Effects of certain anthelmintics on the survival and reproduction of Euoniticellus intermedius (Reiche) (Coleoptera: Scarabaeidae)

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1 Effects of certin nthelmintics on the survivl nd reproduction of Euoniticellus intermedius (Reiche) (Coleopter: Scrbeide) By Crmen Tin Jcobs Submitted in prtil fulfilment of the requirements for the degree Mgister Scientie (Entomology) in the Fculty of Nturl nd Agriculturl Science Deprtment of Zoology nd Entomology University of Pretori South Afric April, 2014

2 Declrtion I, Crmen Tin Jcobs, declre tht this thesis/disserttion, which I hereby submit for the degree Mster of Science (Entomology) t the University of Pretori, is my own work nd hs not previously been submitted by me for degree t this or ny other tertiry institution. SIGNATURE: DATE: ii

3 This mnuscript is dedicted you my Mrm. Thnk you for lwys believing in me, never giving up on me nd giving me more support thn I know wht to do with. Thnk you for lwys being my number 1 fn! iii

4 Acknowledgements I like nonsense; it wkes up the brin cells. Fntsy is necessry ingredient in living; it's wy of looking t life through the wrong end of telescope. Which is wht I do, nd tht enbles you to lugh t life's relities Dr. Seuss. I would like to thnk the following people for being prt of this incredible journey: First nd foremost, my collbortor, my mentor nd my supervisor, Prof. Clrke Scholtz, for your guidnce, support, dvice nd ptience. Sometimes the questions re complicted nd the nswers re simple. Dr. Seuss. To the Deprtment of Zoology nd Entomology, nd especilly Prof. Chris Chimimb. So be sure when you step. Step with cre nd gret tct nd remember tht life s Gret Blncing Act Dr. Seuss. The Scrb Reserch Group: Dr. Ctherine Sole, Dr. Adrin Dvis, Angelik Switl, Christin Deschodt nd Werner Strumpher. Your input nd dvice ws invluble. Think left nd think right nd think low nd think high. Oh, the thinks you cn think up if only you try! Dr. Seuss. My lb rts: Isbelle Buyens, Louwtjie Snymn, Christin Deschodt nd Werner Strumpher. If things strt hppening, don t worry. Don t stew. Just go right long. You ll strt hppening too Dr. Seuss. iv

5 I m very grteful to my fmily: My mom nd dd Rocky nd Muritz, Amnd, Rymond nd Nichols, Junice nd Pul, Grnny June nd Oup Ponnie, for your unending support, love nd ptience. I've herd there re troubles of more thn one kind; some come from hed, nd some come from behind. But I've brought big bt. I'm ll redy, you see; now my troubles re going to hve troubles with me! Dr. Seuss. My extended fmily: Ptrick nd Bernice Mills, Mts nd Rit du Plessis, Angelique nd John Groenewld, Bridgitte, John, Siloh nd Sinise Frser. A little morl support goes long wy. Why fit in when you were born to stnd out? Dr. Seuss. An enormous thnk you to my fntstic friends: Michel Dvite, Jybee vn der Linde nd Kilyn Joubert, Andries Lbschgne nd Mdeleine Venter, Melize du Preez, Donvin Ersmus, Dlize, Json nd Ashton, Anj le Grnge, Angelik Switl, Rolnd Julius, Kendll Crous, Ursul Struss, Rquel Vieri nd Dieter Schulz, Din Fgir, Frnk Venter, Alid de Flmingh, Andrew nd Luren Dvies, Edith Mertz, Lezel Beetge, Otto Schutte, Brdley Reynolds, Sonj Ful nd Vriol vn Zyl. Tody you re You, tht is truer thn true. There is no one live who is You-er thn You Dr. Seuss. My two wonderful children: Ninj nd Nguni, for your unconditionl love nd the best therpy round. Tody ws good. Tody ws fun. Tomorrow is nother one Dr. Seuss. v

6 And lst, but not by ny mens lest, specil thnk you to Jen- Pierre du Plessis. Your love nd support is wht got me through it ll. All the blood, swet nd ters, the frustrtion nd the pin ws worth it! Thnk you helping me fill bgs of poo when no one else wnted to. Thnk you for giving up your free time fter hours, over weekends nd holidys. Thnk you for being my pillr nd my friend. I love you too much. You know you're in love when you cn't fll sleep becuse relity is finlly better thn your drems Dr. Seuss. vi

7 Effects of certin nthelmintics on the survivl nd reproduction of Euoniticellus intermedius (Reiche) (Coleopter: Scrbeide) Student: Supervisor: Deprtment: Degree: Crmen T. Jcobs Prof. Clrke H. Scholtz Deprtment of Zoology nd Entomology, University of Pretori, Pretori, 0002, South Afric Msters of Science (Entomology) Abstrct Avermectins nd milbemycins re commonly used in gro-ecosystems for the control of prsites in domestic livestock. As integrl members of gro-ecosystems with importnce in mintining psture helth through dung buril behviour, dung beetles re n excellent, non-trget, bio-indictor txon for exmining potentil detrimentl effects of pesticide ppliction. The current study uses the dung beetle species, Euoniticellus intermedius (Reiche), s bio-indictor to test the reltive toxicity of four different nthelmintics in dung residues. There hve been numerous lbortory nd field trils on these four nthelmintics but there hs never been lbortory tril compring ivermectin, eprinomectin, dormectin nd moxidectin under precisely the sme conditions. The effects of vermectin nd milbemycin toxicity re not confined to prsitic nemtodes nd rthropods, but lso extend to lrge vriety of beneficil invertebrte species, which use the dung s microhbitt nd breeding resource. Over time, pesticide usge indirectly ffects the rte of dung degrdtion through dverse effects on dung beetles. This potentil problem constitutes the min focus of this study. The present nd previous studies hve indicted no significnt effect on the survivl of dults but significnt reduction in reproductive rte nd reproductive success. Over time, reduced reproductive rte would result in decresed popultion sizes in the dung beetle community nd, ultimtely, decrese in the rte of dung degrdtion nd dung buril. It is, thus, vitlly importnt to crete wreness bout the importnce of dung beetles nd sound frming prctices for helthy gro-ecosystems. Keywords: nthelmintics; endectocides; ivermectin; moxidectin; eprinomectin; dormectin; dung beetles; Euoniticellus intermedius; cttle vii

