Neurological risk of prolonged low dose exposure to imidacloprid in zebrafish

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dc.contributor.advisorKellermann, Tracyen_ZA
dc.contributor.advisorSmith, Carineen_ZA
dc.contributor.authorMcCulloch, Meganen_ZA
dc.contributor.otherFaculty of Medicine and Health Sciences. Dept. of Medicine. Division of Clinical Pharmacology.en_ZA
dc.date.accessioned2022-11-16T07:32:55Zen_ZA
dc.date.accessioned2023-01-16T12:47:45Zen_ZA
dc.date.available2022-11-16T07:32:55Zen_ZA
dc.date.available2023-01-16T12:47:45Zen_ZA
dc.date.issued2022-11en_ZA
dc.descriptionThesis (MMed) -- Stellenbosch University, 2022.en_ZA
dc.description.abstractENGLISH ABSTRACT: Imidacloprid (IMI) is a systemic neonicotinoid insecticide intended to replace the organophosphate pesticides in agriculture. Extensive use of these pesticides increases the risk to the environment and non-target organisms such as humans due to their potential bioaccumulation and toxicity. Researchers studying the effect of IMI on human nicotinic receptors (α4β2) have reported that IMI may have more substantial side effects on humans than originally anticipated. This research project aimed to assess the possibility of long-term neurological risks following prolonged, low dose IMI exposure within an in vivo zebrafish model. Methods A protein precipitation extraction from zebrafish brain, liver and gill homogenate was applied followed by LC-MS/MS detection of neurotransmitters and IMI and its primary metabolites. Protein precipitation was conducted using methanol:acetonitrile (1:1 v/v) as the precipitating solvent. Phenethylamine-d4 and IMI-d4 were used as internal standards for the neurotransmitter and IMI LC-MS/MS methods, respectively. For the neurotransmitters, chromatographic separation was achieved using a Poroshell column (3.0 x 100 mm, 2.7 μm) using a gradient elution mode at a flow rate of 0.45 mL/min and an analysis time of 7 min. Mobile phase A and B consisted of water with 0.1% formic acid and acetonitrile, respectively. For IMI and its metabolites, chromatographic separation was achieved using a biphenyl column (2.1 x 100 mm, 2.7 μm) with gradient elution at a flow rate of 0.4 mL/min. The total analysis time was 8.5 min. Mobile phase A and B consisted of water and methanol respectively, both with 5 mM ammonium formate and 0.1% formic acid. The two developed methods underwent a partial validation to certify that both methods were precise, accurate and reliable. Zebrafish larvae were exposed to IMI at four and five days post fertilisation to determine the no observed adverse effect level (NOAEL) of IMI. After this, adult zebrafish were exposed to the NOAEL concentration for 21 days. Key endpoints included behaviour indicative of neurocognitive decline and possible bioaccumulation in the adult zebrafish brain, liver and gills. Neurotransmitter concentrations were measured in the adult zebrafish brain tissue at the end of the treatment period to evaluate changes in neurotransmitter signalling and potential neurological risks using the developed LC-MS/MS method. Bioaccumulation of IMI and its metabolites in zebrafish brain, liver and gills was evaluated using LC-MS/MS. Results The calibration curve fits a quadratic (weighted 1/C) regression over the concentration range of 31.3 - 1000 ng/mL for acetylcholine, gamma-aminobutyric acid, serotonin and dopamine. The calibration curve for IMI and its metabolites fits a quadratic (weighted 1/C) regression over the concentration range of 1.95 - 125 ng/Ml for imidacloprid-urea and IMI, 0.244 - 125 ng/mL for desnitro-imidacloprid and 3.91 - 125 ng/mL for 5-hydro imidacloprid. The NOAEL of IMI in zebrafish larvae was determined to be 2.5 μg/L. No significant