SECOND GENERATION ANTICOAGULANT RODENTICIDES IN BIRDS OF PRAY OF ESTONIA TEISE GENERATSIOONI ANTIKOAGULANTSED RODENTSITSIIDID EESTI RÖÖVLINDUDES

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1 ESTONIAN UNIVERSITY OF LIFE SCIENCES Institute of Veterinary Medicine and Animal Sciences Priit Peetris SECOND GENERATION ANTICOAGULANT RODENTICIDES IN BIRDS OF PRAY OF ESTONIA TEISE GENERATSIOONI ANTIKOAGULANTSED RODENTSITSIIDID EESTI RÖÖVLINDUDES Final Thesis in Veterinary Medicine Curriculum in Veterinary Medicine Supervisors: Madis Leivits, DVM Tõnu Püssa, PhD Tartu 2019

2 Estonian University of Life Sciences Abstract of Final Thesis in Veterinary Fr. R. Kreutzwaldi 1, Tartu Author: Priit Peetris Medicine Curriculum: Veterinary Medicine Title: Second generation anticoagulant rodenticides in birds of prey of Estonia Pages: 55 Figures: 16 Tables: 2 Appendixes: - Department / Chair: Chair of Veterinary Clinical Medicine Field of research and CERC S code: Veterinary medicine, surgery, physiology, pathology, clinical studies, B750 Supervisors: Madis Leivits, Tõnu Püssa Place and date: Tartu, 2019 The usage of anticoagulant rodenticides has been increasing in the last few decades consequently endangering wild carnivorous species, the birds of prey, through secondary poisoning. In a similar study conducted in Denmark they found that 92% of bird samples tested had residues of rodenticides, 82% of animal samples from Finland had residues and from Norway 67% of eagle owls had detectable residues in their liver. It shows that this is a quite vital topic here, in Northern European countries. The purpose of this study was to evaluate the impact of second generation anticoagulant rodenticides (SGAR) in Estonian birds of prey. The samples were collected from diseased or euthanized birds who were seen in a wild animal hospital. In total, 63 liver samples from 11 different raptor species were tested with liquid chromatography. Three second-generation anticoagulant rodenticides: brodifacoum, difenacoum, and bromadiolone, and one first generation anticoagulant rodenticide, warfarin, were searched for. From the samples, at least one sample from each studied species had SGAR residues and 70% from all birds had residues. Statistical significance with brodifacoum was found from nocturnal species having higher concentrations than diurnal raptors. It seems that there is a significant intoxication among Estonian wildlife and the extent of that needs further investigation. The Estonian University of Life Sciences laboratory compound is appropriate to study the concentrations of anticoagulant rodenticides from liver, and can be used to search for other similar compounds. Keywords: LC-MS, poisoning, SGAR, brodifacoum, bromadiolone

3 Eesti Maaülikool Veterinaarmeditsiini õppe lõputöö Fr. R. Kreutzwaldi 1, Tartu Autor: Priit Peetris lühikokkuvõte Õppekava: veterinaarmeditsiin Pealkiri: Teise generatsiooni antikoagulantsed rodentsitsiidid Eesti röövlindudes Lehekülgi: 55 Jooniseid: 16 Tabeleid: 2 Lisasid: - Osakond/õppetool: kliinilise veterinaarmeditsiini õppetool ETIS-e teadusvaldkond ja CERC S-i kood: veterinaarmeditsiin, kirurgia, füsioloogia, patoloogia, kliinilised uuringud, B750 Juhendajad: Madis Leivits, Tõnu Püssa Kaitsmiskoht ja -aasta: Tartu, 2019 Antikoagulantsete rodentitsiidide kasutamine on suurenenud viimaste aastate jooksul ja sellega seoses kasvab sekundaarse mürgistuse oht looduses elavatele kiskjatele, röövlindudele. Teistes sarnastes uuringutes, mis on tehtud Põhjamaades, leiti rodentitsiidide jääke: Taanis 92% lindudest, Soomes 82% loomadest ning Norras 67% kassikakkudest. Selle alusel võiks teha järelduse, et rodentitsiidide teema on Põhja-Euroopas väga aktuaalne. Käesoleva uuringu eesmärk oli hinnata antikoagulantsete rodentitsiidide saastatuse ulatust Eesti röövlindudes. Uuringus kasutati metsloomade haiglasse saabunud hukkunud või eutaneeritud lindude maksaproove, kokku koguti 11 erinevalt röövlinnu liigist 63 proovi. Igast maksaproovist uuriti vedelikkromatograafia abil kolme teise generatsiooni antikoagulantse rodentitsiidi: brodifaakumi, difenaakumi ja bromadiolooni; ning ühe esimese generatsiooni rodentitsiidi, varfariini kontsentratsiooni. Eestis elevate lindude proovidest leiti 70% juhul rodentitsiidide jääke ning kõikides liikides oli vähemalt üks positiivne proov. Statistiline olulisus brodifaakumiga seoses esines öise eluviisiga lindude ja kõrgema rodentitsiidisisalduse vahel. Ilmnes, et Eestis on teise generatsiooni antikoagulantsete rodentitsiidide mürgistus metsloomade hulgas olemas ja selle levimus vajaks laiemat uurimist. Eesti Maaülikooli labori sisseseade on sobiv selleks, et uurida rodentitsiidide kontsentratsioone maksast ning oleks võimeline uurima ka muid sarnaseid toksilisi aineid. Märksõnad: LC-MS, mürgistus, brodifaakum, bromadioloon

4 TABLE OF CONTENTS 1. LIST OF ABBREVIATIONS INTRODUCTION ACKNOWLEDGEMENTS LITERATURE ANALYSIS Anticoagulant rodenticides History of anticoagulant rodenticides Use of anticoagulant rodenticides Classification Effect of anticoagulant rodenticides Relation to bone metabolism Treatment of anticoagulant rodenticide poisoning Recent studies Species studied Situation in Denmark Situation in Scotland Situation in Scandinavia About Europe Situation in Estonia Detection of residues Possible methods Common methods used AIMS OF THE STUDY MATERIALS AND METHODS

5 3.1. Sample collection Species overview Sample preparation Preparation of standard curves and spiking solutions Analytical method Statistical analysis RESULTS DISCUSSIONS CONCLUSIONS SUMMARY REFERENCES ÜLDKOKKUVÕTE

