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Marzaro, M (2013) Corn seed coated with neonicotinoids: environmental contamination and bee losses in spring. [Tesi di dottorato]

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Abstract (inglese)

The last ten years have witnessed, both at a national and an international level, a crisis in the beekeeping sector due to the death of bees often causing a complete depopulation of hives. This phenomenon has assumed a particularly serious intensity in the three years 2005-2008 with a loss of up to 40% of hives in some states in Europe, amongst these Italy, and losses up to 40-60% in the United States. The name “Colony Collapse Disorder has been coined to describe these generalised bee deaths and a number of questions have been raised as to the causes, amongst these are the spread of parasites such as Varroa destructor or Nosema spp., ever more extreme beekeeping techniques and the widespread environmental pollution due in great measure the use of phyto-sanitary products. The colony losses that have been observed in the Autumn in particular, are generally due to the instance of varroa, while the Spring losses, before the banning of neonicotinoids in 2008, appeared at the same time as the Spring sowing of maize seed where seed coated with insecticide of the neonicotinoid family were sown using pneumatic seed drills.
This text studies in depth aspects which are concerned with the death of bees, particularly in the Spring period. It is possible, in fact, to distinguish the causes which differentiate the losses in the Autumn, caused essentially by the bee mite Varroa destructor from the Spring losses the cause of which is associated with the sowing of maize with neonicotinoids. The means by which poisoning occurred were virtually unknown until the start of the present thesis. Up to this point the deaths had in fact been attributed to poisoning by sub-lethal doses which bees had picked up from the self-sown vegetation surrounding the sowing areas. Instead, the hypothesis has been considered that there exist sources of acute poisoning connected to the sowing of maize. Two different poisoning mechanisms were hypothesised. The first consisted of the release of droplets containing substantial quantities of active ingredient with which the bees could come into contact through a physiological phenomenon called guttation; the second mechanism considered was poisoning with insecticide through the sowing of coated maize seed, whereby large quantities of dust, containing high concentrations of insecticide, poisoned the bees in flight.
Young plants which develop raised root pressure frequently show an emission of liquid around the edge of the leaves, a phenomenon called guttation. The “drops of dew” seen on the leaves of graminaceous plants in the morning are, for most part, guttation drops, in particular on the points of the leaves. The drops are produced and remain on the lamina of the leaves of maize seedlings, often for some hours, but they can drop off or dry out rapidly if wind is present. Guttation can even remain in the calyx of the plant for the whole day. During the Spring period such drops can be used by bees as a water source with which to moisten the hive
The first two studies in this thesis illustrate and show the results of the first year of doctoral activity, that is the discovery that the droplets of guttation contain elevated concentrations, some hundreds of ppm, of inscticides (neonicotinoids) employed in the seed coating. This insecticide, being systemic (water soluble), enters into the circulation of the plant, and is thus released through guttation. Such concentrations, if we consider a bees capacity to ingest (estimated around 20-30µl), proves to be well above the lethal dose for ingestion or, even for contact (ca. 20-40µl of a.i. per bee). However, the implication of guttation in the catastrophic Spring deaths has been excluded through the observations in the field, in which it came to light that such droplets do not constitute a normal water source for bees, unless we assume a long period of drought.
Therefore, in subsequent studies the hypothesis was considered that the toxic effects of neonicotinoids used for the coating of maize seed could be directly related the exposure of bees to the dust emitted during the maize sowing. Field trials were undertaken, both with regard to the emission of particulates, and to determine the mechanisms and the factors that caused the lethal poisoning of bees.
From chemical analysis (conducted by the Department of Chemistry of the University of Padua) it was shown that, even after a single flight in the vicinity of the seed drill, that quantities from between about 50 and 1000 ng per bee were present with a maximum encountered equal to 11µg/bee. In parallel with the field trials steps were taken to quantify the amount of dust emitted by the seed drill with great precision (six trials), and at the same time to determine the dimensions of the “toxic cloud” made up of particulates projected into the air during the sowing. In the fifth study in this thesis, the presence of a toxic cloud around the machine was demonstrated which, in the absence of a sustained wind, had an ellipsoidal form of approximately 20 metres in diameter and at least 3 metres in height. Among the various results obtained, it is also important to underline the setting up of a simple biological test to establish the toxic effect to bees flying near the seed drill. These results are in precise agreement with the analytical data of emissions and/or the fall of particulates emanating from the seed drill.
The chemical analysis, in particular, allowed the putting on place of an innovative protocol to determine the amount of neonicotinoid insecticide in a single bee (seventh study); up to that point all the analysis reported in the literature concerned the examination of at least several hundreds of bees (for example kg of dead insects). It follows that the more refined chemical analysis allows the quantification of contamination in a single insect with an evident advantage in the understanding and evaluation of the cause of poisoning.