morphological changes were observed in the adult zebrafish during the treatment period. Behavioural changes observed during the pesticide exposure period included decrease in appetite of the treatment group. The treatment group was also observed swimming at the bottom of the tank in comparison to the control group. Although IMI, IMI-urea and desnitro-IMI could not be detected in any of the tissue specimens, 5-hydro IMI was detected at relatively high concentrations in the liver (0.793 ng/mg tissue) and gill epithelial tissue (117 ng/mg tissue). Only concentrations of gamma-aminobutyric acid and acetylcholine were detected and quantified in both the treated and control group. The treated group showed a 1.4-fold decrease and 1.9-fold increase in acetylcholine and gamma-aminobutyric acid, respectively in comparison to the control. Serotonin and dopamine could not be detected due to their levels being below the limit of quantitation of this method. Conclusion Robust LC-MS/MS methods were developed for the detection and quantitation of IMI, desnitro-imidacloprid, imidacloprid-urea and 5-hydro-imidacloprid, as well as serotonin, dopamine, acetylcholine and gamma-aminobutyric acid neurotransmitters in 200 μL zebrafish brain, liver and gill epithelial tissue homogenate. The behavioural changes observed in the adult zebrafish could be an indication of one of two things, anxiety or sedative effect. This is verified by the increase in gamma-aminobutyric acid neurotransmitter levels. Together with the evaluation of bioaccumulation of imidacloprid and its metabolites within the brain, liver and gill epithelial tissue of zebrafish, this study provides an indication of the potential risk to human health following chronic neonicotinoid exposure. Overall, our findings further contribute to existing literature and suggest that IMI does pose a threat to more than just insects and therefore requires further investigation.en_ZA
dc.description.abstractAFRIKAANS OPSOMMING: Imidacloprid (IMI) is ‘n sistemiese neonikotinoïed insekdoder wat geoormerk is om die organofosfaat plaagdoders in die landbou te vervang. Die wydverspreide gebruik van hierdie plaagdoders verhoog die risiko vir die omgewing en nie-teiken organismes soos mense, as gevolg van hul potensiële bioakkumluasie en toksisiteit. Narvorsers wat die effek van IMI op menslike nikotienreseptore (α4β2) bestudeer, het gerapprteer dat IMI meer aansienlike newe-effekte op mense kan hê as wat oorspronklik verwag is. Metodes ‘n Proteïen presipitasie van gehomogeniseerde sebravisbrein, lewer en kieu monsters is toegepas, gevolg deur LC-MS/MS-meting van neuro-oordragstowwe, IMI en sy primêre metaboliete. Proteïen presipitasie is uitgevoer met die gebruik van metanol:asetonitriel (1:1, v/v) as die presipiterende oplosmiddel. Phenethylamine-d4 en IMI-d4 is gebruik as interne standaarde vir die neuro-oordragstowwe en IMI LC-MS/MS metodes, onderskeidelik. Vir die neuro-oordragstowwe is chromatografiese skeiding bereik met behulp van n Poroshell-kolom deur gebruik te maak van ‘n gradiënt-eleuringsmodus teen ‘n vloeispoed van 0.45 mL/min en ‘n ontledingstyd van 7 min. Mobiele fase A en B het bestaan uit water met 0.1% mieresuur en asetonitriel onderskeidelik. Vir IMI en sy metaboliete is chromatografiese skeiding bereik met behulp van n biphenyl kolom met gradiënt-eleuring teen ‘n vloeispoed van 0.4 mL/min. Die totale ontledingstyd was 8.5 min. Mobiele fase A en B het onderskeidelik uit water en metanol bestaan beide met 5 mM ammoniumformaat en 0.1% mieresuur. Die twee ontwikkelde metodes het 'n gedeeltelike validering ondergaan om te sertifiseer dat beide metodes presies, akkuraat en betroubaar was. Sebravislarwes is vier en vyf dae na bevrugting aan IMI blootgestel