8 Tble of Contents Declrtion... ii Acknowledgements... iv Abstrct... vii List of tbles nd figures... x Chpter 1: Toxicity testing of nthelmintics using the dung beetle species Euoniticellus intermedius (Reiche) (Coleopter: Scrbeide) Avermectins nd Milbemycins... 1 Introduction... 1 Previous Studies Ivermectin Lethl nd sub-lethl effect studies... 6 b. Survivl nd reproduction studies... 7 c. Dung decomposition studies... 9 d. Community structure studies e. Dung ttrctiveness studies Eprinomectin Dormectin Moxidectin Comprtive studies: comprisons of two products Comprtive studies: comprisons of ll four products Methodologies of dministrtion Pour-on b. Injection c. Comprisons Resistnce Introduction Records of resistnce The wy forwrd References Figures nd Tbles viii

9 Chpter 2: Testing for reltive toxicity of four nthelmintics (ivermectin, eprinomectin, dormectin nd moxidectin) using the dung beetle species, Euoniticellus intermedius (Reiche) (Coleopter: Scrbeide), s bio-indictor Introduction Mterils nd Methods Bio-indictor species Anthelmintic tretment nd bio-indictor protocol Results Overll dult mortlity Overll reproductive output Week 1 versus week 2 reproductive output Control dung results only Pesticide dung results only Discussion Generl trends Specific trends Conclusions References Figures nd Tbles Chpter 3: Survivl nd reproduction of Euoniticellus intermedius (Reiche) (Coleopter: Scrbeide) in dung following tretment of cttle with n unregistered ectoprsiticide.. 67 Introduction Mterils nd Methods Results Adult survivl F1 brood bll production nd F2 emergence F2 brood bll production nd F3 emergence Residue concentrtion in dung Overll sttisticl nlysis Discussion Conclusion References Figures nd Tbles ix

10 List of tbles nd figures Chpter 1 Tble 1. Totl cttle numbers Tble 2. Anthelmintics: resistnce free yers Chpter 2 Figure 1. Men number ( SD) of F1 dult Euoniticellus intermedius surviving 15 dys in untreted control dung or dung contining different types of pesticide residues. Figures 2A, 2B. Overll numbers of F1 brood blls ( SE) nd F2 E.intermedius emergences ( SE) from untreted control dung nd dung contining four different pesticide residues. Tble 1. Percentge overll mortlity of F2 immtures. Tble 2. Percentge F2 immture mortlity over dys. Figures 3A, 3B. Dt for F1 brood blls ( SE) nd F2 E. intermedius emergences ( SE) from untreted control dung. Figures 4A, 4B. Combined dt for F1 brood blls ( SE) nd F2 E. intermedius emergences ( SE) from dung treted with Dormectin, Eprinomectin, Ivermectin or Moxidectin. Appendix 1. Figures 5A 8D. Summries of the vriblity shown by F1 brood bll production nd F2 E. intermedius emergences over week 1 nd week 2 for from untreted control dung or dung contining Moxidectin, Ivermectin, Eprinomectin or Dormectin residues. x

11 Chpter 3 Figures 1A-5A. Comprisons between results for Euoniticellus intermedius (F1 genertion) from dung of untreted cttle (C1 = control) nd those from dung of cttle treted with product x using four different routes of dministrtion (T1-T4). Figures 1B-5B. Comprisons between results for Euoniticellus intermedius from dung 1, 7, 14, 21 nd 28 dys fter tretment with product x. Figures 1A, 1B. Men number of F1 dult surviving 15 dys. Figures 2A, 2B. Men number of F1 brood blls produced during week one. Figures 3A, 3B. Men number of F1 brood blls produced during week two. Figures 4A, 4B. Men number of F2 dults emerging from week one brood blls. Figures 5A, 5B. Men number of F2 dults emerging from week two brood blls. Figures 6A-7A. Comprisons between results for Euoniticellus intermedius (F2 genertion) from dung of untreted cttle (C1 = control) nd those from dung of cttle treted with product x in four different routes of dministrtion (T1-T4). Figures 6B-7B. Comprisons between results for Euoniticellus intermedius from dung voided 1, 7, 14, 21 nd 28 dys fter tretment with product x. Figures 6A, 6B. Men number of F2 brood blls produced during weeks one nd two. Figures 7A, 7B. Men number of F3 dults emerging from brood blls produced in weeks one nd two. Figure 8. Concentrtion of product x residues remining in dung voided 1-28 dys fter tretment in four different routes of dministrtion (T1-T4). Residue concentrtions mesured in ng/g nd log 10 trnsformed. Figures 9A 15D. Ptterns of dung beetle response to product x residues in dung voided from 1-28 dys fter tretment in four different routes of dministrtion (T1-T4). Both xi

12 numbers for beetles nd residue concentrtions (ng/g) log10 trnsformed. GLM one wy ANOVA results for dung beetles only. Tble 1. F-vlues obtined for comprisons between results for Euoniticellus intermedius (F1-F3 genertions) from dung of untreted cttle (control) nd those from dung of cttle treted with product x (T1-T4), which ws voided on five different occsions over time fter tretment (1, 7, 14, 21 nd 28 dys). Tble 2. F-vlues obtined for comprisons between results for Euoniticellus intermedius (F1-F3 genertions) from dung of cttle treted with product x (T1-T4), which ws voided on five different occsions over time fter tretment (1, 7, 14, 21 nd 28 dys). Appendix 1. Averge numbers from control versus tretment dung (ech of the F1 brood, F2 nd F3 vlues represents men dt from five replictes). Appendix 2. Regression pictures showing similrities between tretments nd differences between tretments nd controls. xii