Abstract (italiano)

Negli ultimi dieci anni si è assistito, a livello nazionale ed internazionale, ad una crisi nel settore dell’apicoltura dovuto a morie di api che portano spesso ad un completo spopolamento degli alveari. Questo fenomeno ha assunto intensità particolarmente gravi nel triennio 2005-2008, con perdite fino al 40% del totale degli alveari produttivi in alcuni stati europei, tra cui l’Italia e sino al 40-60% negli Stati Uniti. Questa generalizzata moria delle api, denominata “Colony Collapse Disorder” (CCD) ha comportato una serie di interrogativi sulle cause, tra le quali la diffusione di parassiti come la Varroa destructor oppure il Nosema spp., le tecniche apistiche sempre più spinte e il diffuso inquinamento ambientale dovuto in gran parte all’uso di prodotti fitosanitari. In particolare, la perdita di colonie che si osserva in autunno è dovuta nella generalità dei casi alla varroa, mentre le perdite primaverili, osservate prima del bando dei neonicotinoidi nel 2008, avvengono in corrispondenza delle semine primaverili del mais, dove si impiegano sementi conciate con insetticidi appartenenti alla famiglia dei neonicotinoidi e seminatrici pneumatiche.
In questa tesi si sono approfonditi gli aspetti che riguardano la moria delle api, soprattutto in riferimento al periodo primaverile. Si possono infatti distinguere le cause che differenziano le perdite delle colonie nel periodo autunnale provocate essenzialmente dall’acaro Varroa destructor, dalle perdite primaverili, le cui cause, pur essendo associate alle semine del mais con neonicotinoidi, erano praticamente sconosciute nel modalità di intossicazione sino all’inizio della presente tesi; si attribuiva infatti la morte delle api a dosi sub letali acquisite dalla vegetazione spontanea circostante i seminativi. Si è considerata invece l’ipotesi che esistessero fonti di intossicazione acuta legate alla semina del mais. Si sono ipotizzati due diversi meccanismi d’azione nell’avvelenamento delle api. Il primo consiste nel rilascio, sottoforma di gocce, attraverso un fenomeno fisiologico chiamato guttazione, di ingenti quantità di principio attivo con le quali le api potrebbero venire a contatto; l’altro meccanismo considera l’intossicazione da insetticidi attraverso la semina di mais conciato con la liberazione nell’aria di particolato contenente concentrazioni altissime di insetticida con il quale le api si intossicano in volo.
Le giovani piante che sviluppano una pressione radicale elevata, mostrano frequentemente la fuoriuscita di liquido dai margini delle foglie, un fenomeno detto guttazione. Le “gocce di rugiada” osservate sulle foglie delle graminacee al mattino sono per lo più gocce di guttazione, in particolare se presenti sulla punta delle foglie. Le gocce sono prodotte e permangono sulla lamina delle foglie delle piantine di mais anche per parecchie ore, ma possono cadere o asciugarsi rapidamente in presenza di vento. Nel calice, la guttazione permane anche per tutto il giorno. Tali gocce durante il periodo primaverile possono venir utilizzate dalle api come fonte idrica anche per umettare l’alveare.
