efficacious against bed bugs, includingboth adult and nymphal stages. A complete kill is achieved with DED no matter theinsecticidal dose rate, although is quicker at higher concentrations, and thisdemonstrates that application dose rate is not critical. The time to death isrelatively slow for an insecticide, although this is due to its mode of action. DED isvery different to other insecticides; the waxy surface of the epicuticle is absorbed,meaning that the insect can not maintain moisture levels and the insect dies fromdehydration (St. Aubin 1991). The DED readily adheres to the insect as it movesabout (Figures 7 & 8). Thus the mode of action is more physical rather thanaffecting biochemical and metabolic pathways of the insect like most otherinsecticides.In studies investigating water conservation within the bed bug, it has been foundthat first instar nymphs lose water at a faster rate relative to other stages (Benoitet al. 2007). This would account for the quicker mortality observed in Experiment2. The modified test chamber for this experiment was required in order to containthe insects to the treated area.It is well known that DED is less effective at higher humidities (Quarles & Winn1996, Fields & Korunic 2000). As bed bugs are particularly problematic in tropicalregions, it was thus important that testing was undertaken at a high relativehumidity to examine if the product was still insecticidal. The longer time taken toachieve a complete kill was not unexpected in Experiment 3, however 100%mortality was still obtained with all treatment rates. The higher humidity however,does not affect the insecticide dust itself, rather the insect takes longer to die fromdesiccation. In Australia, humidity levels tend to be greater in the north of thecountry and it thus may be expected that DED would be less efficacious in theseregions, however it is well known that DED is more effective at highertemperatures and this may counteract the higher humidities (Quarles & Winn1996).DEF is a more coarsely ground formulation of DED, with a variable particle sizediameter between 10-1200um, whereas DED has a diameter around 10um.Presumably the more coarse powder of DEF meant that the same surface area ofactive insecticide was not available to the bed bugs and hence the longer timerequired in achieving a complete kill.It is worth noting that there are many different types of DED and not all haveinsecticidal properties. A study undertaken in South Africa against a wide range ofpests including the Common bed bug, found that the DED tested had little to noefficacy (Martindale & Newlands 1981). In contrast, the DED supplied by MountDepartment of Medical Entomology 4

Efficacy Testing of Diatomaceous Earth for Bed BugsSylvia Diatomite Mines was highly efficacious. This implies that any DiatomaceousEarth that is submitted for registration should be accessed for its efficacy.The DED dust used for testing was observed to clump and so there were concernsthat the dust may not flow properly via an insecticide delivery system. Preliminarytesting with a termite duster found that this was not the case and that the productflowed without causing any blockage to the nozzle. It was probable that theclumping resulted from moving the test containers from the darkened to a lightenvironment to read the trialsAs noted above DED has a number of advantages:• Indefinite shelf life,• Long residual action,• No chemical residues,• Low probability of resistance, &• Very low mammalian toxicity.Additionally, as the experiments testify, the dose rate is not critical as alltreatments yielded a complete kill. However, an application rate around 2-4g/m 2would be a good compromise to achieve a moderate kill rate.As DED is a grey dust, it would only be applied to non-obvious locations, which areareas where bed bugs normally harbour. The dust’s unique properties mean that itcould be applied as a barrier treatment, or as a prophylactic insecticide to minimisethe spread of an infestation or to help contain potential new infestations.With the lack of registered products in Australia and the high degree of insecticidalresistance reported to those that are registered, new insecticidal compounds areurgently required for bed bug control. The experiments show that DED is anefficacious product and would be an additional useful tool in the arsenal ofproducts available to the pest manager for bed bug eradication.ConclusionDED was found to be highly efficacious against both nymphs and adults of theCommon bed bug, Cimex lectularius, at a variety of doses and under differenthumidity levels.ReferencesBenoit J.A., Del Grosso N.A., Yoder J.A. and Denlinger D.L. (2007). Resistanceto dehydration between bouts of blood feeding in the bed bug, CimexDepartment of Medical Entomology 5

Efficacy Testing of Diatomaceous Earth for Bed Bugslectularius, is enhanced by water conservation, aggregation, andquiescence. American Journal of Tropical Medicine and Hygiene, 76(5): 987-993.Boase C., Small G., and Naylor R. (2006). Interim report of insecticidessusceptibility status of UK bedbugs. Professional Pest Controller, Summer:12-13.Doggett S.L. and Russell R.C. (2007). Bed Bugs – Latest Trends &Developments. Synopsis of The Australian Environmental Pest ManagersAssociation National Conference, Pacific Bay Resort, Coffs Harbour, 4-6 th July 2007,pg 22-37.Doggett S.L. (2005). Bed bug ecology and control. Chapter 7, Pests ofDisease & Unease. Synopsis of Papers of the Symposium ‘Pests of Disease &Unease’, held 22 April 2005, Westmead Hospital.Doggett S.L, Geary M.J., Crowe W.J., Wilson P. and Russell R.C. (2003). Has theTropical Bed Bug, Cimex hemipterus (Hemiptera: Cimicidae), invadedAustralia? Environmental Health, 3: 80-82.Doggett S.L, Geary M.J. and Russell R.C. (2004). The resurgence of bed bugs inAustralia, with notes on their ecology and control. Environmental Health, 4:30-38.Fields P. and Korunic Z. (2000). The effect of grain moisture content andtemperature on the efficacy of diatomaceous earths from differentgeographical locations against stored-product beetles. Journal of StoredProducts Research, 36: 1-13.Fletcher M.G. and Axtell R.C. (1993). Susceptibility of the bed bug, Cimexlectularius, to selected insecticides and various treated surfaces. Medicaland Veterinary Entomology, 7: 69-72.Martindale C.B. and Newlands G. (1981). ‘Nature’s bug-killer’ doesn’t. SouthAfrican Journal of Science, 77: 352.Quarles W. and Winn P.S. (1996). Diatomaceous earth and stored productPests. The IPM Practitioner, 13(5/6): 1-10.Romero A., Potter M.F., Potter D.A., and Haynes K.F. (2007). Insecticideresistance in the bed bug: a factor in the pest’s sudden resurgence?Journal of Medical Entomology, 44(2): 175-178.Department of Medical Entomology 6

Efficacy Testing of Diatomaceous Earth for Bed BugsSt. Aubin F. (1991). Everything old is new again. Pest Control Technology,June: 50-102.Tarshis I.B. (1962). The use of silica aerogel compounds for the control ofectoparasites. Proceedings of the animal care panel, 217-258.Department of Medical Entomology 7

Efficacy Testing of Diatomaceous Earth for Bed BugsFigure 1. Photograph of the experimental set up for measuring the efficacy of Diatomaceous Earth dust against the adultbed bug, Cimex lectularius.Department of Medical Entomology 8

Efficacy Testing of Diatomaceous Earth for Bed BugsFigure 2. Photograph of the experimental set up for measuring the efficacy of Diatomaceous Earth dust against firstinstar bed bugs.Department of Medical Entomology 9