13 Chpter 1: Toxicity testing of nthelmintics using the dung beetle species Euoniticellus intermedius (Reiche) (Coleopter: Scrbeide). Crmen T. Jcobs * * Scrb Reserch Group, Deprtment of Zoology nd Entomology, University of Pretori Avermectins nd Milbemycins Introduction The importnce plced on nthelmintics to bring prsite popultions under control hs become chllenging rms rce to produce product tht exhibits the perfect blnce between host nd non-trget orgnism toxicity, nd pest resistnce. The need for better product is becoming incresingly importnt becuse indictions re tht s fst s they re being produced, the pests re becoming resistnt. Pest resistnce is rgubly one of the top chllenges s fr s protecting livestock goes nd probbly the min driving force behind prsite control reserch in the livestock industries (Sngster 1999; Wolstenholme et l. 2004) since resistnce hs been reported in mny countries, in vriety of nemtodes nd ginst ll currently vilble nthelmintics (Sutherlnd & Lethwick 2011). Anthelmintics re brod-spectrum drugs tht control helminth pests by removing or killing them; they re grouped ccording to their common chemistry nd mode of ction (Sngster & Dobson 2002; Vercruysse & Rew 2002). Currently, the best nthelmintics on the mrket re the vermectins (ivermectin, eprinomectin nd dormectin) nd the milbemycin (moxidectin), which re collectively known s mcrocyclic lctones. The vermectins nd milbemycins re nturlly produced by strin of soil-dwelling ctinomycete, Streptomyces (Burg et l. 1979; Shoop & Soll 2002). All the vermectins hve unique phrmcophore which consists of 16-membered mcrocyclic lctone 1

14 bckbone (Shoop & Soll 2002) with discchride chin t C.13 (Steel 1993; Vercruysse & Rew 2002). Although the vermectins re glycosidic derivtive of the pentcyclic 16- membered lctone (Chbl et l. 1980; Albers-Schoenberg et l. 1981), they do not possess the ntifungl nd ntibcteril properties ssocited with the mcrolide ntibiotics (Burg et l. 1979; Chbl et l. 1980; Albers-Schoenberg et l. 1981). They ct by interfering with invertebrte neurotrnsmission rther thn inhibiting protein synthesis (Chbl et l. 1980; Albers-Schoenberg et l. 1981). As -minobutyric cid (GABA) gonists ( chemicl substnce cpble of ctivting receptor to induce full or prtil phrmcologicl response), the vermectins ct by eliminting the GABA-medited inhibitory postsynptic potentils nd the excittory postsynptic potentils t the neuromusculr junction in the trget orgnism (Fritz et l. 1979; Cmpbell 1985) thereby inhibiting nervous signl trnsmissions (Putter et l. 1981). Insted of competing with GABA by binding to its receptor, the vermectins stimulte the relese of GABA from nerve-endings which in turn enhnces the binding of GABA to its receptor which is situted on the post-synptic membrne of the excittory motorneuron in nemtodes nd on the post-junction membrne of the muscle cell in rthropods (Cmpbell 1985). This enhncement results in n incresed flow of chloride ions into the cell, which in turn, results in hyperpolristion nd elimintion of signl trnsmission (Cmpbell 1985). Different chloride chnnel subunits in nemtodes show vrible sensitivity to the mcrocyclic lctones nd different sites of expression, which is possibly wht ccounts for the prlytic effects on different neuromusculr systems t different concentrtions ( ). So, in nemtodes, the vermectins block trnsmission signls from the ventrl interneurons to the excittory motorneurons in the ventrl nerve chord (Fritz et l. 1979; Putter et l. 1981; Cmpbell 1985) which prlyses them without cusing hypercontrction or flccid prlysis (Kss et l. 1980). The prlysis occurs in the phrynx, body wll, nd uterine muscles nd even though prlysis of phryngel muscle is more sensitive, prlysis (flccid) of the body wll muscle my be criticl to the host for rpid removl of the prsite ( ). As the concentrtion of the mcrocyclic lctone decreses, it is possible tht motility my be regined; however, prlysis of the phrynx nd s result, inhibition of feeding, my lst longer thn body muscle prlysis nd is wht ultimtely contributes to the prsites deth ( ). 2

15 In certin species of filril nemtodes, the femles living in the tissue move very little s nutrients re bsorbed through the cuticle. A mjor effect of mcrocyclic lctones on these species is most likely prlysis of uterine muscles, resulting in disruption of reproduction s opposed to deth ( ). However, in orgnisms which lck GABA system, like cestodes nd tremtodes, the vermectins hve no effect on their cholinergic nervous systems which renders them non-toxic (Putter et l. 1981). Ivermectin ws the first vermectin to be introduced in 1981 (Steel 1993; Vercruysse & Rew 2002). Ivermectin (22, 23-dihydrovermectin) is discchride derivtive of the pentcyclic 16-membered lctone (Burg et l. 1979; Chbl et l. 1980; Cmpbell 1985; Römbke et l. 2010). The ntiprsitic effect of ivermectin is extremely potent ginst rthropods, nemtodes nd crines but hs no effect on cestodes nd tremtodes due to them lcking GABA system (Putter et l. 1981; Cmpbell 1985). Although potent, ivermectin is not eqully ctive ginst ll species nd is often very stge specific (Cmpbell 1985) which mens tht genus which is known to be susceptible to ivermectin, my not be susceptible t ll life stges (Cmpbell & Benz 1984). Abmectin, combintion of 80% vermectin B 1 nd 20% vermectin B 1b, is the strting mteril for ivermectin (Shoop et l. 1995). It is effective ginst nemtodes s well s crines nd, to dte remins the only vermectin or milbemycin to be used in both the niml helth nd crop industries (Shoop et l. 1995). Eprinomectin ws introduced to the niml helth industry in 1997 s n lterntive to ivermectin since it ws considered to be the only topicl endectocide sfe for use in lctting diry nimls (Shoop et l. 1996b; Vercruysse & Rew 2002). Though ivermectin hs no side effects on the host nd it hs such brod spectrum of ctivity, it cnnot be used in lctting diry nimls due to the levels of residue tht remin in the milk which my result in the diry products being discrded (Shoop et l. 1996, 1996b; Vercruysse & Rew 2002). 3

