Types of pesticides involved in domestic and wild animal poisoning in Italy

Journal Pre-proof Types of pesticides involved in domestic and wild animal poisoning in Italy

Alessia Bertero, Mario Chiari, Nicoletta Vitale, Mariagrazia Zanoni, Elena Faggionato, Alberto Biancardi, Francesca Caloni PII:

S0048-9697(19)36125-X

DOI:

https://doi.org/10.1016/j.scitotenv.2019.136129

Reference:

STOTEN 136129

To appear in:

Science of the Total Environment

Received date:

20 September 2019

Revised date:

20 November 2019

Accepted date:

13 December 2019

Please cite this article as: A. Bertero, M. Chiari, N. Vitale, et al., Types of pesticides involved in domestic and wild animal poisoning in Italy, Science of the Total Environment (2019), https://doi.org/10.1016/j.scitotenv.2019.136129

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2019 Published by Elsevier.

Journal Pre-proof Types of pesticides involved in domestic and wild animal poisoning in Italy Alessia Berteroa,1, Mario Chiarib,*,1, Nicoletta Vitalec, Mariagrazia Zanonic, Elena Faggionatoc, Alberto Biancardic, Francesca Calonia. a

Department of Veterinary Medicine (DIMEVET), Università degli Studi di Milano, Via Celoria 10, 20133 Milan, Italy. b D.G. Welfare, Regional Health Authority of Lombardy, 20124 Milan, Italy. c Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna (IZSLER) “Bruno Ubertini”, Via Bianchi 7/9, 25124 Brescia, Italy.

ro of

* Corresponding author 1 These authors contributed equally to this work.

Jo

ur

na

lP

re

-p

Abstract Data obtained from samples of poisoned domestic and wild animals sent for toxicological evaluation during the period between 2005 and 2014 have been analyzed. Among the 4606 tested samples, the majority of which were collected in the northern regions of Italy, 2006 (43.55%) were found positive for pesticides. Analytical detections were performed via solvent extraction followed by separation and chromatographic characterization and all the methods applied for the toxicological investigations were developed by IZSLER. Insecticides, mainly represented by acetylcholinesterase inhibitors (carbamates 17.55%, n=352; organophosphates 15.15%, n=304) and organochlorines (29.21%, n=586), were found to be the first category of pesticides involved in intoxications, in both domestic and in wild animals, followed by rodenticides (anticoagulant rodenticides 21.09%, n=423; zinc phosphide 2.59%, n=52; chloralose 0.95%, n=19 and thallium 0.15%, n=3) and molluscicides (metaldehyde 6.63%, n=133). Second and third generation anticoagulants (bromadiolone and brodifacoum) were the most represented (10.52%, n=211) but also first generation compounds (i.e. coumatetralyl and warfarin) were still found responsible of intoxications. Even if some pesticides are frequently involved in domestic animal poisoning (i.e. metaldehyde and strychnine), they did not show the same diffusion in wild animals. In particular, unlike domestic species, cyanide and pyrethroids have not been found responsible of intoxications in wild animals. Interestingly, a great number of positive samples involved banned pesticides like α- (14.41%, n=289) and β- (14.16%, n=284) endosulfan, carbofuran (5.73%, n=115), methamidophos (9.47%, n=190), strychnine (6.23%, n = 125) but, on the other hand, many positives were due to the exposure to commercially available products (i.e. metaldehyde and anticoagulant rodenticides). Thus, together with measures aimed to reduce illegal uses, educational campaigns and a wider range of compounds to detect would be beneficial in order to address the issue of animal poisoning, which besides has also repercussions on environmental and public health.

Keywords (max 6) Pesticides; domestic and wild animals; poisoning; samples; Italy.