Efficacy Testing of Diatomaceous Earth for Bed BugsTable 1. Cumulative percentage mortality of adult Cimex lectularius exposed to various doses of Diatomaceous Earthdust, summary data (Experiment 1).Dose Day1Day2Day3Day4Day5Day6Day7Day8Day9Day10Day11Day12Day13Day14Day158g/m 2 37.5 62.5 80.0 85.0 92.5 95.0 97.5 97.5 100 100 100 100 100 100 1004g/m 2 15.0* 32.5 62.5 82.5 95.0 95.0 95.0 97.5 97.5 97.5 97.5 97.5 100 100 1002g/m 2 10.0* 35.0 47.5 90.0 95.0 95.0 95.0 95.0 95.0 100 100 100 100 100 1001g/m 2 0.0* 5.0* 22.5 32.5 60.0 60.0 65.0 77.5 80.0 90.0 95.0 95.0 95.0 97.5 1000g/m 2 2.5 2.5 2.5 2.5 5.0 5.0 7.5 7.5 10.0 15.0 17.5 22.5 22.5 22.5 27.5*Not statistically different to the controls.Table 2. Cumulative percentage mortality of first instar Cimex lectularius exposed to various doses of DiatomaceousEarth dust, summary data (Experiment 2).Dose Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 98g/m 2 92.5 95.3 99.1 100.0 100.0 100.0 100.0 100.0 100.04g/m 2 96.5 99.1 100.0 100.0 100.0 100.0 100.0 100.0 100.02g/m 2 41.1 66.1 87.5 91.1 96.4 98.2 98.2 98.2 100.01g/m 2 17.1 64.3 81.4 92.9 97.1 97.1 98.6 98.6 100.00g/m 2 0.0 0.0 3.6 5.5 5.5 5.5 5.5 9.1 14.5Note: all treatments on all days were statistically different to the controls.Department of Medical Entomology 10

Efficacy Testing of Diatomaceous Earth for Bed BugsTable 3. Cumulative percentage mortality of adult Cimex lectularius exposed to various doses of Diatomaceous Earth athigh humidity, summary data (Experiment 3).DoseDay1Day2Day3Day4Day5Day6Day7Day8Day9Day10Day11Day12Day13Day14Day15Day16Day17Day18Day19Day208g/m 2 20.0 45.0 52.5 62.5 65.0 72.5 82.5 90.0 95.0 97.5 100 100 100 100 100 100 100 100 100 1004g/m 2 10.0 30.0 50.0 60.0 67.5 72.5 77.5 85.0 87.5 95.0 100 100 100 100 100 100 100 100 100 1002g/m 2 2.5* 7.5* 15.0 22.5 32.5 42.5 47.5 52.5 60.0 75.0 85.0 85.0 87.5 87.5 87.5 90.0 95.0 97.5 97.5 1001g/m 2 2.5* 10.0* 27.5 30.0 35.0 42.5 47.5 55.0 57.5 65.0 72.5 75.0 80.0 85.0 87.5 90.0 95.0 100 100 1000g/m 2 0.0 2.5 2.5 5.0 10.0 15.0 17.5 17.5 17.5 22.5 25.0 25.0 25.0 30.0 35.0 45.0 47.5 47.5 50.0 52.5*Not statistically different to the controls.Table 4. Cumulative percentage mortality of adult Cimex lectularius exposed to various doses of Diatomaceous Earth‘fines’, summary data (Experiment 4).DoseDay1Day2Day3Day4Day5Day6Day7Day8Day9Day10Day11Day12Day13Day14Day15Day16Day178g/m 2 10.0* 25.0* 35.0* 65.0 65.0 75.0 75.0 80.0 85.0 90.0 100 100 100 100 100 100 1004g/m 2 0.0* 5.0* 20.0* 35.0* 45.0 60.0 70.0 80.0 85.0 85.0 100 100 100 100 100 100 1002g/m 2 0.0* 0.0* 10.0* 20.0* 30.0* 30.0* 30.0* 60.0 75.0 85.0 95.0 100 100 100 100 100 1001g/m 2 0.0* 0.0* 0.0 0.0 5.0* 10.0* 15.0* 35.0* 45.0 70.0 75.0 85.0 90.0 95.0 95.0 95.0 1000g/m 2 0.0 0.0 0.0 0.0 5.0 5.0 15.0 15.0 20.0 20.0 25.0 35.0 35.0 40.0 50.0 55.0 65.0*Not statistically different to the controls.Department of Medical Entomology 11

Efficacy Testing of Diatomaceous Earth for Bed Bugs10090808g/m24g/m22g/m21g/m20g/m2Figure 3. Cumulative mortality of adult Cimex lectularius exposedFig 3. Cumulative Mortality of Adult Cimex lectulariusExposed to various to Various doses Doses of Diatomaceous of Earth Earth dust.Cumulative Percent Mortality706050403020100Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15Days Post ExposureDepartment of Medical Entomology 12

Efficacy Testing of Diatomaceous Earth for Bed BugsFigure 4. Cumulative Mortality of 1st Instar Bed bugs, Cimex lectularius ,Figure 4. Cumulative mortality of first instar Cimex lectulariusexposed to varying doses of Diatomaceous Earth.exposed to various doses of Diatomaceous Earth dust.100Cumulative Percent Mortality806040208g/m24g/m22g/m21g/m20g/m20Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9Days Post ExposureDepartment of Medical Entomology 13

Efficacy Testing of Diatomaceous Earth for Bed Bugs100Figure 5. Cumulative mortality of adult Cimex lectularius exposedFig 5. Cumulative Mortality of Adult Cimex lectularius Exposed toto Various various Doses doses of Diatomaceous Earth at dust high at humidity high humidity.Cumulative Percent Mortality90807060504030208g/m24g/m22g/m21g/m20g/m2100Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9Day10Day11Day12Day13Day14Day15Day16Day17Day18Day19Day20Days Post ExposureDepartment of Medical Entomology 14

Efficacy Testing of Diatomaceous Earth for Bed Bugs100Figure 6. Cumulative mortality of adult Cimex lectularius exposedFig 6. Cumulative Mortality of Adult Cimex lectularius Exposed toVarious to various Doses doses of of Diatomaceous Earth Earth 'fines' ‘fines’.Cumulative Percent Mortality908070605040308g/m24g/m22g/m21g/m20g/m220100Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15 Day 16 Day 17Days Post ExposureDepartment of Medical Entomology 15

Efficacy Testing of Diatomaceous Earth for Bed BugsFigure 7. Dead adult common bed bugs, Cimex lectularius, after exposure to Diatomaceous Earth dust.Department of Medical Entomology 16

Efficacy Testing of Diatomaceous Earth for Bed BugsFigure 8. Dead first instar common bed bugs, Cimex lectularius, after exposure to Diatomaceous Earth dust.Department of Medical Entomology 17