16 Dormectin ws commercilised in 1993 (Vercruysse et l. 1993) nd is the friendliest vermectin s fr s discomfort during dministrtion goes. In study done by Grndin et l. (1998), 61 red Angus-cross, two yer-old heifers were injected with ivermectin, dormectin or sline solution nd their rections were recorded. The nimls willingness to be re-injected ws lso recorded. They found tht dormectin cused significntly less discomfort during dministrtion thn ivermectin, nd concluded tht the use of product which cuses the lest discomfort during dministrtion improves the ese of hndling in the future nd reduces stress in the niml (Grndin et l. 1998). The milbemycins, lthough structurlly similr to the vermectins, differ in substituents t few of the side chins t the C-13 position nd cn bsiclly be thought of s deglycosylted vermectins (Steel 1993; Sngster & Dobson 2002; Vercruysse & Rew 2002). The milbemycins, lthough discovered in 1973, long before the discovery of ivermectin, were originlly developed for use in crop protection nd only used in veterinry prctices from bout 1986 (Tkiguchi et l. 1980; McKellr & Benchoui 1996). Moxidectin, the only milbemycin vilble on the mrket s n endectocide, ws introduced in 1989 nd commercilised worldwide by the erly 1990 s (Steel 1993; McKellr & Benchoui 1996). The milbemycins re highly lipophilic (moxidectin is bout 100 times more lipophilic thn the vermectins), soluble in orgnic solvents nd insoluble in wter, nd, fter n initil increse in its plsm concentrtion, it is redistributed throughout the body ft reserves, which ct s reservoir from which it is slowly relesed (McKellr & Benchoui 1996). Vrious studies (Cmpbell 1985; Wrdhugh & Rodriguez-Menendez 1988) show tht n unusul chrcteristic of the vermectins is, regrdless of the niml or method of dministrtion, most of the dose is excreted lrgely unltered in the dung where it retins its insecticidl ctivity (Cmpbell & Cmpbell 1989; Steel 1993; Strong 1993) nd this is where the focus of this study lies. The problem is tht the vermectins nd milbemycins mode of ction is not confined to prsitic nemtodes nd rthropods, but lso to very lrge vriety of beneficil invertebrte species which use the dung s microhbitt nd breeding ground nd indirectly ffect the rte of dung degrdtion over time (Strong 1993). 4

17 Previous Studies There hve been numerous lbortory nd field studies done on the effects of vermectins nd milbemycin in cttle dung on non-trget orgnisms. I hve selected few studies from round the globe to illustrte the vrious studies being done on different spects of dung beetle biology nd the effects tht the vermectins nd milbemycin hve on them. I hve specificlly chosen the countries where some of the highest concentrtions of cttle (Tble 1) re found ( ). Texs (Fincher 1992; Fincher & Wng 1992); Argentin (Suárez et l. 2003, 2009; Iglesis et l. 2011); Mexico (Cruz Rosles et l. 2012); Austrli (Ridsdill-Smith 1988; Doherty et l. 1994; Ddour 2000; Wrdhugh et l. 2001); Frnce (Lumret et l. 2005; Errouissi & Lumret 2010); South Afric (Krüger & Scholtz 1997, 1998, 1998b; Kryger et l. 2005); Cnd (Flote et l. 2002; Flote 2006, 2007); the United Kingdom (Wll & Strong 1987; Strong 1993; Strong & Wll 1994; Strong et l. 1996; Webb et l. 2010); Spin (Wrdhugh & Rodriguez-Menendez 1988; Lumret et l. 1993; Römbke et l. 2010); Jpn (Iws et l. 2008) nd Denmrk (Mdsen et l. 1990; Sommer et l. 1992). Although the methods re different in ech country nd hve chnged somewht over the yers, the results hve remined more or less consistent. 1. Ivermectin Ivermectin is the most extensively studied of ll the vermectins nd the first study tht set the scene for interest in the field ws Wll nd Strong (1987). They conducted n experiment in the UK to investigte the environmentl consequences of treting cttle with ivermectin. Four Friesin clves were given ivermectin in bolus form t concentrtion of 40µg/kg/dy nd four clves were given plcebo s the control group. The recommended dose t the time ws one, or repeted subcutneous injections of 200µg/kg ivermectin. A bolus ws chosen s it remins ctive for mny months nd releses ivermectin directly into the gut. Eleven dys fter tretment, fresh dung ws collected for further six dys. Artificil pts were mde from ivermectin-treted s well s control dung nd evenly spced t 1m intervls in n lternting sequence in 5m x 10m enclosure in psture. The pts nd soil smples were then collected fter 20, 30, 40, 50, 60, 80 nd 100 dys nd serched for invertebrtes. In contrst to the control pts, the experimentl pts contined few to no 5

18 Coleopter or Dipter. The results lso indicted tht there ws no visible dung degrdtion in the ivermectin-treted dung when compred to the controls showing tht even t 100 dys the experimentl pts were lrgely intct compred to the control pts which hd mostly disppered. This field tril showed tht treting with rumenl bolus, which delivers 40µg/kg ivermectin per dy, ws enough to disrupt the entire dung-inhbiting insect community (Wll & Strong 1987). Vrious subsequent studies hve simulted or repeted this experiment with vrible results.. Lethl nd sub-lethl effect studies Lumret et l. (1993) studied the effects of ivermectin residues on dung beetles by running field tril on frm in Spin in spring. Dung ws collected from group of six steers treted with single dose of ivermectin t the recommended dose rte of 200µg/kg body weight ivermectin 2, 4, 7, 10, 17, 24 nd 31 dys fter tretment s well s from control group of untreted steers. Forty dung pts of 1kg ech were deposited for ech series t 1m intervls in line cross 100 x 20m plot. At the sme time, four pitfll trps contining 50% ethylene glycol per series were set out nd bited with dung from treted steers followed by dung from untreted steers. Two pts nd soil core from ech series were collected 1, 3, 6, 9, 16 nd 23 dys fter the strt of the experiment nd trnsferred to the lb where the number nd species of beetles were recorded. Dung toxicity ws ssessed by recording the mortlity of the dung beetles feeding on the dung nd fter 29 dys, the numbers of lrve nd pupe were recorded. No dult mortlity ws recorded for the durtion of the study; however, 100% lrvl nd pupl mortlity ws observed in dung collected on the dy of tretment. No differences in offspring numbers between treted nd untreted dung were observed from dy six onwrds. Interestingly, dely in development ws observed for beetles bred in treted dung. This ws most pprent when it ws found tht most of the offspring in treted dung showed delyed development when compred to the control offspring. Another curious observtion cme from the pitfll trp dt, specificlly for those trps bited with dung collected 10 nd 17 dys fter tretment where in both cses, ttrction to treted nd untreted dung ws similr for the first three dys, nd then pek of ttrction occurred between dys 4-6, when the dung ws most ttrctive nd still reltively fresh. The interesting prt is, from dy six onwrds, the ttrction to the treted dung persisted for 30 dys while the untreted dung becme unttrctive fter dy 6

