Valentin Mingo Vom Fachbereich VI (Raum- und Umweltwissenschaften) der Universität Trier zur Erlangung des akademischen Grades Doktor der Naturwissenschaften (Dr. rer. nat.) genehmigte Dissertation The use of plant protection products and its impact on reptiles Betreuender: Prof. Dr. Stefan Lötters Berichterstattende: Prof. Dr. Stefan Lötters, Prof. Dr. Michael Veith, Dr. Carsten Brühl Datum der wissenschaftlichen Aussprache: 28.05.2018 Trier, Mai 2018 Table of contents Acknowledgements ...................................................................................................... III Abstract .......................................................................................................................... 1 Thesis structure .............................................................................................................. 2 1 Summary ................................................................................................................ 7 1.1 The global decline of reptiles ......................................................................... 7 1.2 Causes for reptile declines .............................................................................. 8 1.3 Reptiles in agricultural landscapes ............................................................... 11 1.3.1 Risk evaluation of pesticide use to protected European reptile species ... 13 1.3.2 Risk of pesticide exposure for reptile species in the European Union ..... 14 1.4 The current status of reptiles in pesticide ecotoxicology ............................. 16 1.4.1 The use of buccal swabs as a minimally-invasive method to detect effects of pesticide exposure in the Common wall lizard ................................................ 18 1.4.2 Validating buccal swabbing as a minimal-invasive method to detect pesticide exposure in reptiles. ............................................................................... 20 1.5 Effects of pesticide exposure and exposure pathways in reptiles ................ 22 1.5.1 Determination of Pesticides Adsorbed on Arthropods and Gastropods by a Micro‑QuEChERS Approach and GC–MS/MS .................................................. 27 1.5.2 The impact of land use intensity and associated pesticide applications on fitness and enzymatic activity in reptiles — A field study ................................... 29 1.6 The current status of reptiles in legislation concerning the admission of plant protection products in the European Union .................................................. 31 I 1.7 Conclusion .................................................................................................... 34 1.8 References .................................................................................................... 36 CHAPTER I ................................................................................................................. 48 Risk evaluation of pesticide use to protected European reptile species ................... 49 Risk of pesticide exposure for reptile species in the European Union ..................... 75 CHAPTER II .............................................................................................................. 95 The use of buccal swabs as a minimal-invasive method for detecting effects of pesticide exposure on enzymatic activity in common wall lizards .......................... 96 Validating buccal swabbing as a minimal-invasive method to detect pesticide exposure in reptiles ................................................................................................. 125 CHAPTER III ............................................................................................................ 166 Determination of pesticides adsorbed on arthropods and gastropods by a micro- QuEChERS approach and GC-MS/MS .................................................................. 167 The impact of land use intensity and associated pesticide applications on fitness and enzymatic activity in reptiles – A field study ......................................................... 181 Supplements ............................................................................................................... 218 Supplementary material CHAPTER I ........................................................................ 219 Supplementary material CHAPTER II ....................................................................... 254 Supplementary material CHAPTER III ...................................................................... 262 Curriculum Vitae ........................................................................................................ 270 Declaration ................................................................................................................. 272 II Acknowledgements I wish to express my special thanks to Prof. Dr. Stefan Lötters and to Dr. Norman Wagner for supervising this thesis, and particularly for their critical suggestions which have been a considerable contribution to my work, and without whose expertise this thesis would not have been possible. Furthermore, I want to express my gratitude towards the Konrad-Adenauer- Stiftung, who thankfully granted a PhD scholarship to fund this thesis. My thanks go to all people who helped me during fieldwork: Corrado Modica, Maria Weinrich, Daniela Rößler and Luis Fernando Marin da Fonte. I also want to thank the Biogeography working group for the pleasant time and coffee break conversations. My sincere thanks go to all of my friends, without which the last years would not have been as enjoyable. Last but not least, my special thanks go to my family, especially my parents, who have always supported me. III Abstract Reptiles belong to a taxonomic group characterized by increasing worldwide population declines. However, it has not been until comparatively recent years that public interest in these taxa has increased, and conservation measures are starting to show results. While many factors contribute to these declines, environmental pollution, especially in form of pesticides, has seen a strong increase in the last few decades, and is nowadays considered a main driver for reptile diversity loss. In light of the above, and given that reptiles are extremely underrepresented in ecotoxicological studies regarding the effects of plant protection products, this thesis aims at studying the impacts of pesticide exposure in reptiles, by using the Common wall lizard (Podarcis muralis) as model species. In a first approach, I evaluated the risk of pesticide exposure for reptile species within the European Union, as a means to detect species with above average exposure probabilities and to detect especially sensitive reptile orders. While helpful to detect species at risk, a risk evaluation is only the first step towards addressing this problem. It is thus indispensable to identify effects of pesticide exposure in wildlife. For this, the use of enzymatic biomarkers has become a popular method to study sub-individual responses, and gain information regarding the mode of action of chemicals. However, current methodologies are very invasive. Thus, in a second step, I explored the use of buccal swabs as a minimally invasive method to detect changes in enzymatic biomarker activity in reptiles, as an indicator for pesticide uptake and effects at the sub-individual level. Finally, the last part of this thesis focuses on field data regarding pesticide exposure and its effects on reptile wildlife. Here, a method to determine pesticide residues in food items of the Common wall lizard was established, as a means to generate data for future dietary risk assessments. Subsequently, a field study was conducted with the aim to describe actual effects of pesticide exposure on reptile populations at different levels. 1 Thesis structure This thesis addresses the question of how pesticide applications affect wild reptile populations, by evaluating exposure probability of reptiles to pesticides, establishing new methods to detect exposure scenarios using sub-individual biomarkers and quantifying contamination of food items, as well as studying the impacts of exposure at the sub- individual, individual and population level. According to these topics, this thesis is divided into three chapters. The first chapter addresses the risk of pesticide exposure for reptile species natively occurring within the European Union (EU). It consists of two GIS-based approaches through which the probability of exposure to plant protection products (PPP) was evaluated. The first part of Chapter I focuses on the exposure risk of protected European reptile species listed under Annex II of the habitats directive, within their special areas of conservation, and can be found in: Wagner, N., Mingo, V., Schulte, U., Lötters, S. 2015. Risk evaluation of pesticide use to protected European reptile species. Biological Conservation 191, 667-673. In the second part, the focus shifts towards evaluating the risk of pesticide exposure of reptiles at the entire EU level, and encompasses an assessment of 102 natively occurring reptile species. The study further elucidates differences in exposure sensitivity between different reptile orders and suborders, as well as exposure probability for different reptile species inhabiting different pesticide admission zones (“Mutual Recognition Zones”) within the EU, and was published in: Mingo, V., Lötters, S., Wagner, N. 2016. Risk of pesticide exposure for reptile species in the European Union. Environmental Pollution