Efficacy Testing of Diatomaceous Earth for Bed BugsTable 5. Mortality of adult Cimex lectularius exposed to various doses of Diatomaceous Earth dust, raw data, resultsDays 1 – 4 (Experiment 1).Day 1 Day 2 Day 3 Day 4Dose ReplicateDust%%%No.No. % No. % Cum.No. % Cum.No. % Cum.AmountCum.Cum.Cum.TestedDead Mortality Dead Mortality Dead* Dead Mortality Dead* Dead Mortality Dead*(g)Dead**Dead**Dead**a 10 0.052 7 70 1 10 8 80 1 10 9 90 0 0 9 90b 10 0.051 3 30 4 40 7 70 2 20 9 90 1 10 10 1008g/m 2 c 10 0.051 1 10 5 50 6 60 3 30 9 90 0 0 9 90d 10 0.052 4 40 0 0 4 40 1 10 5 50 1 10 6 60Total 40 15 37.5 10 25.0 25 62.5 7 17.5 32 80.0 2 5.0 34 85.0a 10 0.025 0 0 1 10 1 10 1 10 2 20 4 40 6 60b 10 0.025 3 30 2 20 5 50 4 40 9 90 1 10 10 1004g/m 2 c 10 0.025 1 10 3 30 4 40 3 30 7 70 3 30 10 100d 10 0.025 2 20 1 10 3 30 4 40 7 70 0 0 7 70Total 40 6 15.0 7 17.5 13 32.5 12 30.0 25 62.5 8 20.0 33 82.5a 10 0.013 2 20 3 30 5 50 0 0 5 50 4 40 9 90b 10 0.013 2 20 2 20 4 40 1 10 5 50 4 40 9 902g/m 2 c 10 0.013 0 0 4 40 4 40 1 10 5 50 4 40 9 90d 10 0.013 0 0 1 10 1 10 3 30 4 40 5 50 9 90Total 40 4 10.0 10 25.0 14 35.0 5 12.5 19 47.5 17 42.5 36 90.0a 10 0.006 0 0 1 10 1 10 2 20 3 30 2 20 5 50b 10 0.006 0 0 1 10 1 10 1 10 2 20 0 0 2 201g/m 2 c 10 0.006 0 0 0 0 0 0 2 20 2 20 1 10 3 30d 10 0.006 0 0 0 0 0 0 2 20 2 20 1 10 3 30Total 40 0 0.0 2 5.0 2 5.0 7 70.0 9 22.5 4 10.0 13 32.50g/m 2(Control)a 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0b 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0c 10 0 1 10 0 0 1 10 0 0 1 10 0 0 1 10d 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0Total 40 1 2.5 0 0.0 1 2.5 0 0.0 1 2.5 0 0.0 1 2.5*Cumulative mortality, **Percent cumulative mortalityDepartment of Medical Entomology 18

Efficacy Testing of Diatomaceous Earth for Bed BugsTable 5, cont. Mortality of adult Cimex lectularius exposed to various doses of Diatomaceous Earth dust, raw data,results Days 5 – 8.Day 5 Day 6 Day 7 Day 8Dose ReplicateNo. % Cum. % Cum. No. % Cum. % Cum. No. % Cum. % Cum. No. % Cum. % Cum.Dead Mortality Dead* Dead** Dead Mortality Dead* Dead** Dead Mortality Dead* Dead** Dead Mortality Dead* Dead**a 0 0 9 90 1 10 10 100 10 100 10 100b 10 100 10 100 10 100 10 1008g/m 2 c 1 10 10 90 10 100 10 100 10 100d 2 20 8 80 0 0 8 80 1 10 9 90 0 0 9 90Total 3 30.0 37 92.5 1 2.5 38 95.0 1 2.5 39 97.5 0 0.0 39 97.5a 2 20 8 80 0 0 8 80 1 10 9 90 0 0 9 90b 10 100 10 100 10 100 10 1004g/m 2 c 10 100 10 100 10 100 10 100d 3 30 10 100 10 100 10 100 10 100Total 5 12.5 38 95.0 0 0.0 38 95.0 1 2.5 39 97.5 0 0.0 39 97.5a 0 0 9 90 0 0 9 90 0 0 9 90 0 0 9 90b 1 10 10 100 10 100 10 100 10 1002g/m 2 c 1 10 10 100 10 100 10 100 10 100d 0 0 9 90 0 0 9 90 0 0 9 90 0 0 9 90Total 2 5.0 38 95.0 0 0.0 38 95.0 0 0.0 38 95.0 0 0.0 38 95.0a 2 20 7 70 0 0 7 70 0 0 7 70 1 10 8 80b 3 30 5 50 0 0 5 50 1 10 6 60 0 0 6 601g/m 2 c 3 30 6 60 0 0 6 60 1 10 7 70 2 20 9 90d 3 30 6 60 0 0 6 60 0 0 6 60 2 20 8 80Total 11 27.5 24 60.0 0 0.0 24 60.0 2 5.0 26 65.0 5 12.5 31 77.50g/m 2(Control)a 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0b 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0c 1 10 2 20 0 0 2 20 1 0 3 30 0 0 3 30d 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0Total 1 2.5 2 5.0 0 0.0 2 5.0 1 2.5 3 7.5 0 0.0 3 7.5*Cumulative mortality, **Percent cumulative mortalityDepartment of Medical Entomology 19

Efficacy Testing of Diatomaceous Earth for Bed BugsTable 6. Mortality of first instar Cimex lectularius exposed to various doses of Diatomaceous Earth dust, raw data, resultsDays 1-6 (Experiment 2).Dose8g/m24g/m22g/m21g/m20g/m2Day 1 Day 2 Day 3 Day 4 Day 5ReplicateDust%%%%No.No. % No. Cum.No. Cum.No. Cum.No. CumAmountCum.Cum.Cum.Cum.TestedDead Mortality Dead Dead*Dead Dead*Dead Dead*Dead Dead**(g)Dead**Dead**DeadDead**a 41 0.052 37 90.2 2 39 95.1 2 41 100.0 41 100.0 41 100.0b 20 0.051 17 85.0 1 18 90.0 2 20 100.0 20 100.0 20 100.0c 17 0.051 17 100.0 17 100.0 17 100.0 17 100.0 17 100.0d 28 0.052 27 96.4 0 27 96.4 0 27 96.4 1 28 100.0 28 100.0Total 106 98 92.5 3 101 95.3 4 105 99.1 1 106 100.0 106 100.0a 21 0.025 20 95.2 0 20 95.2 1 21 100.0 21 100.0 21 100.0b 25 0.025 23 92.0 2 25 100.0 25 100.0 25 100.0 25 100.0c 37 0.025 37 100.0 0 37 100.0 37 100.0 37 100.0 37 100.0d 31 0.025 30 96.8 1 31 100.0 31 100.0 31 100.0 31 100.0Total 114 110 96.5 3 113 99.1 1 114 100.0 114 100.0 114 100.0a 23 0.013 15 65.2 3 18 78.3 1 19 82.6 1 20 87.0 1 21 91.3b 7 0.013 0 0.0 3 3 42.9 3 6 85.7 0 6 85.7 1 7 100.0c 11 0.013 5 45.5 3 8 72.7 3 11 100.0 11 100.0 11 100.0d 15 0.013 3 20.0 5 8 53.3 5 13 86.7 1 14 93.3 1 15 100.0Total 56 23 41.1 14 37 66.1 12 49 87.5 51 91.1 3 54 96.4a 14 0.006 4 28.6 3 7 50.0 4 11 78.6 1 12 85.7 1 13 92.9b 27 0.006 5 18.5 16 21 77.8 2 23 85.2 3 26 96.3 0 26 96.3c 19 0.006 3 15.8 7 10 52.6 5 15 78.9 2 17 89.5 2 19 100.0d 10 0.006 0 0.0 7 7 70.0 1 8 80.0 2 10 100.0 10 100.0Total 70 12 17.1 33 45 64.3 12 57 81.4 8 65 92.9 3 68 97.1a 17 0 0 0.0 0 0 0.0 0 0 0.0 0 0 0.0 0 0 0.0b 10 0 0 0.0 0 0 0.0 0 0 0.0 1 1 10.0 0 1 10.0c 14 0 0 0.0 0 0 0.0 0 0 0.0 0 0 0.0 0 0 0.0d 14 0 0 0.0 0 0 0.0 2 2 14.3 0 2 14.3 0 2 14.3Total 55 0 0.0 0 0 0.0 2 2 3.6 1 3 5.5 0 3 5.5*Cumulative mortality, **Percent cumulative mortalityDepartment of Medical Entomology 22