Nei primi due lavori presenti in questa tesi sono illustrati i risultati del primo anno dell’attività di dottorato, cioè la scoperta che le gocce di guttazione contengono concentrazioni elevate, anche centinaia di ppm, di insetticidi (neonicotinoidi) impiegati nella concia dei semi: queste molecole essendo idrosolubili entrano in circolo nella pianta e sono quindi parzialmente rilasciati attraverso le guttazioni. Tali concentrazioni, se si considera la capacità di ingestione di un’ape ( stimata attorno a 20-30 µl), risultano ben al di sopra della dose letale sia per ingestione ed anche per contatto (circa 20-40 ng di p.a./ape). Si è tuttavia esclusa l’implicazione delle guttazioni nelle catastrofiche morie primaverili attraverso osservazioni di campo che hanno messo in luce come tali gocce non costituiscano di norma una fonte idrica per le api a meno che, si suppone, non perduri un lungo periodo di siccità.
Nei lavori successivi si è quindi considerata l’ipotesi che l’effetto tossico dei neonicotinoidi usati per la concia del seme di mais, fosse direttamente connesso all’esposizione delle api alle polveri emesse durante la semina. Si sono svolte prove in campo, sia per quanto riguarda l’emissione del particolato, sia sul meccanismo e i fattori che provocavano un intossicazione letale alle api.
Dalle analisi chimiche (eseguite dal Dipartimento di Scienze Chimiche dell’Università di Padova), le quantità di insetticidi rinvenuti su api morte, anche dopo un solo volo vicino alla seminatrice, sono comprese tra circa 50 e 1000 ng per ape, con una punta massima riscontrata pari a 11 µg/ape.
Parallelamente alle prove in campo, si è provveduto a quantificare con maggior precisione la quantità di polvere che viene emessa dalla seminatrice (sesto lavoro) e contemporaneamente a determinare la dimensione della “nube tossica” formata dal particolato proiettato in aria durante la semina. Nel quinto lavoro presente nella tesi, è stata dimostrata così la presenza di una nube tossica attorno alla macchina, che in assenza di vento sostenuto, ha la forma di un elissoide di circa 20 metri di diametro e di almeno 3 metri d’altezza. Tra i vari risultati ottenuti è rilevante sottolineare anche la messa a punto un semplice test biologico per saggiare l’effettiva intossicazione delle api in vicinanza delle seminatrici i cui risultati sono in ottimo accordo con i dati analitici di emissione e/o di ricaduta delle particelle originate con la semina.
Le analisi chimiche, in particolare, hanno consentito di mettere a punto un protocollo innovativo per la determinazione di insetticidi neonicotinoidi in singole api (settimo lavoro); sino ad ora tutte le analisi riportate in letteratura riguardano l’esame di campioni di almeno alcune decine di api (es. kg di insetti morti). Ne consegue che l’analisi chimica più raffinata permette di quantificare la contaminazione del singolo insetto con un evidente vantaggio per la comprensione e valutazione delle cause di intossicazione.

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Tipo di EPrint:Tesi di dottorato
Relatore:Girolami, V
Dottorato (corsi e scuole):Ciclo 24 > Scuole 24 > SCIENZE DELLE PRODUZIONI VEGETALI > PROTEZIONE DELLE COLTURE
Data di deposito della tesi:21 Gennaio 2013
Anno di Pubblicazione:31 Gennaio 2013
Parole chiave (italiano / inglese):guttation, neonicotinoids, honeybee, Apis mellifera, seed coating, toxic powder, humidity influence, clothianidin, imidacloprid, driller modification, drift of dust evaluation, colony losses
Settori scientifico-disciplinari MIUR:Area 07 - Scienze agrarie e veterinarie > AGR/11 Entomologia generale e applicata
Struttura di riferimento:Dipartimenti > Dipartimento di Agronomia Animali Alimenti Risorse Naturali e Ambiente
Codice ID:5398
Depositato il:15 Ott 2013 14:16
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