19 seven. Lumret et l. (1993) proposed tht incresed ttrctiveness is result of biochemicl modifictions in the dung composition, most likely due to protein degrdtion relesed by ivermectin therpy (Lumret et l. 1993). Krüger nd Scholtz (1997) rn lbortory tril to determine the lethl nd sub-lethl effects of ivermectin residues in dung from nimls treted with single stndrd injection of ivermectin t 200µg/kg. A group of three steers ws treted with ivermectin while nother three were left untreted s controls. Dung ws collected from the steers on 1, 2, 3, 4, 7, 14, 21 nd 28 dys fter tretment. Lbortory-rered Eoniticellus intermedius were provided with 250ml of dung twice week for two weeks nd monitored for lrvl mortlity s well s for brood bll numbers. Brood blls were counted, removed nd incubted to monitor for emergence. No results regrding lrvl survivl were reported. There ws no significnt difference between treted nd control popultions in the number of brood blls formed; however, on verge, the number of dults emerging from treted brood blls ws significntly lower thn in the controls (similr findings in Fincher 1992). Ivermectin cused 100% mortlity in offspring from 2-7 dys fter tretment nd significntly fewer emergences from dy 14 fter tretment when compred to the controls. Prolonged development in treted broods (similr findings Lumret et l. 1993) ws lso recorded, roughly 2.5 times longer for dung collected 1, 7 nd 14 dys fter tretment nd lrvl developmentl time of 5 weeks compred to the control of 3.5 weeks for dung collected 28 dys fter tretment (Krüger & Scholtz 1997). b. Survivl nd reproduction studies Ridsdill-Smith (1988) studied the effect of ivermectin on the survivl nd reproduction of the dung beetle Onthophgus binodis in Austrli. Hlf herd of 600 cttle ws given 200µg/kg dose of ivermectin; the other hlf ws treted with different nthelmintic. Dung ws collected 1, 2, 4, 8 nd 11 weeks fter tretment. Five pirs of dung beetles, collected from the field, were plced on ech of five replicte one-litre pts of treted dung on dmp snd in plstic boxes. After eight dys, the snd ws sieved nd the surviving beetles were gin plced on one-litre pts of treted dung on dmp snd in plstic boxes. After further eight dys the snd ws sieved gin nd the brood blls nd surviving beetles were recovered nd counted. Survivl of the immtures ws determined from 7

20 rndom smple of 100 from ll replictes of ech dung type. Ridsdill-Smith found tht ivermectin hd no influence on dult dung beetle survivl. Immture survivl, however, ws zero for week one fter tretment but stedily rose to equl tht of the other nthelmintic by week eight fter tretment. There ws no control group to compre tretments to (Ridsdill- Smith 1988). Fincher (1992) compred the effect of 20µg/kg nd 200µg/kg ivermectin on some dung-inhbiting insects, including the introduced Africn dung beetle Euoniticellus intermedius in Texs, USA. Dung ws collected from six steers; two treted with 20µg/kg ivermectin, two treted with 200µg/kg ivermectin nd two untreted, the dy before tretment s well s weekly fter tretment for 10 weeks. Two pirs of beetles were plced in continers with soil nd dung from ech tretment group. After one week the surviving dults nd number of brood blls were recorded. The brood blls were trnsferred to seprte continer nd monitored for emergence. The results reveled tht neither dosge hd ny significnt effect on dult survivl, the sme results s Ridsdill-Smith (1988) nd Wrdhugh nd Rodriguez-Menendez (1988), or brood bll production, when compred to the controls; however, emergence of dult E. intermedius from brood blls mde with dung from cttle tht received 200µg/kg ivermectin ws reduced, but for no more thn two weeks fter tretment (Fincher 1992). Cruz Rosles et l. (2012) evluted the effect of ivermectin on the survivl nd fecundity of Euoniticellus intermedius dults s well s on the survivl nd development of E. intermedius from egg to dult in Mexico. Ivermectin ws dded to cttle dung (spiked) t three different concentrtions (0.01ppm (10µg/kg), 1.0ppm (1 000µg/kg) nd 100ppm ( µg/kg)) nd given to lbortory-rised beetles. The dung ws replced every three dys nd weight nd number of brood msses, fecundity of femles, mortlity of offspring nd developmentl time (egg dult) were mesured. They found tht t low concentrtions (10µg/kg), the ivermectin hd no effect on the survivl or fertility of the dults or on the survivl of the lrve, but did record n increse in the lrvl development time. At the medium concentrtion (1 000µg/kg), which is five times the recommended dose, the survivl of dults ws reduced to lmost hlf nd no lrve emerged. At the highest concentrtion ( µg/kg) 100% mortlity ws observed nd no oviposition ws performed. They concluded tht the prolonging of development time my cuse phse lg in the field 8

21 ctivity cycle which my reduce the number of E. intermedius individuls nd the efficiency of the environmentl services tht they provide nd tht more nlyses with higher concentrtions between 0.01 nd 0.1ppm of ivermectin re needed to estblish lethl concentrtions for lrve nd dults of E. intermedius (Cruz Rosles et l. 2012). c. Dung decomposition studies Wrdhugh nd Rodriguez-Menendez (1988) studied the effect of ivermectin on the development nd survivl of the dung beetles Copris hispnus, Bubs bublus nd Onitis belil in southern Spin. Eight clves were injected with ivermectin t 200µg/kg. Dung from the treted clves ws collected the dy before the tretment (control), then 1, 2, 3, 4, 8, 16 nd 32 dys fter tretment. Ten pirs of beetles were housed in one-litre continers nd supplied with control dung, nd 10 pirs were supplied with dung collected on dy three. Ech pir ws exmined on weekly bsis for dult mortlity s well s oviposition ctivity, for 60 dys. Prentl nd immture mortlity were then estimted by exmining the nests. Similr procedures were followed in second tril to compre the effects of dung collected 0, 8 nd 16 dys fter tretment. The results showed no dult mortlity, reduced egg-lying nd reduced juvenile survivl s did Ridsdill-Smith (1988). Interestingly, mrked reduction in dult feeding ctivity ws observed in tretments suffering the highest mortlities, nmely dy 1-8 dung, nd the inference ws mde tht mortlity ws due to the ccumulting toxic effects which suppressed feeding (Wrdhugh & Rodriguez-Menendez 1988). Mdsen et l. (1990) conducted field s well s lbortory experiments in Denmrk to show how treting cttle with ivermectin ffects the fun nd decomposition of dung pts. Dung ws collected from three heifers on 1, 10, 20 nd 30 dys fter tretment with the recommended dose of 200µg/kg ivermectin s well s from three untreted heifers. Sixty 1kg pts of dung from treted cttle were plced t 2m intervls in n lternting sequence cross field nd insect ctivity ws observed for 5-15 min fter deposition. Smples were tken within 2-3 months nd the insects in the dung pts were identified nd counted in the lb. The disppernce rte of the dung pt ws estimted s percentge on five collection occsions 3, 14, 35, 53, nd 86 dys fter being deposited. For the lbortory tril, dung ws collected from the sme ivermectin-treted heifers t the sme time intervls nd exposed t the sme field site for 7, 45 nd 62 dys fter which they were collected nd biossyed with 9