6 1. LIST OF ABBREVIATIONS AR anticoagulant rodenticide BDL bromadiolone BDF brodifacoum dspe dispersive solid phase extraction DFN - difenacoum FGAR first generation anticoagulant rodenticide FLD fluorescence detection GC-MS gas chromatography mass spectrometry H-ESI-LC-MS/MS heated electrospray ionization liquid chromatography tandem mass spectrometry HF-LPME hollow fibre liquid phase micro extraction HPLC high-performance liquid chromatography HPLC-(AJS ESI)-qTOF-MS/MS high performance liquid chromatography (Agilent Jet Stream Electrospray Ionisation) quadrupole time of flight tandem mass spectrometry HPLC-UV high-performance liquid chromatography ultraviolet spectrometry IC ion chromatography IQR inter quartile range LC-MS liquid chromatography mass spectrometry LDS-DLLME low-density solvent-based dispersive liquid-liquid micro extraction LLE liquid-liquid extraction LOD limit of detection LOQ limit of quantitation PBMS predatory birds monitoring scheme QuEChERS quick, easy, cheap, effective, rugged, safe SGAR second generation anticoagulant rodenticide SPE solid phase extraction UHPLC-MS ultra-high-performance liquid chromatography mass spectrometry UPLC-CSH ultra-performance liquid chromatography charged surface hybrid UPLC-MS/MS ultra-performance tandem mass spectrometry 6

7 UPLC-TOFMS ultra-performance liquid chromatography time of flight mass spectrometry ALB Haliaeetus albicilla, white-tailed sea eagle, merikotkas ALU Strix aluco, tawny owl, kodukakk BUB Bubo bubo, eagle owl, kassikakk BUT Buteo buteo, common buzzard, hiireviu CHR Aquila chrysaetus, golden eagle, kaljukotkas FLA Asio flammeus, short-eared owl, sooräts GEN Accipiter gentilis, goshawk, kanakull NIS Accipiter nisus, sparrow hawk, raudkull POM Clanga pomarina, lesser spotted eagle, väike-konnakotkas TIN Falco tinnuculus, common kestrel, tuuletallaja URA Strix uralensis, Ural owl, händkakk 7

8 2. INTRODUCTION Rodent infestations in urban and rural settings are usually controlled with anticoagulant rodenticides. These have been for sale in the markets for the last 70 years (Hadler, Buckle 1992), but their impact on the ecological system has only been studied in the last years (Nakayama et al. 2019), and it still not a widely known issue for the common consumer. Only in the recent 10 years, countries all over the world have eagerly started to investigate the possible causes of the near extinction of the most majestic creatures living in our skies, the birds of prey, by anticoagulant rodenticides (Albert et al. 2010, Christensen et al. 2012, Hughes et al. 2013, Koivisto et al. 2018, Lohr et al. 2018, Walker et al. 2017). None of these studies have been conducted in Estonia nor in other Baltic States. While most of the birds of prey living in Estonia are under the species protection category (I ja II kaitsekategooriana kaitse alla võetavate liikide loetelu 2004, 4, section 2, 8 section 2, III kaitsekategooria liikide kaitse alla võtmine 2014, 4, section 2) their nesting numbers are still decreasing (Elts et al. 2013). The impact of and intoxication by anticoagulant rodenticides has been studied in different wild vertebrates, birds (Albert et al. 2010, Christensen et al. 2012, Hughes et al. 2013, Koivisto et al. 2018, Lohr et al. 2018, Walker et al. 2017), mammals (Geduhn et al. 2015, Koivisto et al. 2018, Elliott et al. 2013), fish (Kotthoff et al. 2018) and reptiles (Lohr, Davis 2018). Detecting and quantifying residues of rodenticides is challenging, as the substance concentrations are often lower than the detectable limits. Moreover, the concentrations acquired from eating an affected rodent are too low for a lethal outcome, hence causing cumulative and chronic effects on the organism. For the animals to end up in a wildlife hospital, they need to be alive when found, while the diseased ones are usually consumed by nature, and therefore, cannot be tested. This study looks at the broad picture of anticoagulant rodenticides in birds of prey from different countries from Europe and elsewhere, and the local aspects of intoxication in 11 species of Estonian birds of prey. The study was conducted to estimate the occurrence and exposure of the second generation anticoagulant rodenticide poisoning in birds of prey, to assess the need for further studies on this topic, and the possibilities of detecting biocide liver values using available laboratory capacity of Estonian University of Life Sciences. 8

9 3. ACKNOWLEDGEMENTS Thank you, Madis Leivits (DVM) for giving me the opportunity to study and present this important topic and for collecting all the samples throughout the years. Thank you, Tõnu Püssa (PhD) for helping me in understanding liquid chromatography and its possibilities. Thank you, Dea Anton (DVM) for helping me with sample preparation techniques. Thank you, Linda Rusalepp (MSc) for the collaboration, sample analysis in chromatography and initial calculations. Thank you Tanel Kaart, (PhD) for helping with statistical analysis. Thank you, Chair of Food Hygiene and Veterinary Public Health of Estonian University of Life Sciences for allowing me to use your laboratory and its machines. Thank you, all of my co-students with whom I have been honoured to study and graduate together. 9

10 1. LITERATURE ANALYSIS 1.1. Anticoagulant rodenticides History of anticoagulant rodenticides The first rodenticides were concentrated for the treatment of thrombosis, one of which was dicoumarin. In an attempt to increase the potency of the drug, the workers at the Wisconsin Alumni Research Foundation finally arrived to the most active compound, warfarin. It was registered as a rodenticide for sale in the USA in 1950 (Hadler, Buckle 1992). In recent years, it has been estimated that rodents have a global impact as high as $50 billion annually, meaning there is a worldwide need to control rodent pests from spreading diseases and destructing infrastructure (Elliott et al. 2016) Use of anticoagulant rodenticides Nowadays worldwide rodent pest control relies largely on the use of anticoagulant rodenticides (ARs) (Stone et al. 2003). Baits are dropped near households and crop fields, to attract rodents to consume these, which causes them to be intoxicated to death. When weakened, rodents become food more easily for predators, who in turn become intoxicated (Figure 1). 10

11 Figure 1. Exposure patterns for ARs. Many smaller non-targeted species can potentially consume the original product, and the exposure to predators can be secondary or even tertiary. The bold arrows indicate the most probable routes of transport. Blue colour (square) indicates the first level of nontarget species, who have access to bait (brown oval). Violet colour (hexagon) indicates the primary poisoning of the targeted animals. Green colour (oval) indicates secondary poisoning by consuming intoxicated animals. Source: Elliott et al. 2016: 402. Ironically, humans are unknowingly killing the predators, who usually would hunt for these pests (Elliott et al. 2016). The ARs are easily absorbed through ingestion in the gastrointestinal tract, and diminishing synthesis of the vitamin K1-dependent clotting factors (II, VII, IX, X) by inhibiting hepatic vitamin K1 epoxide reductase. The symptoms of anticoagulant poisoning start with mild blood loss that causes anaemia, pale mucous membranes, weakness, and tachycardia, eventually leading to severe haemorrhage with poor coagulation. External bleeding may be noted, but internal bleeding is more common. (Vandenbroucke et al. 2008). The central component of haemostasis is blood coagulation. When investigating coagulation at a cellular level, it is initiated through an extrinsic pathway, called tissue factor pathway. The generation of tissue factor, complexed with carboxylated factor VII, activates factor X mostly 11