Efficacy Testing of Diatomaceous Earth for Bed BugsTable 6, cont. Mortality of first instar Cimex lectularius exposed to various doses of Diatomaceous Earth dust, raw data,results Days 6-9.Dose Replicate8g/m24g/m22g/m21g/m20g/m2No.TestedNo.DeadDay 6 Day 7 Day 8 Day 9% Cum. No. Cum. % Cum. No. Cum. % Cum. No. Cum.Dead** Dead Dead* Dead** Dead Dead* Dead** Dead Dead*Cum.Dead*% Cum.Dead**a 41 41 100.0 41 100.0 41 100.0 41 100.0b 20 20 100.0 20 100.0 20 100.0 20 100.0c 17 17 100.0 17 100.0 17 100.0 17 100.0d 28 28 100.0 28 100.0 28 100.0 28 100.0Total 106 106 100.0 106 100.0 106 100.0 106 100.0a 21 21 100.0 21 100.0 21 100.0 21 100.0b 25 25 100.0 25 100.0 25 100.0 25 100.0c 37 37 100.0 37 100.0 37 100.0 37 100.0d 31 31 100.0 31 100.0 31 100.0 31 100.0Total 114 114 100.0 114 100.0 114 100.0 114 100.0a 23 1 22 95.7 22 95.7 0 22 95.7 1 23 100.0b 7 7 100.0 7 100.0 7 100.0 7 100.0c 11 11 100.0 11 100.0 11 100.0 11 100.0d 15 15 100.0 15 100.0 15 100.0 15 100.0Total 56 1 55 98.2 55 98.2 55 98.2 1 56 100.0a 14 0 13 92.9 1 14 100.0 14 100.0 14 100.0b 27 0 26 96.3 0 26 96.3 0 26 96.3 1 27 100.0c 19 19 100.0 19 100.0 19 100.0 19 100.0d 10 10 100.0 10 100.0 10 100.0 10 100.0Total 70 0 68 97.1 1 69 98.6 69 98.6 1 70 100.0a 17 0 0 0.0 0 0 0.0 0 0 0.0 0 0 0.0b 10 0 1 10.0 0 1 10.0 0 1 10.0 2 3 30.0c 14 0 0 0.0 0 0 0.0 1 1 7.1 1 2 14.3d 14 0 2 14.3 0 2 14.3 1 3 21.4 0 3 21.4Total 55 0 3 5.5 0 3 5.5 2 5 9.1 3 8 14.5*Cumulative mortality, **Percent cumulative mortalityDepartment of Medical Entomology 23

Efficacy Testing of Diatomaceous Earth for Bed BugsTable 7. Mortality of adult Cimex lectularius exposed to various doses of Diatomaceous Earth dust at high humidity, rawdata, Days 1-4 (Experiment 3).Dose8g/m24g/m22g/m21g/m20g/m2ReplicateNo.TestedDustAmount(g)No.DeadDay 1 Day 2 Day 3 Day 4%MortalityNo.Dead%MortalityCum.Dead*%Cum.Dead**No.Dead%MortalityCum.Dead*%Cum.Dead**No.Dead%Mortalitya 10 0.052 0 0 1 10 1 10 0 0 1 10 2 20 3 30b 10 0.051 3 30 3 30 6 60 0 0 6 60 1 10 7 70c 10 0.051 2 20 2 20 4 40 3 30 7 70 0 0 7 70d 10 0.052 3 30 4 40 7 70 0 0 7 70 1 10 8 80Total 40 8 20.0 10 25.0 18 45.0 3 7.5 21 52.5 4 10.0 25 62.5a 10 0.025 1 10 2 30 3 30 2 20 5 50 2 20 7 70b 10 0.025 0 0 3 30 3 30 0 0 3 30 2 20 5 50c 10 0.025 2 20 0 0 2 20 4 40 6 60 0 0 6 60d 10 0.025 1 10 3 30 4 40 2 20 6 60 0 0 6 60Total 40 4 10.0 8 20.0 12 30.0 8 20.0 20 50.0 4 10.0 24 60.0a 10 0.013 0 0 0 0 0 0 1 10 1 10 1 10 2 20b 10 0.013 1 10 0 0 1 10 0 0 1 10 1 10 2 20c 10 0.013 0 0 1 1 1 10 1 10 2 20 1 10 3 30d 10 0.013 0 0 1 1 1 10 1 10 2 20 0 0 2 20Total 40 1 2.5 2 5.0 3 7.5 3 7.5 6 15.0 3 7.5 9 22.5a 10 0.006 0 0 0 0 0 0 3 30 3 30 0 0 3 30b 10 0.006 0 0 2 2 2 20 3 30 5 50 0 0 5 50c 10 0.006 0 0 0 0 0 0 0 0 0 0 1 10 1 10d 10 0.006 1 10 1 1 2 20 1 10 3 30 0 0 3 30Total 40 1 2.5 3 7.5 4 10.0 7 17.5 11 27.5 1 2.5 12 30.0a 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0b 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0c 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0d 10 0 0 0 1 1 1 10 0 0 1 10 1 10 2 2Total 40 0 0.0 1 2.5 1 2.5 0 0.0 1 2.5 1 2.5 2 5.0*Cumulative mortality, **Percent cumulative mortalityCum.Dead*%Cum.Dead**Department of Medical Entomology 24

Efficacy Testing of Diatomaceous Earth for Bed BugsTable 7, cont. Mortality of adult Cimex lectularius exposed to various doses of Diatomaceous Earth dust at highhumidity, raw data, Days 4-8.Dose8g/m24g/m22g/m21g/m20g/m2ReplicateNo.Dead%MortalityDay 5 Day 6 Day 7 Day 8Cum.Dead*%Cum.Dead**No.Dead%MortalityCum.Dead*%Cum.Dead**No.Dead%MortalityCum.Dead*%Cum.Dead**No.Dead%Mortalitya 0 0 3 30 1 10 4 40 2 20 6 60 2 20 8 80b 1 10 8 80 0 0 8 80 0 0 8 80 1 10 9 90c 1 10 8 70 2 20 10 100 10 100 10 100d 0 0 8 80 0 0 8 80 1 10 9 90 0 3 9 90Total 2 5.0 27 65.0 3 7.5 30 72.5 3 7.5 33 82.5 3 7.5 36 90.0a 1 10 8 80 0 0 8 80 0 0 8 80 1 10 9 90b 0 0 5 50 1 10 6 60 2 20 8 80 1 10 9 90c 1 10 7 70 1 10 8 80 0 0 8 80 1 10 9 90d 1 10 7 70 0 0 7 70 0 0 7 70 0 0 7 70Total 3 7.5 27 67.5 2 5.0 29 72.5 2 5.0 31 77.5 3 7.5 34 85.0a 0 2 2 20 0 0 2 20 0 0 2 20 1 10 3 30b 2 20 4 40 2 20 6 60 1 10 7 70 1 10 8 80c 0 0 3 30 0 0 3 30 0 0 3 30 0 0 3 30d 2 20 4 40 2 20 6 60 1 10 7 70 0 0 7 70Total 4 10.0 13 32.5 4 10.0 17 42.5 2 5.0 19 47.5 2 5.0 21 52.5a 0 0 3 30 1 10 4 40 1 10 5 50 1 10 6 60b 0 0 5 50 1 10 6 60 0 0 6 60 0 0 6 60c 2 20 3 30 0 0 3 30 1 10 4 40 0 0 4 40d 0 0 3 30 1 10 4 40 0 0 4 40 2 20 6 60Total 2 5.0 14 35.0 3 7.5 17 42.5 2 5.0 19 47.5 3 7.5 22 55.0a 0 0 0 0 1 10 1 10 0 0 1 10 0 0 1 10b 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0c 1 10 1 10 0 0 1 10 0 0 1 10 0 0 1 10d 1 10 3 30 1 10 4 40 1 10 5 50 0 0 5 50Total 2 5.0 4 10.0 2 5.0 6 15.0 1 2.5 7 17.5 0 0.0 7 17.5*Cumulative mortality, **Percent cumulative mortalityCum.Dead*%Cum.Dead**Department of Medical Entomology 25