22 Musc utumnlis. In nother tril, fresh dung ws collected from the ivermectin treted heifers t different intervls (1-33 dys) fter tretment nd tested ginst M. domestic s well s M. utumnlis. The results from the field tril showed tht ivermectin hd n effect on beetle lrve 1-10 dys fter tretment but tht the number of lrve ws not ffected by ivermectin pplied dys before collection. The decomposition rte ws significntly delyed when compred to control dung but lso depended on vribles such s climte, seson, soil type, funl inhbitnts nd microclimte. The results from the lbortory biossys showed % mortlity rte in M. domestic s well s M. utumnlis for dung collected one dy fter tretment. There ws no cler reduction in excreted ivermectin plced in the field for 7-62 dys nd the 62-dy ssy ws obscured by nturl mortlity. Most of the vrince found in this experiment ws ttributed to sesonl conditions (Mdsen et l. 1990). Sommer et l. (1992) rn field tril in Denmrk to ssess the impct of ivermectin residues on dung fun nd the resulting effect on dung degrdtion. Twenty-four heifers were divided into three groups of eight. One group ws treted with 0.2 mg/kg subcutneous ivermectin, nother group with 0.5 mg/kg pour-on formultion of ivermectin nd the third group ws left untreted, s the control. Dung ws collected from the heifers on 1, 2, 5, 13, 14, 28, 29, 42 nd 43 dys fter tretment. Twelve 1kg pts of dung from ech tretment were plced in n ungrzed field nd three replictes were smpled fter 3, 17, 31 nd 45 dys of field exposure to check for rthropods. According to the rthropods found in the treted dung, Sommer et l. (1992) found no significnt difference between the residues found in the pour-on nd injectble formultions even though the injectble formultion ws 2.5 times the dose of the pour-on formultion; however, dung collected from cttle 1-2 dys fter tretment with the injectble formultion led to delyed dung degrdtion for up to 45 dys but no effect ws observed from dung collected dys fter tretment. Dung collected from cttle 1-2 dys fter tretment with the pour-on formultion led to delyed dung degrdtion for up to dys fter tretment which ws similr result to tht of Wrdhugh nd Rodriguez-Menendez (1988) nd Mdsen et l. (1990). Iglesis et l. (2011) evluted the locl effects of ivermectin on dung fun nd degrdtion under different meteorologicl nd biologicl conditions in the sme re in Argentin in The experiment ws crried out s tril on four nturlly prsitized 10

23 clves. Two were treted with 0.2 mg/kg ivermectin nd the other two were left untreted s the control. Dung ws collected 3, 7, 14, 21 nd 28 dys fter tretment nd the ivermectin concentrtion ws determined chemiclly by high-pressure liquid chromtogrphy (HPLC), the orgnic mtter percentges were nlysed nd the orgnisms in the pts were collected nd counted. Meteorologicl dt were simultneously recorded t weekly intervls for the durtion of the tril. Ivermectin concentrtions were highest in smples tken three dys fter tretment nd lowest in smples tken dys fter tretment. The results showed fewer rthropods, however not sttisticlly significnt, were found in the dung of the clves treted with ivermectin (Iglesis et l. 2011). d. Community structure studies Krüger nd Scholtz (1998, b) conducted lrge-scle field study to determine the ecotoxicologicl effect of ivermectin on the dung beetle community structure under drought (Krüger & Scholtz 1998) nd high rinfll (Krüger & Scholtz 1998b) conditions. Both studies were crried out in the sme summer-rinfll re in South Afric. A breeding herd of 80 cows ws split into two nd hlf the herd ws treted with single recommended dose of 200µg/kg ivermectin while the other hlf remined untreted. Insects were collected directly from dung pts, s opposed to pit-fll trps, to simulte more nturl sitution, s well s from rtificil 1kg dung pts tht were stndrdized to eliminted s mny vribles s possible. Collection occurred t monthly intervls for three months. The results showed lrge effect on the dung beetle community in the form of significntly lower species richness nd evenness s well s incresed species dominnce in treted dung during drought (Krüger & Scholtz 1998). During high rinfll, however, fewer beetle nd fly lrve were found in the pts fter seven dys, but no effect of ivermectin ws detected fter yer (Krüger & Scholtz 1998b). This suggests tht these ecotoxicologicl effects re likely to be more severe in times of drought thn under more fvourble conditions. Kryger et l. (2005) crried out long-term, lrge-scle field study in South Afric to ssess the effect of ivermectin on the structure of dung beetle communities. One herd of 25 heifers ws treted with ivermectin t 200µg/kg nd the other herd of 25 heifers ws left untreted. Dung beetle communities were monitored over the entire summer by pitfll trp 11