12 in the common pathway, and in some degree, in the intrinsic pathway, called the contact activation pathway, where factor IX is activated. In several avian species, the factors XI and XII of the intrinsic pathways are missing. A number of reactions are needed for prothrombin to activate and form thrombin. Measuring clotting time of citrated plasma has been used as a routine diagnostic tool for anticoagulant intoxication in people, while its application to diagnose rodenticide poisoning in captive and free-ranging wildlife is rare. Clotting time assays, being precise and inexpensive, considering its sensitivity and linking to pathogenesis of toxicity, are applicable as biomarkers of exposure. In wild animals, the prolongation of prothrombin time by more than 25% is suggestive of anticoagulant exposure, connecting it with analytical detection of rodenticides in blood or tissue, intoxication can be confirmed (Rattner et al. 2014). The mortality of pests occurs several days after bait consumption, which makes them particularly effective against neophobic species, such as the Norway rat (Rattus norvegicus). Because of the rapid use of ARs, stains of resistance in the Norway rats, roof rats (Rattus rattus) and house mice (Mus musculus and Mus domesticus) has been noted (Langford et al. 2013) Classification ARs are divided into two generations. First generation anticoagulant rodenticides (FGARs) include warfarin, chlorophacinone, coumatetralyl, and diphacinone, they require several days and multiple feedings to be fully active. After a short period of being on the market, rodents started showing resistance to FGARs, and thus came the need for more potent ARs (Langford et al. 2013, Nakayama et al. 2019). Second generation anticoagulant rodenticides (SGARs) are more potent and therefore need only one feeding to have a lethal action (Berny et al. 2014). SGARs are derivates of 4-hydroxycoumarins, including bromadiolone, difenacoum, brodifacoum, flocoumafen and diphethialone (Langford et al. 2013, Nakayama et al. 2019, Figure 2). 12

13 Figure 2. Chemical structure of nine typical ARs (A to I) and vitamin K (J). FGARs are represented by warfarin (A), coumatetralyl (B), diphacinone (C), and chlorophacinone (D). SGARs are represented by brodifacoum (E), bromadiolone (F), difenacoum (G), difethialone (H), and flocoumafen (I). The main structure of AR is similar to that of vitamin K (J). Source: Nakayama et al. 2019: 299. There have been descriptions of some resistance to two of the less potent SGARs, bromadiolone and difenacoum, while there is no field evidence of resistance to the three other SGARs (Berny et al. 2014) Effect of anticoagulant rodenticides The solubility and volatility of anticoagulant rodenticides in water is low. For the firstgeneration anticoagulant rodenticides and bromadiolone, the octanol-water partition 13

14 coefficients (log KW), are less than 5, and thus the bioaccumulation potential is moderate to low. However, for SGARs the log KOW ranges from 5.17 to 8.5 for difethialone, difenacoum and brodifacoum, meaning the bioaccumulation of these products exhibit greater potential (Rattner et al. 2014). Once ingested, the clotting factors are lost by normal attrition over a day or few days, depending on the AR. Animals will be vulnerable to fatal haemorrhage precipitated by minor trauma, exertion, and possibly other factors. Single exposures of high amount of ARs might be lethal and multiple sub lethal doses may produce accumulation of toxins, resulting in a fatal chronic dose (Stone et al. 2003). Within a few hours of administering brodifacoum, animals might develop a dose-dependent transient haemoglobinuria, which will resolve in a day. Afterwards the animal will develop true haematuria, associated with anticoagulation (Feinstein et al. 2016). Based on structure-activity relationship models, the toxicity of rodenticides is related to the length and hydrophobicity of the side chain at carbon 13, with the less active compounds having less volume and more active compounds having greater volume and large lipophilic groups (Rattner et al. 2014). Lethal dose of different anticoagulant rodenticides has a wide variation in the literature due to different testing methods. The biggest variations occur when the test has been carried out using single dose as opposed to repeated (5-day) doses. LD50 doses depend on the animal, species, strain, and sex. For mice, the reported oral LD50 dose is 1 mg/kg body weight for SGAR, to 374 mg/kg body weight for warfarin. For warfarin, the bait needs to be ingested multiple times over several days before any clinical signs occur (Vandenbroucke et al. 2008). The toxic dose of brodifacoum or difenacoum for humans is 1 mg for an adult, meaning that an adult must eat 20g of SGAR pellets with a concentration of 0.005% (Caravati et al. 2007). It might also be possible that the exposure to rodenticides changes the behaviour of rats by making them more susceptible to become live prey to diurnal predators, for an example, kestrels. In a clinical trial, rats, who were fed with anticoagulant rodenticides, spent more time outdoors in the daytime, than normal rats. Rats showed more motion in the daytime by moving out in the open, rather than in contact with a wall. All rats in the study, who were about to die, emerged from their house box, and went into an open area (Cox, Smith 1992). This suggests that poisoned rats are more susceptible to diurnal predators by being less cautious, thus increasing the risk of secondary poisoning. 14

15 Relation to bone metabolism In bone metabolism, there is an important role for vitamin K as being required for the formulation of γ-carboxyglutamyl, a component of bone proteins such as osteocalcin (Knopper et al. 2007). Human studies have shown that bone density, osteoporosis, and the frequency of bone fractures are all linked to anticoagulant therapy and low dietary vitamin K intake (Barnes et al. 2005). However, a study conducted by Knopper et al. (2007) on 28 barn owls and 20 kestrels showed that bone strength and density did not correlate with the amount of SGARs residues found in the liver. They analysed 48 samples of liver, humerus and femur bones from birds found dead in the forest. However, they did find that age and sex of the birds are responsible for bone density. Considering: the magnitude of trace amounts found in the liver, timing, and duration from ingestion to death; it might have been that the long-term effect of rodenticides had not yet shown up in the bones. None of the examined birds had died because of haemorrhage, but rather from collision with a vehicle or starvation. It might be possible, that once the exposure to SGARs wears off, the normal levels of vitamin K are restored (Knopper et al. 2007) Treatment of anticoagulant rodenticide poisoning The antidote for these anticoagulants is vitamin K1 (phytomenadione) and it is used to treat humans, companion animals, and, occasionally, wildlife (Vandenbroucke et al. 2008). The administration results in the formation of the vitamin K hydroquinone by DT-diaphorase, which is an enzyme resistant to anticoagulant rodenticides, thus restoring carboxylation of clotting factors (Rattner et al. 2014). Depending on the dose and kinetics of the specific AR involved, the length of vitamin K1 treatment can vary. The type of anticoagulant rodenticide, with the clinical and pathological signs of intoxication, might be required for diagnosis especially in case of malicious poisonings of companion animals. Because the residues of rodenticides in casualties are very low in the tissue, especially in blood, sensitive analytical techniques are required for the chemical diagnosis of poisoning. For identifying the rodenticide responsible for poisoning, liver is the most suitable tissue, as it has the highest accumulation of toxins in unchanged form. Liver is commonly used because of its ability to accumulate anticoagulants for days and even weeks (Vandenbroucke et al. 2008). Although the haemorrhagic syndrome associated with vitamin K deficiency in chickens and warfarin 15