Efficacy Testing of Diatomaceous Earth for Bed BugsTable 7, cont. Mortality of adult Cimex lectularius exposed to various doses of Diatomaceous Earth dust at highhumidity, raw data, Days 8-12.Dose8g/m24g/m22g/m21g/m20g/m2ReplicateNo.Dead%MortalityDay 9 Day 10 Day 11 Day 12Cum.Dead*%Cum.Dead**No.Dead%MortalityCum.Dead*%Cum.Dead**No.Dead%MortalityCum.Dead*%Cum.Dead**No.Dead%Mortalitya 0 0 8 80 1 10 9 90 1 10 10 100 10 100b 1 10 10 100 10 100 10 100 10 100c 10 100 10 100 10 100 10 100d 1 10 10 100 10 100 10 100 10 100Total 2 5.0 38 95.0 1 2.5 39 97.5 1 10.0 40 100.0 40 100.0a 0 0 9 90 1 10 10 100 10 100 10 100b 0 0 9 90 1 10 10 100 10 100 10 100c 0 0 9 90 0 0 9 90 1 10 10 100 10 100d 1 10 8 80 1 10 9 90 1 10 10 100 10 100Total 1 2.5 35 87.5 3 7.5 38 95.0 2 5.0 40 100.0 40 100.0a 2 20 5 50 2 20 7 70 2 20 9 90 0 0 9 90b 0 0 8 80 0 0 8 80 0 0 8 80 0 0 8 80c 1 10 4 40 3 30 7 70 0 0 7 70 0 0 7 70d 0 0 7 70 1 10 8 80 2 20 10 100 10 100Total 3 7.5 24 60.0 6 15.0 30 75.0 4 10.0 34 85.0 0 0.0 34 85.0a 0 0 6 60 1 10 7 70 1 10 8 80 0 0 8 80b 0 0 6 60 1 10 7 70 0 0 7 70 0 0 7 70c 1 10 5 50 1 10 6 60 2 20 8 80 0 0 8 80d 0 0 6 60 0 0 6 60 0 0 6 60 1 10 7 70Total 1 2.5 23 57.5 3 7.5 26 65.0 3 7.5 29 72.5 1 2.5 30 75.0a 0 0 1 10 0 0 1 10 0 0 1 10 0 0 1 10b 0 0 0 0 1 10 1 10 1 10 2 20 0 0 2 20c 0 0 1 10 1 10 2 20 0 0 2 20 0 0 2 20d 0 0 5 50 0 0 5 50 0 0 5 50 0 0 5 50Total 0 0.0 7 17.5 2 5.0 9 22.5 1 2.5 10 25.0 0 0.0 10 25.0*Cumulative mortality, **Percent cumulative mortalityCum.Dead*%Cum.Dead**Department of Medical Entomology 26

Efficacy Testing of Diatomaceous Earth for Bed BugsTable 7, cont. Mortality of adult Cimex lectularius exposed to various doses of Diatomaceous Earth dustat high humidity,raw data, Days 13-16.Dose8g/m24g/m22g/m21g/m20g/m2ReplicateNo.Dead%MortalityDay 13 Day 14 Day 15 Day 16Cum.Dead*%Cum.Dead**No.Dead%MortalityCum.Dead*%Cum.Dead**No.Dead%MortalityCum.Dead*%Cum.Dead**No.Dead%Mortalitya 10 100 10 100 10 100 10 100b 10 100 10 100 10 100 10 100c 10 100 10 100 10 100 10 100d 10 100 10 100 10 100 10 100Total 40 100.0 40 100.0 40 100.0 40 100.0a 10 100 10 100 10 100 10 100b 10 100 10 100 10 100 10 100c 10 100 10 100 10 100 10 100d 10 100 10 100 10 100 10 100Total 40 100.0 40 100.0 40 100.0 40 100.0a 1 10 10 100 10 100 10 100 10 100b 0 0 8 80 8 80 0 0 8 80 8 80c 0 0 7 70 7 70 0 0 7 70 1 1 8 80d 10 100 10 100 10 100 10 100Total 1 2.5 35 87.5 35 87.5 0 0.0 35 87.5 1 2.5 36 90.0a 0 0 8 80 0 0 8 80 0 0 8 80 0 0 8 80b 1 10 8 80 1 10 9 90 1 10 10 100 10 100c 1 10 9 90 1 10 10 100 10 100 10 100d 0 0 7 70 0 0 7 70 0 0 7 70 1 1 8 80Total 2 5.0 32 80.0 2 5.0 34 85.0 1 2.5 35 87.5 1 2.5 36 90.0a 0 0 1 10 1 10 2 20 0 0 2 20 1 10 3 30b 0 0 2 20 1 10 3 30 0 0 3 30 1 10 4 40c 0 0 2 20 0 0 2 20 2 20 4 40 1 10 5 50d 0 0 5 50 0 0 5 50 0 0 5 50 1 10 6 60Total 0 0.0 10 25.0 2 5.0 12 30.0 2 5.0 14 35.0 4 10.0 18 45.0*Cumulative mortality, **Percent cumulative mortalityCum.Dead*%Cum.Dead**Department of Medical Entomology 27

Efficacy Testing of Diatomaceous Earth for Bed BugsTable 7, cont. Mortality of adult Cimex lectularius exposed to various doses of Diatomaceous Earth dust at highhumidity, raw data, Days 17-20.Dose8g/m24g/m22g/m21g/m20g/m2ReplicateNo.Dead%MortalityDay 17 Day 18 Day 19 Day 20Cum.Dead*%Cum.Dead**No.Dead%MortalityCum.Dead*%Cum.Dead**No.Dead%MortalityCum.Dead*%Cum.Dead**No.Dead%Mortalitya 10 100 10 100 10 100 10 100b 10 100 10 100 10 100 10 100c 10 100 10 100 10 100 10 100d 10 100 10 100 10 100 10 100Total 40 100.0 40 100.0 40 100.0 40 100.0a 10 100 10 100 10 100 10 100b 10 100 10 100 10 100 10 100c 10 100 10 100 10 100 10 100d 10 100 10 100 10 100 10 100Total 40 100.0 40 100.0 40 100.0 40 100.0a 10 100 10 100 10 100 10 100b 2 20 10 100 10 100 10 100 10 100c 0 0 8 80 1 10 9 90 0 0 9 90 1 10 10 100d 10 100 10 100 10 100 10 100Total 2 5.0 38 95.0 1 2.5 39 97.5 0 0.0 39 97.5 40 100.0a 1 10 9 90 1 10 10 100 10 100 10 100b 10 100 10 100 10 100 10 100c 10 100 10 100 10 100 10 100d 1 10 9 80 1 10 10 100 10 100 10 100Total 2 5.0 38 95.0 2 5.0 40 100.0 40 100.0 40 100.0a 1 10 4 40 4 40 4 40 4 40b 4 40 4 40 4 40 4 40c 5 50 5 50 1 10 6 60 1 10 7 70d 6 60 6 60 6 60 6 60Total 1 2.5 19 47.5 0 0.0 19 47.5 1 2.5 20 50.0 1 2.5 21 52.5*Cumulative mortality, **Percent cumulative mortalityCum.Dead*%Cum.Dead**Department of Medical Entomology 28