24 smpling. No observble effects of ivermectin on the dung beetle communities ws found s the disprities between treted nd untreted dung were insignificnt nd most probbly due to differences in microclimte. Species richness nd diversity were lso unffected nd ecologiclly similr to the control communities. This study showed tht tretment with ivermectin under extensive frming conditions in the South Africn Highveld cn be considered sfe with regrd to the dung beetle communities under high rinfll (Kryger et l. 2005). Strong et l. (1996) crried out comprtive field tril to exmine the effects of ivermectin nd fenbendzole on dung colonizing Dipter nd Coleopter in the UK. Twelve clves were divided into three groups of four nd ech group ws treted with either ivermectin, fenbendzole or left untreted. Dung ws collected 7, 14, 21 nd 42 dys fter tretment nd serched for invertebrtes. Forty-five dys fter tretment, 18 pitfll trps contining wter nd sop were set out nd bited with dung from treted clves followed by dung from untreted clves. Although there were no significnt differences in dult beetle numbers between the treted nd untreted dung, not only ws there significnt difference in lrvl nd pupl numbers found between the ivermectin nd fenbendzole treted nd untreted dung, but the lrve found in the ivermectin treted dung were inhibited in their development. Pitfll trpping showed no significnt difference in dult beetle numbers between treted nd untreted dung, lthough trend towrd higher numbers of beetle ttrctions to the treted dung ws noted (Strong et l. 1996). Römbke et l. (2010) crried out field study in Spin to see the effects of ivermectin on the structure nd function of dung nd soil invertebrte communities. Dung ws collected 2, 3, 4 nd 7 dys fter treting cttle with 200µg/kg ivermectin s well s from untreted cttle. Stndrdised, rtificil dung pts were distributed in field nd collected 2, 4, 7, 14 nd 28 dys lter. The dung fun ws smpled nd ll nimls were identified nd recorded. They observed significntly lower bundnce of dult dung beetles on the dung from cttle treted with ivermectin compred to the control group. They lso noted tht lthough dult dung beetles were ttrcted to the ivermectin-spiked dung, the rte of degrdtion ws slower thn for the control dung (Römbke et l. 2010). 12

25 e. Dung ttrctiveness studies Errouissi nd Lumret (2010) studied the effects tht ivermectin hve on the ttrctiveness of dung treted with ivermectin to dung beetles. They performed two-yer study in Frnce to ssess the field effects of ivermectin residues on the ttrctiveness of dung to dung-colonizing insects. They used pitfll trps bited with dung from cttle treted with slow-relese bolus of ivermectin s well s dung from untreted cttle. The pitfll trps were plced out t one-week intervls for totl of five weeks. They found tht the ivermectin-contminted dung showed significnt ttrctive effect which highlighted the dnger of wide-spred ivermectin use s this potentilly puts the dung beetles offspring, nd indirectly, future beetle genertions survivl t risk (Errouissi & Lumret 2010). 2. Eprinomectin Lumret et l. (2005) exmined the lrvicidl ctivity of eprinomectin residues on the dung-inhbiting fly Neomyi cornicin in Frnce. Five heifers were treted with topicl pour-on eprinomectin solution t the recommended dose of 500µg/kg nd five remined untreted. Fecl smples were collected from the heifers before tretment, on the dy of tretment nd dily for seven dys, s well s 9, 12, 15, 20, 25, 29 nd 41 dys fter tretment. Eprinomectin concentrtions were mesured using HPLC. Biossys were performed by depositing N. cornicin eggs in continers filled with 250g of dung nd snd. The eggs were left to incubte in the lbortory nd left until the emergence of dult flies. Eprinomectin concentrtion in the dung ws highest on dy three fter tretment nd slowly dropped to lmost undetectble on dy 29. Eprinomectin residues in dung hd significnt effect on N. cornicin s no emergences were observed on the dung from dys 1 11 but fter dy 12 the first flies emerged. No significnt differences were observed from dy post tretment (Lumret et l. 2005). 13

26 3. Dormectin There re very few publictions on the toxicity of dormectin ginst non-trget dung-inhbiting orgnisms nd they re comprisons with ivermectin (Ddour 2000; Suárez et l. 2003; Webb et l. 2010) nd moxidectin (Suárez et l. 2009) which is discussed in section five. I could not find ny studies done on dormectin lone. 4. Moxidectin Fincher nd Wng (1992) tested the effects of moxidectin on two introduced Africn species of dung beetle nmely, Euoniticellus intermedius nd Onthopgus gzell. In Texs, Holstein steers were injected with moxidectin t the recommended dose of 200µg/kg nd dung ws collected 0, 1, 2, 3, 7, 10, 14, 21, 28, 35 nd 42 dys fter tretment. Two pirs of ech dung beetle species were plced in 40l buckets filled with moist soil nd 400g dung pt from ech steer. Fresh dung ws dded fter week nd the prents were removed. The buckets were monitored for progeny nd sex nd numbers were recorded. After five weeks the buckets were emptied nd the number of complete brood blls ws recorded to clculte percentge emergences. There were no significnt differences between the men number of brood blls produced by either species or on the emergence of progeny between treted nd untreted dung. There lso seemed to be no effect on the sex rtio for either species. They concluded by stting tht moxidectin seemed to be compound which is comptible with beneficil dung-burying beetles when used t the recommended dose (Fincher & Wng 1992). Iws et l. (2008) exmined the effects of moxidectin on non-trget coprophilous insects in cttle dung in field s well s lbortory trils in Jpn. Dung from cttle treted with the recommended dose of 500µg/kg moxidectin ws collected 1, 3, 7, 14, 21, 28 nd 35 dys fter tretment nd ws plced in continer with three mting pirs of the dung beetle Cccobius jessoensis. The sme ws done for dung from untreted cttle. Brood blls were collected, counted, weighed nd incubted to record dult emergences. Artificil dung pts, mde from dung collected from cttle treted with 500µg/kg moxidectin s well s untreted dung, were plced in field nd collected fter month, from which brood blls were 14