16 resistance in rats has been studied in length, the role of vitamin K in raptor species has not yet been evaluated. Most studies have been focused on the signs of toxicity and mortality of ARs, but rarely on measuring blood-clotting factors (Rattner et al. 2014). Feinstein et al. (2016), reviewing studies, conducted and observed on humans, found that combining K1 supplementation with oral binding resin (e.g., cholestyramine) can capture the ARs from the intestines and increase faecal excretion right after the ingestion, therefore decreasing the amount of ARs absorbed. They also found that administering of early intravenous infusion of lipid emulsion would disperse the ARs to skeletal muscles and liver, rather than major critical organs such as the brain, kidneys, and heart. With that, it would minimize the systemic effects, and accelerate its excretion. Ware et al. (2015) tested co-treatment with antioxidant N- acetylcysteine, which fully prevented the brodifacoum induced early haemoglobinuria. The half-life of anticoagulants is usually determined after single oral dosage. The highest concentration of difenacoum in mice were established in the liver at 24h after ingestion, the half-life of difenacoum in the liver is 118 days. For warfarin, the half-life reported in a rat s liver is 7-10 days, and for second generation anticoagulant rodenticides half-life exceeds 100 days. The half-life of brodifacoum in dogs is about 3 days in plasma (Vandenbroucke et al. 2008). The significance of sub-lethal doses of ARs and other environmental contaminants is a longstanding issue. Predatory wildlife exposed to contaminants can either die of acute poisoning or survive with seemingly low visible signs, with no known long-term effect to the organism. However, it is possible that ARs trigger a cascade in which the animal gets ill in the long run. The animal can be hit by a moving vehicle without any signs of coagulopathy or previous AR intoxication, but still have a moderate degree of AR poisoning. In a study by Maureen Murray conducted in the years in her Wildlife Clinic in Massachusetts, USA, no significant connection was found in the amount of brodifacoum in liver and signs of toxicosis or the cause of death. The reason for death or euthanasia of these birds was mostly a consequence of trauma (Murray 2011) Recent studies In recent years there have been several studies conducted in Europe to monitor the second generation anticoagulant rodenticide exposure to non-target predatory species from mammals 16

17 to birds (Christensen et al. 2012, Hughes et al. 2013, Koivisto et al. 2018, Walker et al. 2017). Non-target animals consume anticoagulant rodenticides by direct consumption of baits (primary poisoning) or by consuming contaminated animals (secondary poisoning) (Koivisto et al. 2018) Species studied Koivisto et al. (2018), among mammalian species, studied birds like eagle owl (Bubo bubo), goshawk (Accipiter gentilis), hooded crow (Corvus cornix), hen harrier (Circus cyaneus), Eurasian magpie (Pica pica), sparrow hawk (Accipiter nisus), white-tailed sea eagle (Haliaeetus albicilla), and tawny owl (Strix aluco). Hughes et al. (2013) studied raptors like red kite (Milvus milvus), buzzard (Buteo buteo), common kestrel (Falco tinnunculus), barn owl (Tyto alba), tawny owl, sparrow hawk, peregrine falcon (Falco peregrinus). Langford et al. (2013) also studied osprey (Pandion haliaetus) and gyrfalcon (Falco rusticolus). Christensen et al. (2012) studied exposure rates also in little owl (Athene noctua), long-eared owl (Asio otus), Marsh harrier (Circus aeruginosus), rough-legged buzzard (Buteo lagopus) and shorteared owl (Asio flammeus) Situation in Denmark In Denmark, municipalities and landowners are obligated to keep rat infestations under control. The preferred method is using second generation anticoagulant rodenticides. They come in contained bait boxes or rodent holts outside the building to minimize the risk of primary poisoning of non-target species, and people. The AR products that are used for mice and vole eradication are also available to private homeowners. During the past years, AR sales have been going down but the amount of SGARs used has remained the same. Christensen et al. (2012) conducted a study on 430 raptors and owls combined, found from all over Denmark, in Europe. They found that 92% of samples tested had AR concentration higher than the limit of detection (LOD), from 84% to 100% within species, difenacoum, bromadiolone, and brodifacoum being the most prevalent substance. In the majority of the birds, the mean value of ARs was low, but in six core species, the summed AR concentration per liver sample was more than 100 ng/g, which may cause haemorrhages and other clinical signs in predatory birds. 17

18 Potentially lethal concentration of ARs over 200 ng/g wet weight of liver were detected in more than 13% of kestrels and barn owls. The highest concentrations were determined in scavengers, red kite and eagle owl, who prey mostly on rats and other birds who habit in agricultural areas. This indicates a very high level of general exposure of anticoagulant rodenticides in farm areas. Species living in more natural habitats, further away from humans, did not have critical levels of AR concentrations in their liver Situation in Scotland Hughes et al. (2013) wrote a study to analyse rodenticide residues in non-target birds of prey in Scotland. They concluded that red kites had significantly higher exposure to SGARs than common buzzards because of their dietary differences. Red kites have an opportunistic diet, which contains carrions and rodents from near farm buildings. On the other hand, buzzards, while still consuming carrion and rodents, have a broader range in diet from also feeding on insects and other birds. The exposure for kestrels, barn owls, and tawny owls were lower, although they mostly prey on live rodents. An interesting finding was that sparrow hawks and peregrine falcons, whose diet consists almost exclusively of other birds, had exposure rates of 37% and 24% respectively, meaning birds may provide secondary exposure routes of ARs as well as the target species, rodents Situation in Scandinavia From a study conducted in Norway, it was discovered that most of the deceased birds found were near densely populated areas with higher concentrations of rodenticides. This might be because there are more people using AR baits. It cannot be ruled out that in more densely populated areas, the probability of finding a dead bird is higher than in deep forests or higher mountains. In eagle owls, 67% of samples had residues of SGARs, but none was detected from osprey, peregrine falcon or gyrfalcon, which might be due to their feeding habits. Ospreys feed almost exclusively on fish, gyrfalcons and peregrine falcons eat mostly other birds and, only occasionally, small mammals. It was also stated that there is a significant species difference in the lethal sensitivities to ARs and in some species, there might be a significant poisoning in concentrations below 100 ng/g in liver wet weight (Langford et al. 2013). 18