Efficacy Testing of Diatomaceous Earth for Bed BugsTable 8. Mortality of adult Cimex lectularius exposed to various doses of Diatomaceous Earth ‘fines’, raw data, Days 1-4(Experiment 4).Dose8g/m24g/m22g/m21g/m20g/m2ReplicateNo.TestedDustAmount(g)*No.DeadDay 1 Day 2 Day 3 Day 4%MortalityNo.Dead%MortalityCum.Dead*%Cum.Dead**No.Dead%MortalityCum.Dead*%Cum.Dead**No.Dead%Mortalitya 10 0.052 0 0 0 0 0 0 1 10 1 10 4 40 5 50b 10 0.052 2 20 3 30 5 50 1 10 6 60 2 20 8 80Total 20 2 10 3 15 5 25 2 10 7 35 6 30 13 65a 10 0.025 0 0 1 10 1 10 0 0 1 10 2 20 3 30b 10 0.025 0 0 0 0 0 0 3 30 3 30 1 10 4 40Total 20 0 0 1 5 1 5 3 15 4 20 3 15 7 35a 10 0.013 0 0 0 0 0 0 1 10 1 10 1 10 2 20b 10 0.013 0 0 0 0 0 0 1 10 1 10 1 10 2 20Total 20 0 0 0 0 0 0 2 10 2 10 2 10 4 20a 10 0.06 0 0 0 0 0 0 0 0 0 0 0 0 0 0b 10 0.06 0 0 0 0 0 0 0 0 0 0 0 0 0 0Total 20 0 0 0 0 0 0 0 0 0 0 0 0 0 0a 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0b 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0Total 20 0 0 0 0 0 0 0 0 0 0 0 0 0 0*Cumulative mortality, **Percent cumulative mortalityCum.Dead*%Cum.Dead**Department of Medical Entomology 29

Efficacy Testing of Diatomaceous Earth for Bed BugsTable 8, cont. Mortality of adult Cimex lectularius exposed to various doses of Diatomaceous Earth ‘fines’, raw data,Days 5-8.Dose8g/m24g/m22g/m21g/m20g/m2ReplicateNo.Dead%MortalityDay 5 Day 6 Day 7 Day 8Cum.Dead*%Cum.Dead**No.Dead%MortalityCum.Dead*%Cum.Dead**No.Dead%MortalityCum.Dead*%Cum.Dead**No.Dead%Mortalitya 0 0 5 50 1 1 6 60 0 0 6 60 1 10 7 70b 0 0 8 80 1 10 9 90 0 0 9 90 0 0 9 90Total 0 0 13 65 2 11 15 75 0 0 15 75 1 5 16 80a 1 10 4 40 2 20 6 60 2 20 8 80 1 10 9 90b 1 10 5 50 1 10 6 60 0 0 6 60 1 10 7 70Total 2 10 9 45 3 15 12 60 2 10 14 70 2 10 16 80a 0 0 2 20 0 0 2 20 0 0 2 20 4 40 6 60b 2 20 4 40 0 0 4 40 0 0 4 40 2 20 6 60Total 2 10 6 30 0 0 6 30 0 0 6 30 6 30 12 60a 1 10 1 10 0 0 1 10 2 20 3 20 0 0 3 30b 0 0 0 0 1 10 1 10 0 0 1 10 3 30 4 40Total 1 5 1 5 1 5 2 10 2 10 3 15 30 15 7 35a 0 0 0 0 0 0 0 0 2 20 2 20 0 0 2 20b 1 10 1 10 0 0 1 10 0 0 1 10 0 0 1 10Total 1 5 1 5 0 0 1 5 2 10 3 15 0 0 3 15*Cumulative mortality, **Percent cumulative mortalityCum.Dead*%Cum.Dead**Department of Medical Entomology 30

Efficacy Testing of Diatomaceous Earth for Bed BugsTable 8, cont. Mortality of adult Cimex lectularius exposed to various doses of Diatomaceous Earth ‘fines’, raw data,Days 9-12.Dose8g/m24g/m22g/m21g/m20g/m2ReplicateNo.Dead%MortalityDay 9 Day 10 Day 11 Day 12Cum.Dead*%Cum.Dead**No.Dead%MortalityCum.Dead*%Cum.Dead**No.Dead%MortalityCum.Dead*%Cum.Dead**No.Dead%Mortalitya 1 10 8 80 0 0 8 80 2 20 10 100 10 100b 0 0 9 90 1 1 10 100 10 100 10 100Total 1 5 17 85 1 5 18 90 2 10 20 100 20 100a 1 10 10 100 10 100 10 100 10 100b 0 0 7 70 0 0 7 70 3 30 10 100 10 100Total 1 5 17 85 0 0 17 85 20 100 20 100a 3 30 9 90 1 10 10 100 10 100 10 100b 0 0 6 60 1 10 7 70 2 20 9 90 1 10 10 100Total 3 15 15 75 2 10 17 85 2 10 19 95 20 100a 1 10 4 40 3 30 7 70 0 0 7 70 1 10 8 80b 1 10 5 50 2 20 7 70 1 10 8 80 1 10 9 90Total 2 10 9 45 5 25 14 70 10 5 15 75 2 10 17 85a 0 0 2 20 0 0 2 20 0 0 2 20 1 10 3 30b 1 10 2 20 0 0 2 20 1 10 3 30 1 10 4 40Total 1 5 4 20 0 0 2 20 1 5 5 25 2 10 7 35*Cumulative mortality, **Percent cumulative mortalityCum.Dead*%Cum.Dead**Department of Medical Entomology 31

Efficacy Testing of Diatomaceous Earth for Bed BugsTable 8, cont. Mortality of adult Cimex lectularius exposed to various doses of Diatomaceous Earth ‘fines’, raw data,Days 13-16.Dose8g/m24g/m22g/m21g/m20g/m2ReplicateNo.Dead%MortalityDay 13 Day 14 Day 15 Day 16Cum.Dead*%Cum.Dead**No.Dead%MortalityCum.Dead*%Cum.Dead**No.Dead%MortalityCum.Dead*%Cum.Dead**No.Dead%Mortalitya 1 100 10 100 10 100 10 100b 10 100 10 100 10 100 10 100Total 20 100 20 100 20 100 20 100a 1 100 10 100 10 100 10 100b 10 100 10 100 10 100 10 100Total 20 100 20 100 20 100 20 100a 1 100 10 100 10 100 10 100b 10 100 10 100 10 100 10 100Total 20 100 20 100 20 100 20 100a 0 0 8 80 1 10 9 90 0 0 9 90 9 90b 1 10 10 100 10 100 10 100 10 100Total 1 5 18 90 1 5 19 95 19 95 19 95a 0 10 3 30 1 10 4 40 1 10 5 50 0 0 5 50b 0 0 4 40 0 0 4 40 1 10 5 50 1 10 6 60Total 0 0 7 35 1 5 8 40 2 10 1 50 1 5 11 55*Cumulative mortality, **Percent cumulative mortalityCum.Dead*%Cum.Dead**Department of Medical Entomology 32