1. Introduction Poisoning of domestic and wild animals, either intentional or accidental, is anything but rare and has been reported all over the world (Caloni et al., 2016, 2018; Chiari et al., 2017; P. K. Gupta,

Journal Pre-proof

Jo

ur

na

lP

re

-p

ro of

2018; Gwaltney-Brant, 2018). Lots of compounds are responsible for intoxications but, among them, in Europe and in Italy the most often involved are pesticides and in particular insecticides (Bille et al., 2016; Caloni et al., 2018, 2016; VANDENBROUCKE et al., 2008). Intoxications can result from an incorrect use or abuse of these compounds, from poor management to intentional poisoning, even with baits, of species considered harmful for human activities but also of domestic animals (Bille et al., 2016). Moreover, since the widespread application of pesticides to limit the agricultural pests and benefit the production is a common and sometimes uncontrolled practice, direct or indirect poisoning of wild animal species are also likely to occur (Berny, 2007). The incidence of poisonings related to pesticides is influenced by many factors, such as the availability on the local, international and online market, agricultural techniques, cultural background, etc (Chiari et al., 2017). In general pesticides are easy to purchase not only for professional uses but also for domestic applications and, on the other hand, it should be emphasized that restrictions in their use did not directly result in a substantial reduction of animal poisonings. For instance, the use of strychnine in agriculture and as a rodenticide was banned in the European Union in 2006, whereas aldicarb and carbofuran were withdrawn in 2003 (EC, 2003) and in 2007 (EC, 2007), respectively, but they are still frequently found responsible of intentional and accidental intoxications (Boucaud-Maitre et al., 2019; Ruiz-Suárez et al., 2015). Namely, strychnine still account for a large number of dog poisoning episodes in Italy (Caloni et al., 2012) and it is still often used for the preparation of baits (n=60, 13.3% of baits containing rodenticides) (Chiari et al., 2017). In a study performed in Spain (Ruiz-Suárez et al., 2015), aldicarb and carbofuran were isolated in nearly 75% of poisoning cases, percentage that come close to 100% considering the analyzed baits, thus the availability of these compounds still represent an unsolved issue to address in order to reduce the incidence of intoxications. In this context, the continuous and accurate collection of epidemiological data on animal poisoning is crucial to obtain useful information on toxicant trends and rise of new substances in order to carry out and enhance preventive measures for an appropriate risk management, to help veterinarians to manage cases of suspected poisoning, and finally to draw attention towards an issue that has also environmental and human health implications. In this study, we analyzed the incidence of pesticides isolated from animals/carcasses and other materials sent to the Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna (IZSLER) for toxicological evaluations, providing a general overview on animal exposure to pesticides, both domestic and wild species, over a period of 10 years.

2. Material and methods 2.1. Study area and sample collection Lombardy and Emilia Romagna Regions are located in the north of Italy, with an area of 46,290 km2 (17,872 sq. mi) and 10.0 million people (ISTAT, 2019). Lombardy is bordered by Switzerland to the north, to the east by the Italian regions of TrentinoAlto Adige and Veneto, to the west by Piedmont and to the south by Emilia Romagna. The eastern part of Emilia Romagna is located on the Adriatic Sea. Lombardy and Emilia Romagna are distinguished in three zones: mountains, hills and plains. Nearly half of territory (48%) consists of plains (Pianura Padana), characterized by an intensive agriculture enhanced by a well-developed use of fertilizers and animal husbandry. Wild animals are also relevant in the hill and mountain area and in the forest and shrubland area in the hill and higher plains zones. In Italy, in order to safeguarding people, animals and the environment health the detention and the improper use, preparation, mixing and scattering of poison baits are forbidden and enshrined in.a Decree of the Ministry of Labour, Health and Social Policies. This Decree has been in force

Journal Pre-proof

ro of

since January 2009 (Italian Ministerial Decree of December 18, 2008 – Italian Official Journal no. 13, January 17, 2009). In case of the identification of a poison bait and suspected poisoned animal, the competent health authority must be informed and send them to the local public veterinary institute for necropsy and/or toxicological investigations in order to identify the potentially present toxic substance. All the samples must be delivered to the laboratory with data collected through a specific form attached to the aforesaid decree. If the poisoning events are confirmed, the competent authority has to be notified and legal prosecution may follow. Data from suspected poisoned animals collected in the described framework over the period 2005–2014 identified by both citizens and/or veterinarians and collected by local public veterinary heath authorities were presented. All the animal samples were collected from carcasses of dead animals suspected to be poisoned and for this reason delivered. to the local public veterinary institute for necropsy and/or toxicological investigations. The study area is defined as Lombardy and Emilia Romagna Regions, even if same samples came from other two Italian regions.