19 The exposure of anticoagulant rodenticides to non-target predators and scavengers, including mammals and birds, was studied in Finland. As a result, from the 131 samples tested in 17 mammalian and avian species, 82% had detectable residues of ARs (Koivisto et al. 2018) About Europe It seems that using ARs is a problem with a broad range, since studies have been conducted in all over Europe (Christensen et al. 2012, Hughes et al. 2013, Koivisto et al. 2018, Walker et al. 2008, Walker et al. 2017). Receiving different results in exposure rates might be because of the slightly different nature of these studies, whether the animals were mostly found dead or they were to come to a wildlife clinic as patients with different symptoms. The difference can also be dependent on how intensively ARs are being used in the subject country and whether they have any kind of restrictions or regulations. For an example, the United Kingdom has been compelled to compile a Predatory Birds Monitoring Scheme (PBMS) and the Wildlife Incident Monitoring Scheme to analyse the risks of using rodenticides. European Union compiled a Risk Mitigating Measures for Anticoagulant Rodenticides as Biocidal Products that is made by an expert group to set suggestions and recommendations for Risk Mitigating Measures (Berny et al. 2014) Situation in Estonia Estonian listing of protected species of birds of prey under the species protection category I are white-tailed sea eagle (Haliaeetus albicilla), short-toed snake eagle (Circaetus gallicus), lesser spotted eagle (Aquila pomarina), greater spotted eagle (Aquila clanga), golden eagle (Aquila chrysaetos), osprey (Pandion haliaetus), merlin (Falco columbarius), peregrine falcon (Falco peregrinus), eagle owl (Bubo bubo), and great grey owl (Strix nebulosa). The species under the protection category II are goshawk (Accipiter gentilis), short-eared owl (Asio flammeus), and boreal owl (Aegolius funereus) (I ja II kaitsekategooriana kaitse alla võetavate liikide loetelu 2004, 4, section 2, 8 section 2). Under the species protection category III are European honey buzzard (Pernis apivorus), black kite (Milvus migrans), marsh harrier (Circus aeruginosus), hen harrier (Circus cyaneus), Montagu s harrier (Circus pygargus), sparrow 19

20 hawk (Accipiter nisus), common buzzard (Buteo buteo), rough legged buzzard (Buteo lagopus), common kestrel (Falco tinnunculus), red-footed falcon (Falco vespertinus), Eurasian hobby (Falco subbuteo), snowy owl (Bubo scandiacus), northern hawk-owl (Surnnia ulula), Eurasian pygmy owl (Glaucidium passerinum), tawny owl (Strix aluco), and Ural owl (Strix uralensis) (III kaitsekategooria liikide kaitse alla võtmine 2014, 4, section 2). All of the birds of prey nesting in Estonia, except the long-eared owl and red kite, are under the species protection regulation, and therefore, it is important to investigate their causes of morbidity and mortality to sustain and protect viable populations and their welfare. Many of the species studied in the aforementioned papers, are the same as the ones under the protection regulations in Estonia. It is possible that the same birds, who migrate alongside with the ones living in our neighbouring countries, are impacted with the residues of anticoagulant rodenticides detected in other studies. In Estonia, it is allowed to use only four active ingredients of anticoagulant rodenticides: brodifacoum, difenacoum, bromadiolone, diphethialone, while there are 35 registered products allowed in the markets for regular users, pest control, and professional users (Eestis väljastatud biotsiidide load seisuga ). Unfortunately, there is no data available for the Estonian government of how much ARs has been sold in recent years Detection of residues Possible methods Sample preparation techniques The determination of anticoagulant rodenticide residues from biological samples is not something routinely done on animal or human patients, and therefore, there is not one correct way of analysing AR residues. Imran et al. (2015) has reviewed different methods used in various papers on ARs. The sample testing consists of two phases: sample preparation and analytical method. For ante mortem, analysis blood is the specimen of choice, since it is easily available. However, the half-life of brodifacoum in a dog s plasma is 6 days while the onset of clinical symptoms is 36h after ingestion. Therefore, it is not the best method for evaluating the intensity of exposure. Liver contains the highest concentration of the rodenticides, which gives the most accurate evaluation on the extent of intoxication, but the liver is not the preferred 20

21 tissue in a living animal from which a surgeon would like to take a biopsy. Sample preparation is one of the most important steps in the methodology. It has been reported that about 30% of errors produced in the analysis is in the preparation. The phases of biological sample extraction are usually either liquid-liquid, solid phase, or a combination of those two. Liquid-liquid extraction (LLE) is done by using organic solvents. One method is to add acetonitrile to the homogenized sample, vortex and centrifuge the sample, then remove the organic layer, add diethylether and mix. The layers should be allowed to separate on standing and then the ether layer should be transferred to another tube, evaporated to dryness with nitrogen, and reconstituted with methanol. This method has a recovery rate of 68-98%. The best recovery rate (92-109%) with LLE was reported to be when using acetonitrile ethyl ether (9:1) in plasma and liver tissue samples. Solid phase extraction (SPE) is useful in the manner that many problems encountered in LLE can be avoided and there is not so large consumption of organic solvents. SPE methods have produced 75-95% of recovery from blood samples, but these are time consuming. The most common SPE sorbent is C18 column. For analysis of pesticide residues in biological samples, dispersive solid-phase extraction (dspe) has been developed. This method is quick, easy, cheap, effective, rugged, and safe (QuEChERS). To overcome LLE and SPE issues, hollow fibre liquid phase microextraction (HF-LPME) has been developed. Depending upon the number of liquid phases, there are two modes of HF-LPME: three- and two-phase HF-LPME. Three-phase mode for extraction of warfarin is being used for human plasma. The advantages are simplicity, insignificant volume of solvents used, high enrichment, inexpensiveness, and excellent sample clean-up ability. The hollow fibres are cheap and therefore can be used once and then disposed. For human plasma, for the extraction of warfarin, a low-density solvent-based dispersive liquid-liquid microextraction (LDS-DLLME), has also been developed that has produced an excellent recovery of 91%, compared to HF-LPME, which gave only 61.4%. Although these are inexpensive and easy to use methods, there has not been any studies using these in order to detect other anticoagulant rodenticides Analytical methods For analytical methods there are studies using high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS) or in tandem LC-MS/MS, gas chromatography-mass spectrometry (GC-MS), ion chromatography (IC). For hydrocoumarin 21