Efficacy Testing of Diatomaceous Earth for Bed BugsTable 8, cont. Mortality of adult Cimex lectularius exposed to various doses of Diatomaceous Earth ‘fines’, raw data, Day17.Dose8g/m24g/m22g/m21g/m20g/m2ReplicateNo.Dead%MortalityDay 17Cum.Dead*%Cum.Dead**a 10 100b 10 100Total 20 100a 10 100b 10 100Total 20 100a 10 100b 10 100Total 20 100a 1 10 10 100b 10 100Total 1 5 20 100a 1 10 6 60b 1 10 7 70Total 2 10 13 65*Cumulative mortality, **Percent cumulative mortalityDepartment of Medical Entomology 33

Efficacy Testing of Diatomaceous Earth for Bed BugsTable 9. P values of the statistical analysis for comparing the treatments with the controls for Experiment 1.Dose Day1Day2Day3Day4Day5Day6Day7Day8Day9Day10Day11Day12Day13Day14Day158g/m 2 * * * * * * * * * * * * * * *4g/m 2 0.12 * * * * * * * * * * * * * *2g/m 2 0.28 * * * * * * * * * * * * * *1g/m 2 0.36 0.54 * * * * * * * * * * * * **>0.05, i.e. statistically different to the controlsTable 10. P values of the statistical analysis for comparing the treatments with the controls for Experiment 2.Dose Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 98g/m 2 * * * * * * * * *4g/m 2 * * * * * * * * *2g/m 2 * * * * * * * * *1g/m 2 * * * * * * * * **>0.05, i.e. statistically different to the controlsDepartment of Medical Entomology 34

Efficacy Testing of Diatomaceous Earth for Bed BugsTable 11. P values of the statistical analysis for comparing the treatments with the controls for Experiment 3.Day Day Day Day Day Day Day Day Day Day Day Day Day Day Day Day Day Day Day DayDose 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 208g/m 2 * * * * * * * * * * * * * * * * * * * *4g/m 2 * * * * * * * * * * * * * * * * * * * *2g/m 2 0.36 0.21 * * * * * * * * * * * * * * * * * *1g/m 2 0.36 0.28 * * * * * * * * * * * * * * * * * **>0.05, i.e. statistically different to the controlsTable 12. P values of the statistical analysis for comparing the treatments with the controls for Experiment 4.Day1Day2Day3Day4Day5Day6Day7Day8Dose8g/m 2 0.42 0.42 0.30 * * * * * * * * * * * * * *4g/m 2 1 1 0.43 0.18 * * * * * * * * * * * * *2g/m 2 1 1 1 1 0.10 0.15 0.31 * * * * * * * * * *1g/m 2 1 1 1 1 1 0.42 0.70 0.11 * * * * * * * * **>0.05, i.e. statistically different to the controlsDay9Day10Day11Day12Day13Day14Day15Day16Day17Department of Medical Entomology 35

Efficacy Testing of Diatomaceous Earth for Bed BugsThe efficacy testing of Diatomaceous Earth Dust to theCommon Bed Bug, Cimex lectularius.Backgroundii. Mesocosm InvestigationsIn the initial laboratory trials, Diatomaceous Earth dust (DED) was found highlyefficacious against the Common bed bug, Cimex lectularius. Ideally the next step intesting the efficacy of a product is to undertake field trials. However, as DED is adust, it would not be applied to all locations where bed bugs may harbour, such ason mattresses. Dusts being obvious are unsightly and it is inappropriate to applysuch insecticide formulations to areas when they can be readily seen and/or quicklyremoved via vacuuming, which would render them ineffective. For bed bug control,dusts should be applied to non-obvious locations such as underneath carpets andstraight edges, behind skirtings, in wall voids, ensemble bases and power points(for more information on appropriate bed bug control consult the ‘Code of Practicefor the Control of Bed Bug Infestations in Australia”, www.bedbug.org.au, whichwas compiled by the author of this report). Thus other insecticide formulations,notably liquids, must always be employed in conjunction with dusts. This meansthat in the field situation it is not possible to assess the efficacy solely of dusts in ascientific manner when bed bugs are the target pest, as the other insecticide/s willhave a confounding effect.To circumvent this issue, mesocosms were constructed for the purpose of furthertesting the efficacy of DED. A mesocosm is essentially an artificial or a simulatedenvironment, and allows for the testing of a product on a larger scale to produceresults that should be indicative of what would occur with a field infestation. Withthe mesocosm, a single product can be tested without the confounding effects ofother insecticides. It was also important to access if DED can be applied withnormal pest control equipment for insecticidal dust applications such as bulb handpuffers.Thus the aims of the experiment were to;• To test the efficacy of Diatomaceous Earth dust in a simulated environment (a‘mesocosm’), and,• To demonstrate that the product can be applied via normal insecticide dustingequipment, namely a bulb hand puffer.Department of Medical Entomology 36

Efficacy Testing of Diatomaceous Earth for Bed BugsMethodsTest AnimalThe bed bugs used are the Common bed bug, Cimex lectularius, from a colonyestablished by the Department of Medical Entomology during 2004. The coloniesheld by the Department are the only bed bug colonies in Australia. The founderspecimens were sourced from various infestations within the Sydney metropolitanarea and have not been profiled for insecticide susceptibility, however are knownfrom in-house efficacy testing to have a degree of insecticide resistance to both thesynthetic pyrethroids and the carbamates. The colony is maintained within 1Lcontainers, provided with a blood meal once per week via anesthetised rats, andheld at 25 o C, with 80%RH. Active unengorged bed bug adults were removed andused for the tests. Sex and age of the bed bugs were not determined.InsecticideThe DED was supplied by Dr Ian Neering of Mount Sylvia Diatomite. No batchnumber or date of manufacture was provided. This was the same DED batch asused in the initial laboratory investigations.Mesocosm ConstructionMesocosms (Fig. 9) were constructed out of materials that are often typicallyencountered in dwellings; notably gyprock and pine, and were designed tosimulate a bed bug infestation in a cavity such as a wall void. The lid of themesocosm consisted of clear Perspex sheeting, which was fixed into position withremovable screws. Perspex was used so that the flow of the DED into themesocosm cavity could be observed, to facilitate the introduction of living bedbugs, to aid in experimental observation, and the removal of dead insects. Foursuch mesocosms were constructed; two tests (which had DED introduced) andtwo controls.Bulb Hand Duster CalibrationA Rega bulb hand insecticide dust puffer was employed to deliver the DED, whichwas purchased from Garrards (www.garrards.com.au). To calculate the deliveryrate of the insecticide with the bulb hand puffer, a calibration trial wasundertaken. This involved charging the bulb hand puffer with DED, weighing theunit, delivering ten puffs of DED, reweighing the unit and calculating the averageamount of dust per puff. This was then repeated a further nine times to achieveten replicates with 100 puffs in total, and the total average per puff was thencalculated. This amount was then used to determine the number of puffsrequired to treat the mesocosms at the target rate.Experimental ProcedureFifty adult unsexed Common bed bugs (Cimex lectularius) were placed into eachmesocosm (Fig. 10). The Perspex lid was then fixed into position and the bedDepartment of Medical Entomology 37