om die “geen waargenome nadelige effek”-vlak van IMI te bepaal. Hierna is volwasse sebravisse vir 21 dae aan die bepaalde konsentrasie blootgestel. Neuro-oordragstof konsentrasies is gemeet in die volwasse sebravis breinweefsel aan die einde van die behandelingsperiode om veranderinge in neuro-oordragstof seinoordrag en potensiële neurologiese risiko's te evalueer deur die ontwikkelde LC-MS/MS metode te gebruik. Bioakkumulasie van IMI en sy metaboliete in sebravisbrein, lewer en kieue is geëvalueer met behulp van LC-MS/MS. Resultate Die kalibrasiekurwe pas by 'n kwadratiese (geweegde 1/C) regressie oor die konsentrasiereeks van 31.3 - 1000 ng/mL vir asetielcholien, gamma-aminobottersuur, serotonien en dopamien. Die kalibrasiekurwe vir IMI en sy metaboliete pas by 'n kwadratiese (geweegde 1/C) regressie oor die konsentrasiereeks van 1.95 - 125 ng/Ml vir imidacloprid-ureum en IMI, 0.244 - 125 ng/mL vir desnitro-Imidacloprid en 3.91 - 125 ng/mL vir 5-hidro-imidakloprid. Die geen waargenome nadelige effekvlak van IMI in sebravislarwes is bepaal as 2.5 μg/L. Belangrike eindpunte het gedrag ingesluit, wat dui op neurokognitiewe agteruitgang. Voorlopige data dui ook op moontlike bioakkumulasie in die volwasse sebravisbrein, lewer en kieue. Geen betekenisvolle morfologiese veranderinge is in die volwasse sebravis tydens die behandelingsperiode waargeneem nie. Gedragsveranderinge wat tydens die blootstelling aan plaagdoders waargeneem is, sluit afname in eetlus van die behandelingsgroep in. Die behandelingsgroep is ook waargeneem wat aan die onderkant van die tenk geswem het in vergelyking met die kontrolegroep. Alhoewel IMI, IMI-ureum en desnitro-IMI nie in enige van die weefselmonsters opgespoor kon word nie, is 5-hidro IMI by relatief hoë konsentrasies in die lewer (0.793 ng/mg weefsel) en kieuepiteelweefsel (117 ng/mg weefsel) opgespoor. Slegs konsentrasies gamma-aminobottersuur en asetielcholien is in beide die behandelde en kontrolegroep opgespoor en gekwantifiseer. Die behandelde groep het 'n 1.6-voudige afname en 1.9-voudige toename in asetielcholien en gamma-aminobottersuur onderskeidelik in vergelyking met die kontrole getoon. Serotonien en dopamien kon nie opgespoor word nie omdat hul vlakke onder die drumpel van kwantifisering van hierdie metode was. Gevolgtrekking Robuuste LC-MS/MS-metodes is ontwikkel vir die opsporing en kwantifisering van IMI, desnitro-IMI, imidacloprid-ureum en 5-hidro imidakloprid, sowel as serotonien, dopamien, asetielcholien en gamma-aminobottersuur neuro-oordragstowwe in 200 μL sebravisse brein, lewer en kieuepiteelweefsel homogeniseer. Die gedragsveranderinge wat by die volwasse sebravis waargeneem word, dui ‘n toksisiteit/verdovend effek aan en word geverifieer deur die verhoging in gamma-aminobottersuur vlakke. Saam met die evaluering van bioakkumulasie van imidacloprid en sy metaboliete binne die brein, lewer en kieuepiteelweefsel van sebravis, verskaf hierdie studie 'n aanduiding van die impak op menslike gesondheid na chroniese neonikotinoïedblootstelling. Oor die algemeen dra ons bevindinge verder by tot bestaande literatuur en stel voor dat IMI wel 'n bedreiging vir meer as net insekte inhou en daarom verdere ondersoek verg.af_ZA
dc.description.versionMastersen_ZA
dc.format.extentxi, 125 pages : illustrationsen_ZA
dc.identifier.urihttp://hdl.handle.net/10019.1/126055en_ZA
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subjectImidacloprid -- Dose-response relationshipen_ZA
dc.subjectNeonicotinoids -- South Africaen_ZA
dc.subjectPesticides -- South Africaen_ZA
dc.subjectUCTDen_ZA
dc.titleNeurological risk of prolonged low dose exposure to imidacloprid in zebrafishen_ZA
dc.typeThesisen_ZA
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