22 detection using HPLC, gradient elution is mostly used. High performance liquid chromatography ultraviolet spectrometry (HPLC-UV) has been mostly used for the detection of SGARs from blood, plasma, and liver. LC-MS has shown good sensitivity and relatively good recovery rates, it has also been the most commonly used method when combined with UV or fluorescence detection (FLD). Taylor et al. (2008) compared UPLC-MS/MS (ultraperformance tandem mass spectrometry) method with UPLC-TOFMS (ultra-performance liquid chromatography time of flight mass spectrometry) on multi-residue detection of pesticides from strawberries and received favourably comparable results Common methods used Studies conducted in the last 5 years have all used LC-MS with variations and with different sample preparations. Ruiz-Suárez et al. (2014) used LLE for sample preparation and heated electrospray ionization liquid chromatography tandem mass spectrometry (H-ESI-LC- MS/MS), the average recovery ranged from 89.2% to 97.4%. Walker et al. (2008) used SPE and quantified the samples with LC-MS/MS. They received recovery of 60-70%. Lopez-Perea et al. (2015) used homogenization, followed by evaporation, dilution, SPE column and another evaporation and dilution. After that, they used LC-ESI-MS for sample analysis, gaining recovery values of over 70%. Koivisto et al. (2018) used LLE with evaporation and analysed the samples with an ultra-high-performance liquid chromatography mass spectrometry (UHPLC-MS). Their received recovery was too low in comparison to pure standards and as a result, matrix added calibration curves were made for each sample set. Geduhn et al. (2016) collected barn owl regurgitated pellets from their nests and tested these using solid supported liquid extraction and evaporation with SPE and for quantitation UHPLC-MS. They received recovery of %. Lohr (2018) used QuEChERS method for extraction and for quantitation LC-MS/MS with ultra-performance liquid chromatography charged surface hybrid (UPLC CSH) C18 column. They received recovery of %. 22

23 2. AIMS OF THE STUDY The aims of this study was to investigate: the capability of monitoring anticoagulant rodenticides in the laboratory of Estonian University of Life Sciences; the occurrence of second generation anticoagulants rodenticides (SGAR) in Estonian prey birds; the possibility of intoxication and possible relations to interspecies correlation; the further need of SGAR monitoring in Estonia. 23

24 3. MATERIALS AND METHODS 3.1. Sample collection Analysed liver samples were collected from dead birds. The birds were brought to Estonian University of Life Sciences Wild Animal Clinic either dead on arrival or with injuries and illnesses unsuitable for release, thus being euthanized on site. All the birds were necropsied afterwards and livers were collected, packed individually with identification number and refrigerated at -20 C. All the samples were collected between 2013 and During the necropsy, no notes were recorded regarding the cause of death. For this study, 11 different species of birds of prey were used, all being native to Estonian wildlife. The birds collected that were under the first species protection category are white-tailed sea eagle (Haliaeetus albicilla, ALB, n = 4 samples), golden eagle (Aquila chrysaetus, CHR, n = 1), lesser spotted eagle (Clanga pomarina, POM, n = 2) and eagle owl (Bubo bubo, BUB, n = 3). Species under the second protection category are goshawk (Anccipiter gentilis, GEN, n = 9), common buzzard (Buteo buteo, BUT, n = 14) and shot eared owl (Asio flammeus, FLA, n = 2). Species under the third category are sparrow hawk (Accipiter nisus, NIS, n = 2), tawny owl (Strix aluco, ALU, n = 9), common kestrel (Falco tinnuculus, TIN, n = 3) and Ural owl (Stix uralensis, URA, n = 4). Some samples in storage were only identifiable by species, but not with individual number so they could not be traced back to their medical history. Simplifying the identification in the laboratory, the used samples got a new ID, being the first three letters of their species epithet and a number in which order they were sampled. For example, the first sample of goshawk, Accipiter gentilis, has a sample ID of GEN Species overview These aforementioned species are widely distributed across Estonia. The species in question are on the lowest level of threat in the IUCN Red List of Threatened Species 2015 as a least concerned (BirdLife International 2015). By the data collected by Elts et al. (2013), it has been noted that all of the studied 11 species are of natural origin and they have been regularly sighted 24

25 between the years 1950 and White-tailed sea eagle is a regular breeder, some of them are passage migrants and even spending the winters here. The number of breeding pairs is around 230 and has been increasing in recent years (Elts et al. 2013). They nest in preferably old pine or mixed forests, away from human activity, although they have been starting to nest near old farms (Nellis, Volke 2018). The number of Goshawk, common kestrel and lesser spotted eagle pairs, who nest in this area, spend the winter and also passage, is around The number of goshawks has been decreasing slightly, while lesser spotted eagles number has been the same, and kestrels have been increasing slightly (Elts et al. 2013). Goshawks live in large old conifer or mixed forest but they can be seen to hunt in cultural landscapes and near urban areas (Väli 2018b). Common kestrels prefer nesting near open cultural landscapes, forest edges, or even in old houses (Tuule, E., Tuule, A. 2018). Lesser spotted eagles live on the mainland, away from sea shores, but loves rivers and large forests (Väli 2018a). A few thousand pairs of sparrow hawks and buzzards nest and winter in Estonia, with the number being similar throughout the years (Elts et al. 2013). Sparrow hawks live mostly in young dense spruce forests and can be seen near urban areas (Jair, Väli 2018a). Buzzards nest in forests that are bordered with a large enough open field. They are specialized in eating small rodents (Jair, Väli 2018b). For tawny owl and Ural owl, the number of nests is around 1000, and they winter frequently. The numbers have stayed about the same (Elts et al. 2013). Tawny owls live mostly in cultural landscapes; they are not afraid of human activity, and often live in city parks, being very loyal to their nesting places (Lelov 2018). Ural owls nest in older conifer and mixed forests, where there are tree cavities (Jair 2018). There are three species, whose nesting pair count is below 100: eagle owl, golden eagle, and short-eared owl, in decreasing order. Only the numbers of golden eagles have been slowly increasing. They all nest and winter in the area (Elts et al. 2013). Golden eagles live near large wild bogs (Sein 2018). Eagle owls nest in conifer forests, away from urban areas (Nellis 2018). Short-eared owls tend to live near open fields, bogs, and they eat mostly small rodents (Tammekänd 2018) Sample preparation Liver samples from birds were homogenised in a mortar with a pestle, scalpel, and a pair of scissors, then weighed (with Ohaus analytical Standard) by 1.00 grams into a 20 ml sample tube and 4 ml of 98% methanol solution was added. The samples were vortexed (Heidorph 25