Efficacy Testing of Diatomaceous Earth for Bed Bugsbugs left for one hour before the introduction of the dust so that the bugs wouldsettle in a harbourage as they do in a typical infestation. In all the mesocosmsthe bugs aggregated in the corners and along the edges of the mesocosm (Fig.11).In each end of the test mesocosms, two holes were drilled to allow for theintroduction of the DED via a bulb hand puffer insecticidal dust applicator (Fig.12). From the results of the initial laboratory investigations, it was decided that atarget dose rate of the lower mid level, namely 2g/m 2 would be used. Theinternal dimensions of the mesocosms were: 50cm (l) x 41cm (w) x 2cm (h),giving a surface area for treatment of approximately 0.23m 2 (this included theside walls but not the Perspex lid). At a dose rate of 2g/m 2 , a total of 0.47g pertest mesocosm was required.Prior and post application, the bulb hand puffer was weighed in order to recordthe insecticidal delivery amount. To each end of the mesocosm, an equivalentnumber of puffs of DED were applied and the holes plugged so that bed bugscould not escape. Mortality was determined daily from 24 hours onwards bycounting and removing bed bugs that did not respond to gentile stimuli (slight prodwith a fresh wooden applicator stick). The experiments were conducted at 22±1 o C,in a darkened environment, relative humidity of 55±5%. For each insecticidal dose,the daily results were statistically compared with the control via one way ANOVA.Results & DiscussionBulb Hand Duster CalibrationTable 13 provides the calibration results of the bulb hand duster. The averagedelivery amount per puff was 0.0325g. It is known that bulb hand dusters arehighly variable in their delivery for various reasons, and this was demonstratedwith the large range in delivery amounts per replicate, with average puffsranging from 0.01g to 0.05g.Application Dose RateAs mentioned in the methods above, for the target dose rate of 2g/m 2 , a total of0.47g of DED per test mesocosm was required. This meant that 14.55 puffs intotal were needed and this was rounded up to 16 with 8 puffs of DED per eachend of mesocosm. This gave a theoretical application amount of 0.52g permesocosm, which equated to 2.2g/m 2 . The 16 puffs were applied to the firstmesocosm (Test 1) and the weight before application was 162.04g and 160.15gafter, with a difference of 1.89g. This amount was well above the target dose,however as bulb hand dusters are notoriously inaccurate in their dose rate, itwas felt justified to continue with the experiment as such variability would benormally experienced with pest controllers in the field. For the second mesocosmDepartment of Medical Entomology 38

Efficacy Testing of Diatomaceous Earth for Bed Bugshowever, the number of puffs was halved to a total of eight, or four each end.Pre application to Test 2, the weight of the bulb hand duster was 160.15g and159.51g after dosing, which give a difference of 0.64g and this was much closerto the target dose.Efficacy ResultsDespite the different application amounts, the results of the test mesocosmswere remarkably similar and hence the test results were combined. Table 14provides the summary data of the percent cumulative mortality over time of theadult Cimex lectularius when exposed to DED within the mesocosm. Table 15 hasthe raw data and Fig. 13 is a graphical representation of the summary data.By 24 hours, test mortality was 21% and this was significantly different to thecontrol mortality of 0%. By Day 9, all bed bugs in the test mesocosms weredead, with only 4% control mortality.In the initial laboratory trials, some clumping of the DED was observed and sothere were concerns that the dust may not flow properly via an insecticide dustapplicator. This was not an issue as the dust flowed smoothly with the bulb handduster and spread throughout the mesocosm, with the result that all bed bugswere dusted. Over the duration of the experiment, the DED was observed toclump, however as the mesocosm was regularly moved from a dark to light areafor observation purposes, it was probable that the clumping was the result ofmovement and this is unlikely to be an issue once the product has been applied inthe field situation. Despite the clumping, a complete kill was achieved.ConclusionDED was found to be highly efficacious against adults of the Common bed bug,Cimex lectularius, in a simulated environment and could be readily applied viatypical pest control equipment employed in insecticidal dust application.ReferencesDoggett S.L. (2007). A Code of Practice for the Control of Bed BugInfestations in Australia. 2 nd Edition. Department of Medical Entomology &The Australian Environmental Pest Managers Association, Westmead Hospital,Sydney. 67pp. ISBN 1740800974.Department of Medical Entomology 39

Efficacy Testing of Diatomaceous Earth for Bed BugsFigure 9. The ‘Mesocosm’ for testing the efficacy of Diatomaceous Earth dust when applied by a bulb hand duster.Department of Medical Entomology 40

Efficacy Testing of Diatomaceous Earth for Bed BugsFigure 10. Bed bugs being placed within the ‘Mesocosm’.Department of Medical Entomology 41

Efficacy Testing of Diatomaceous Earth for Bed BugsFigure 11. Bed bugs one hour after being placed in the ‘Mesocosm’.Department of Medical Entomology 42

Efficacy Testing of Diatomaceous Earth for Bed BugsFigure 12. Applying the Diatomaceous Earth dust to the ‘Mesocosm’.Department of Medical Entomology 43

Efficacy Testing of Diatomaceous Earth for Bed BugsTable 13. Calibration of hand bulb duster through measuring the weight of theduster after puffing ten times with Diatomaceous Earth dust.Weight (g)Replicate* Before After Difference Ave/Puff1 151.15 150.91 0.24 0.022 150.91 150.54 0.37 0.043 150.54 150.09 0.45 0.044 150.09 149.69 0.40 0.045 149.69 149.51 0.18 0.026 149.51 149.37 0.14 0.017 149.37 149.13 0.24 0.028 149.13 148.70 0.43 0.049 148.70 148.24 0.46 0.0510 148.24 147.90 0.34 0.03Total average: 0.0325*Each replicate represents ten puffs of the duster.Department of Medical Entomology 44

Efficacy Testing of Diatomaceous Earth for Bed BugsTable 14. Percent cumulative mortality of adult Cimex lectularius exposed to Diatomaceous Earth dust within themesocosm, summary data*.Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9Test 0 21.0 54.0 74.0 85.0 91.0 97.0 98.0 99.0 100.0Control 0 0.0 1.0 1.0 1.0 1.0 1.0 4.0 4.0 4.0*From and including Day 1, all test results were significantly different to the controls.Table 15. Mortality of adult Cimex lectularius exposed to Diatomaceous Earth dust within the mesocosm, raw data.Day 1 Day 2 Day 3 Day 4 Day 5%%%%Cum Cum No. Cum Cum No. Cum Cum No. Cum Cum No. CumMort* Mort** Dead Mort Mort Dead Mort Mort Dead Mort Mort Dead MortNo.No. DeadTest 1 49 11 11 22.4 12 23 46.9 7 30 61.2 8 38 77.6 4 42 85.7Test 2 51 10 10 19.6 21 31 60.8 13 44 86.3 3 47 92.2 2 49 96.1Control 1 50 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0Control 2 50 0 0 0 1 1 2.0 0 1 2.0 0 1 2.0 0 1 2.0*cumulative mortality, **percentage cumulative mortality.Day 6 Day 7 Day 8 Day 9%%%Cum Cum Cum Cum Cum Cum CumMort Mort Mort. Mort Mort Mort. Mort Mort Mort. Mort%CumMortNo. Mort.Test 1 49 4 46 93.9 1 47 95.9 1 48 98.0 1 49 100Test 2 51 2 51 100 51 100 51 100 51 100Control 1 50 0 0 0 2 2 4.0 0 2 4.0 0 2 4.0Control 2 50 0 1 2.0 1 2 4.0 0 2 4.0 0 2 4.0%CumMortDepartment of Medical Entomology 45

Efficacy Testing of Diatomaceous Earth for Bed Bugs10090TestControlFig 13. Cumulative Figure 13. Mortality Cumulative of Adult mortality Common of adult Bed Bugs, Cimex Cimex lectularius lectularius ,exposed to Diatomaceous Earth in in the the Mesocosm. mesocosm.80Percentage Cumulative Mortality706050403020100Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9Department of Medical Entomology 46