27 collected nd incubted to record dult emergences. Moxidectin concentrtion ws determined using HPLC nd the results showed tht concentrtions were t mximum levels three dys fter tretment, showed mrked decline by dy seven nd none ws detectble by dy 21. No significnt differences were found between the control nd the treted cttle dung with regrds to numbers nd weight of brood blls s well s emergence rtes. Results of the field study, gin, showed no significnt differences between the control nd the treted cttle dung. They concluded tht moxidectin hs no, or t most, the lest effect compred to other vermectins on non-trget coprophgous insects (Iws et l. 2008). 5. Comprtive studies: comprisons of two products The following re comprtive studies between ivermectin nd dormectin (Ddour 2000; Suárez et l. 2003; Webb et l. 2010); ivermectin nd moxidectin (Doherty et l. 1994; Strong & Wll 1994); moxidectin nd dormectin (Suárez et l. 2009); nd moxidectin nd eprinomectin (Wrdhugh et l. 2001). Ddour (2000) exmined the impct tht bmectin nd dormectin hve on the survivl nd reproduction of the dung beetle Onthophgus binodis. This study ws done in Austrli nd bmectin, rther thn ivermectin, ws chosen becuse it ws the first vermectin sold commercilly for the tretment of endoprsites in Austrli. Dung ws collected 1, 3, 6, 9, 18, 24, 34 nd 42 dys fter 150 heifers were divided into 3 groups nd treted with either 200µg/kg dormectin, 200µg/kg bmectin or nothing to serve s the control group. Five pirs of O. binodis were given fresh dung once week from ech tretment nd monitored for 4 weeks for dult survivl nd brood bll numbers. The brood blls were then incubted nd monitored dily for emergence. Dormectin concentrtions were determined using HPLC. Significnt dult mortlity ws observed from bmectintreted dung 3-6 dys fter tretment nd in dormectin-treted dung t nine dys fter tretment. This study confirmed n interesting response in dung beetles. Wheres bmectin residues hd no effect on dult mortlity in sexully mture beetles but rther sexully immture (newly emerged) beetles, which go through period of intense feeding during which they re exposed to mximum bmectin residues, were found to be much more ffected by the residues. In contrst to other studies (Fincher 1992; Krüger & Scholtz 1997) 15

28 brood bll production ws lso significntly lower in beetles fed on dung from cttle treted with bmectin for up to 42 dys fter tretment. Brood bll production ws lso significntly lower in beetles fed on dung from cttle treted with dormectin, but only for 3-6 dys fter tretment. The enhnced brood mss in beetles fed on dung from dormectintreted cttle t dys fter tretment could not be explined. According to the HPLC results, dormectin reched mximum concentrtion on dy three fter tretment following liner decline with n elimintion hlf-life of 15 dys (Ddour 2000). Surez et l. (2003) compred the effects of ivermectin nd dormectin on the invertebrte colonistion of cttle dung in Argentin. Dung ws collected from two groups of steers treted with either the recommended dose of 200µg/kg dormectin or 200µg/kg ivermectin nd from third untreted group 3, 7, 16, nd 29 dys fter tretment. Fifty rtificil pts were formed nd plced in three rows in 2m lternting sequence nd collected fter 7, 14, 21, 42, 100 nd 180 dys for nlysis in the lbortory. Ivermectin nd dormectin residue concentrtions were lso determined using HPLC. No significnt differences were found in the numbers of dult beetles, regrdless of the tretment. Fecl residue concentrtions for both ivermectin nd dormectin were highest in the first few dys nd remined reltively high throughout the experimentl period. Dormectin concentrtions were higher thn ivermectin concentrtions s the results show tht fter 180 dys of exposure to environmentl conditions, dung collected 27 dys fter ivermectin tretment still showed 56% residue compred to dung collected from dormectin tretment which showed 75% residue (Suárez et l. 2003). Webb et l. (2010) ssessed the bundnce nd dispersl of dung beetles in response to ivermectin nd dormectin tretment on pstured cttle in Scotlnd by running two-yer field tril. Three groups of cttle were treted with the recommended dosge of 500µg/kg ivermectin, 500µg/kg dormectin nd the lst group ws left untreted. Dung ws collected from cttle five dys prior to, nd two dys fter tretment. Eight dung-bited pitfll trps were set in two grids of four in ech field, with trps spced pproximtely 8m prt within ech grid. Trps were emptied nd re-bited every 7 10 dys in the first yer nd every 14 dys in the second yer. On ech smpling dte, two trps from ech of the two grids in ech study field were selected, nd the individuls of ech Aphodius species were counted nd pooled cross those four trps. In the field-scle study, significntly more beetles were 16

29 trpped in fields grzed by cttle treted with n vermectin thn in fields where cttle remined untreted. The colonising trils, however, indicted tht Aphodius beetles preferred colonising dung from untreted cttle rther thn dung from cttle treted with dormectin nd tht Aphodius dung beetles cn discriminte between dung from untreted cttle nd dung from cttle treted with dormectin t sptil scle of t lest 70 m (Webb et l. 2010). Doherty et l. (1994) compred the lrvicidl ctivities of different concentrtions of moxidectin nd bmectin on Onthophgus gzell to ssess the level of thret they pose to dung fun nd consequently, dung degrdtion, in Austrli. Dung pts were mde from dung spiked with 4, 8, 16, 32, 64, 128, 256 nd 512µg/kg of either moxidectin or bmectin nd fed to pirs of lb-rered O. gzell. The beetles were removed 18 dys lter, leving only the brood blls for nother 35 dys. Brood blls, lrve, pupe nd dults were then counted. Although oviposition ws not ffected by either tretment, lrvl survivl ws ffected by ll concentrtions of bmectin nd by ll concentrtions of moxidectin over 128µg/kg. In fct, moxidectin t 256 nd 512µg/kg produced survivl comprble to 4 nd 8µg/kg bmectin (Doherty et l. 1994). Strong nd Wll (1994) compred the reltive effects of ivermectin nd moxidectin on the colonistion of dung by dung-inhbiting insects in Englnd. Dung ws collected from cttle 2, 7, 14 nd 21 dys fter being treted with either ivermectin 200µg/kg or moxidectin 200µg/kg s well s from untreted cttle. Ninety-six rtificil dung pts were rndomly plced t 1m intervls in 12 x 8m grid, collected gin 7, 14, 21 nd 28 dys fter deposition, nd nlysed for invertebrtes. There ws no significnt difference between the three tretments in dult Scrbeide numbers showing tht neither ivermectin nor moxidectin residues repel colonizing dult beetles. However, dung collected from ivermectin-treted cttle up to seven dys fter tretment, showed high lrvl mortlity which moxidectin nd the control did not (Strong & Wll 1994). Surez et l. (2009) showed the effects of moxidectin nd dormectin fecl residues on the ctivity of dung colonizing insects by depositing dung from cttle treted with moxidectin, dung from cttle treted with dormectin nd control dung from untreted cttle on field. Comprisons of dung degrdtion were inconclusive; however, totl numbers of 17