26 REAX top) vertically for 15 seconds, and put into a rotator mixer (Biosan Multi RS-60 vortex) for 20 minutes. After that, the samples were held in an ultrasonic bath (Cole-Palmer 8891) for 10 minutes, and then centrifuged at (Eppendorf Centrifuge 5810 R) 20 C 4000 rpm for 10 min. The liquid supernatant was poured to a new clean tube, washed twice with 2 ml of hexane to discard the lipids. The extract was pipetted into SPE C18 column (Agilent SampliQ C18 column) to remove traces of hydrophobic compounds. The column had been previously primed with 100 µl of methanol. The samples were eluted into glass vials that were sealed and labelled with the sample ID, date, sample number, and species. In every patch there were a total of liver samples tested, including two standards Preparation of standard curves and spiking solutions Separately from every studied rodenticide, solution with concentration of 1 mg/ml was prepared, 1 ml of every solution was pooled, and 6 ml of methanol added to receive a mixed solution with a concentration of 100 µg/ml of every compound. This solution was diluted 1000 times to get a solution of 0.1 µg/ml. For the construction of calibration curve, further solutions with concentrations 0.05, 0.025, and µg/ml, were prepared. In every analysis set, there were one blank sample and one spiked sample. For blank and spiked samples, chicken (Gallus gallus domesticus) livers were used that were obtained from refrigerated packages meant for human consumption. 1 ml of 0.01 µg/ml of 0.001% mixed rodenticide methanol solution was added to 1 g of chicken liver and left to stay for at least 15 minutes. Afterwards, 3 ml of methanol was added to that sample, at the same time as to the other samples. After adding methanol to all the 12 samples, the samples were homogenized and processed as analytical samples (see 2.4.). The analytical standards were Bromadiolon PESTANAL 100 mg, Difenacoum PESTANAL 25 mg, Warfarin PESTANAL 250 mg, Brodifacoum PESTANAL 100 mg, made by Merck KGaA, Darmstadt, Germany Analytical method The liquid chromatographic analysis of extracts were carried out by a 1290 Infinity system (Agilent Technologies, Waldbronn, Germany) coupled to an Agilent 6450 Q-TOF mass 26

27 spectrometer equipped with a Jetstream ESI source with a combination name of high performance liquid chromatography (Agilent Jet Stream Electrospray Ionisation) quadrupole time of flight tandem mass spectrometry (HPLC-(AJS ESI)-qTOF-MS/MS). Samples were subjected to Eclipse Plus C18 RRHD column ( mm; particle size 1.8 µm, Agilent Technologies) kept at 40 C. For the elution of the samples a gradient of 0.1% of formic acid in water (A) and acetonitrile (B) was used as follows: 0.0 min 40% B, 10.0 min 100% B, 15.0 min 100% B, 15.1 min 40% B, regeneration time 7 min. The eluent flow rate was set to 0.3 ml/min and the injection size was 5 µl. Mass-spectrometer was working in negative ionization mode in the mass to charge ratio (m/z) range of amu. Data acquisition and initial data processing was carried out by MassHunter software (Agilent Technologies) Statistical analysis Box plots and violin plots were used to visualize the empirical distributions on studied rodenticides. As all concentrations were non-normally distributed (right skewed) the median and inter quartile range were used to describe the most common locations of concentrations. The Kruskal-Wallis test was applied to study the differences of rodenticides concentrations between species, behavioural groups and protection categories. The chi-square test was applied to compare the number of compounds over limit of detection and the number of compounds over limit of quantitation between species, behavioural groups and protection categories. The Spearman correlation analysis was performed to study the relationships between concentrations of different rodenticides. All results were considered statistically significant at p 0.05, statistical analyses were performed, and figures were constructed with R version (R Foundation for Statistical Computing, Vienna, Austria). 27

28 4. RESULTS With the LC-MS method, the chromatograph measures and calculates, the retention times of compound spikes and molecular masses (Table 1). Table 1. Retention times (tr (min)) and mass to charge ratio (m/z) with negatively charged form of molecule ([M-H] - ) of ARs found on chromatogram. Bromadiolone (BDL) has two isotopologues, difenacoum (DFN) has two isomers and brodifacoum (BDF) has two isomers, which both have two isotopologues t R (min) m/z [M-H] - Name warfarin BDL (79Br isotopologue) BDL (81Br isotopologue) cis-dfn trans-dfn cis-bdf (79Br isotopologue) cis-bdf (81Br isotopologue) trans-bdf (79Br isotopologue) trans-bdf (81Br isotopologue) Bromadiolone (BDL) has two bromine (Br) isotopologues, therefore two different molecular masses have separate retention times in a chromatogram. Difenacoum (DFN) has two isomers, cis and trans, but the same molecular weight, and only a slightly different molecular shape. Brodifacoum (BDF) has cis and trans isomers, and both have two brome isotopologues. Isomers were summed, but the different isotopologues were calculated by mean values for the calibration curve. The quantity of any given substance was derived from the surface area of its spike (Figure 3). 28

29 Figure 3. Chromatogram of a spiked chicken liver. The peaks of warfarin, brodifacoum (BDF), difenacoum (DFN), and bromadiolone (BDL) on retention time scale. The areas for isomers and isotopologues were measured separately. LOD was defined as 3 times the standard error of the mean by measuring the surface area of a calibration sample 4 times, calculating standard deviation and dividing it by square root of repetitions (sdev/sqrt(n)). LOQ (limit of quantitation) was defined as 10 times the standard error of the mean. LOD is the smallest amount of analyte that can be detected from the sample that is distinguishable from zero, whereas concentrations below LOD cannot be stated as absent or zero (Evard et al. 2016). LOD values for ARs were 2.1 ng/g for warfarin, 2.7 ng/g for BDL, 5.9 ng/g for DFN and 12.1 ng/g for BDF. LOQ values were 6.9 ng/g warfarin, 9.1 ng/g BDL, 19.6 ng/g DFN and 40.3 ng/g for BDF. As an example, the sample GEN3 had no detectable liver values of warfarin (0.0 ng/g), BDF was with and acquisition time of min and the concentration was ng/g, DFN had acquisition time of min and concentration ng/g, BDL had acquisition time of min and concentration ng/g (Figure 4). Concentrations have been corrected with calibration curves. 29

30 Figure 4. Chromatogram view of the sample GEN3. Brodifacoum (BDF), difenacoum (DFN), and bromadiolone (BDL) spikes with total area coloured. Anticoagulant residues were detected (over LOD) in 70% of the 63 samples analysed and in 100% of the species. From the positive samples where residues were detected, 27% (n = 12) had residues of more than one substance, and only one sample had residues over LOD of all three SGARs. Of all the samples, 49% (n = 31) had concentrations over LOQ. Bromadiolone was the most common SGAR detected, making up 89% from the positive samples, followed by brodifacoum (34%) and difenacoum (7%). None of the samples had a significant amount of warfarin over LOD (2.1 ng/g). The total liver AR concentration over possibly lethal threshold level 100 ng/g was discovered in 10% (n = 6) of samples. The highest result total AR concentration was in sample URA4 (Strix uralensis, Ural owl) measuring 138 ng/g from which 132 ng/g contained of bromadiolone. The distribution of studied rodenticides concentrations is presented in Figure 5. 30

31 Figure 5. Distribution of studied rodenticides' concentrations. The distributions are presented in form of violin plots, grey squares in background denote the inter quartile range (IQR), small horizontal lines devote single birds and strong horizontal lines denote medians; for better visualization the y-axis is presented in logarithmic scale. For each rodenticide also median, IQR and maximum concentration are presented numerically above the plot; red lines with numerical values denote limit of detection (LOD) and limit of quantitation (LOQ). The concentrations of each AR found in samples are presented in Figure 6. It shows that BDL and BDF have the highest concentrations while warfarin is entirely absent and only three samples had detectable and quantifiable residues of DFN. 31

32 Figure 6. Number and proportion of samples with warfarin (A), bromadiolone (B), difenacoum (C) or brodifacoum (D) concentration below limit of detection (<LOD), below limit of quantitation (< LOQ), and over limit of quantitation (> LOQ); LOD and LOQ values of different rodenticides are presented on Figure 5. The total AR concentrations were compared by grouping samples by their species. Kruskal- Wallis test showed no significant difference (p = 0.990; Figure 7). This result did not change when considering only six most frequent species with four or more samples the difference remained statistically non-significant. 32

33 Figure 7. Distribution of total anticoagulant rodenticides' concentration by species. The distributions are presented in form of box plots and violin plots (grey distributions in background), small horizontal lines denote single birds and strong horizontal lines denote medians by species, dotted black line marks the overall median. For each species, the median and number of birds (n) are presented numerically above the plot. Distribution of each separate AR concentration was also compared to species that had four or more samples presented. Kruskal-Wallis test showed that concentration of an AR does not differ statistically significantly between species (Figure 8). 33

34 Figure 8. Distribution of studied anticoagulant rodenticides' concentration by more frequent species (n > 3). The distributions are presented in form of box plots and violin plots (grey distributions in background). Small horizontal lines denote single birds and strong horizontal lines denote medians by species. Dotted black lines mark the overall medians, dotted red lines mark the limits of detection, and solid red lines mark the limits of quantitation. For better visualization, the y-axis is presented in logarithmic scale. For each rodenticide and species, the median is presented also numerically above the plot. Distributing species samples by compound counts over LOD and LOQ showed no significant relationship. However, either of these differences with LOD nor LOQ were not statistically significant when only six species that are more frequent was compared. See Figure 9. 34

35 Figure 9. Distribution of samples by (A) number of compounds over limit of detection (LOD) and (B) number of compounds over limit of quantitation (LOQ) depending on species with four or more samples. When grouping the species regarding their behavioural preference, as nocturnal and diurnal, and comparing their total AR concentrations, no statistically significant difference appeared in present study (p = 0.469). The median value for nocturnal species was 0.04 ng/g lower, while the quartiles and maximum value were slightly higher (Figure 10). There were 43 (68%) samples from diurnal birds, and 20 (32%) samples from nocturnal birds. 35

36 Figure 10. Distribution of total anticoagulant rodenticides' concentration by birds behavioural group. The distributions are presented in form of violin plots, grey squares in background denote the inter quartile range, small horizontal lines denote single birds and strong horizontal lines denote medians by groups, dotted black line marks the overall median. For each group the median is presented numerically above the plot. While no significant relationship between total AR concentration and behavioural preference could be found, there was a strong statistically significant difference in the concentration of brodifacoum between diurnal and nocturnal species (p = 0.005; Figure 11D). There was also statistically significant difference between diurnal and nocturnal birds when it comes to difenacoum concentration (p = 0.043; Figure 11C). 36

37 Figure 11. Distribution of studied anticoagulant rodenticides' concentration by birds' behavioural group. The distributions are presented in the form of violin plots, grey squares on background denote the inter quartile range. Small horizontal lines denote single birds and strong horizontal lines denote medians by groups. Dotted black lines mark the overall medians. Dotted red lines mark the limit of detection, and solid red lines mark the limit of quantitation. For better visualization, the y-axis is presented in logarithmic scale. For each rodenticide and behavioural group, the median is presented also numerically above the plot. The nocturnal birds had proportionally more samples with two compounds over limit of detection, while all samples with three compounds over LOD belonged to diurnal birds. However, these differences were not statistically significant (p = 0.069; Figure 12A). At the 37

38 same time there was no difference between diurnal and nocturnal birds in number of compounds over LOQ (p = 0.424; Figure 12B). Figure 12. Distribution of samples by (A) number of compounds over limit of detection (LOD) and (B) number of compounds over limit of quantitation (LOQ) depending on birds behaviour (diurnal vs nocturnal). SGAR residues were detected from 91% of samples from the first species protection category, 70% from the second, and 63% from the third category. There was no statistically significant relationship between species protection category and the total AR concentration (p = 0.980; Figure 13). 38

39 Figure 13. Distribution of total rodenticides' concentration by birds' protection category. The distributions are presented in form of violin plots, grey squares in background denote the inter quartile range, small horizontal lines denote single birds, and strong horizontal lines denote medians by categories, dotted black line marks the overall median. For each category, the median is presented numerically above the plot. In addition, there was no statistically significant relationship between specific AR concentrations and the species protection category (Figure 14). 39

40 Figure 14. Distribution of each studied rodenticides' concentration by birds' protection category. The distributions are presented in form of violin plots, grey squares in background denote the inter quartile range, small horizontal lines denote single birds, and strong horizontal lines denote medians by categories, dotted black lines mark the overall medians; dotted red lines mark the limits of detection, and solid red lines mark the limits of quantitation; for better visualization the y-axis is presented in logarithmic scale. For each rodenticide and protection category the median is presented also numerically above the plot. When distributing samples by number of compounds over LOD and LOQ, depending on species protection category, there is slight but not statistically significant relationship. It appears that species in the third protection category might have a higher concentration as more 40

41 compounds exceed the LOQ threshold in samples, but overall it is statistically non-significant (p = 0.089; Figure 15). Figure 15. Distribution of samples by (A) number of compounds over limit of detection (LOD) and (B) number of compounds over limit of quantitation (LOQ) depending on species protection category. The correlation analysis revealed weak positive relationships between concentrations of all four studied rodenticides: the values of Spearman correlation coefficients varied between 0.01 (between warfarin and difenacoum) and 0.27 (between bromadiolone and brodifacoum). However, only the last relationship was statistically significant (p = 0.034), indicating that in case of higher concentration of bromadiolone in sample, the concentration of brodifacoum was also higher on an average (Figure 16A). Still, this relationship is weak there are some samples with higher bromadiolone and lower brodifacoum concentration, but in addition to the opposite occurred as well. Only all five samples with brodifacoum concentration over limit of quantitation had also bromadiolone concentration over limit of quantitation (dots in upper right corner of Figure 16A), simultaneously the samples with bromadiolone concentration over limit of quantitation had very different brodifacoum concentration. The relationship between bromadiolone and brodifacoum groups was also not statistically significant (p = 0.137; Figure 16B). 41