Jo

ur

na

lP

re

-p

2.2. Laboratory analyses Positive analytical detections of pesticides in samples were performed using solvent extraction followed by separation and characterization by chromatographic methods. These included qualitative thin-layer chromatography (NP-TLC), qualitative colorimetric methods, semiquantitative gas chromatography mass spectrometry (GC-MS) and semiquantitative liquid chromatography mass spectrometry (LC-MS). All analytical methods used for toxicological investigations were developed by IZSLER. The detection of strychnine was carried out by qualitative NP-TLC (Bogan et al., 1966; Gocan and Cimpan, 2004). The sample was digested in acidic aqueous medium, defatted with n-hexane and the analyte was back-extracted in dichloromethane organic phase by means of a pH-regulated liquid-liquid partition. The final concentrated extract was spotted on a sylicagel TLC (Gocan and Cimpan, 2004). After plate developing, the detection of strychnine was performed by spraying an acidic solution of ammonium metavanadate, which produced a violet complex with strychnine. This complex shortly turned into orange (Bogan et al., 1966; Gocan and Cimpan, 2004). The detection of cyanide was performed by a qualitative colorimetric method (Bark and Higson, 1963): a basic aqueous solution of the sample was treated with ferrous chloride saturated solution, then it was warmed and hydrochloric acid was added dropwise. In the presence of cyanide, a Prussian blue complex appeared (Bark and Higson, 1963). The detection of zinc phosphide was based on a qualitative colorimetric method (Poppenga et al., 2005; Robertson et al., 1945). In a flask containing the sample, concentrated sulfuric acid was added; in presence of zinc phosphide, the phosphine gas developed and made turn iridescent black a paper strip wetted with a silver nitrate solution. In the meanwhile a control strip soaked with lead acetate kept colorless (Poppenga et al., 2005; Robertson et al., 1945). The determination of anticoagulants (coumafuryl, warfarin, coumatetralyl, coumachlor, diphacinone, chlorophacinone, bromadiolone, difenacoum, brodifacoum, flocoumafen, difethialone) and α-chloralose was carried out by means of a semiquantitative LC-MS method (Armentano et al., 2012; VANDENBROUCKE et al., 2008). The LOQ was 0.010 and 1 mg/kg for of anticoagulants and α-chloralose, respectively. The sample was extracted with acetone; an aliquot was dried and reconstituted with 2% ammonia solution in acetonitrile. Two defatting steps with nhexane followed. Finally, an aliquot was stripped to dryness and reconstituted with methanol containing 5 mM ammonium formiate. A 2 µL volume was injected into an LC-MS system (Waters Micromass ZQ, equipped with an Alliance Waters 2695 HPLC). Chromatographic column was XTerra MS C18 (15 cm, i.d. 2.1 mm, 3.5 µm). Column temperature was set at 40° C.

Journal Pre-proof

Jo

ur

na

lP

re

-p

ro of

Chromatographic elution was performed through a linear gradient using as aqueous phase ammonium formiate 5 mM (pH 9.2) and as organic phase methanol containing 5 mM ammonium formiate. Flow rate was set at 0.2 mL/min. Acquisition of [M-H]- ions was carried out in SIM mode (ESI negative) (Armentano et al., 2012; VANDENBROUCKE et al., 2008). The determination of 22 organochlorine (Alpha-hexachlorocyclohexane, Betahexachlorocyclohexane, Gamma hexachlorocyclohexane, hexachlorobenzene, heptachlor, cisheptachlor epoxide, trans-heptachlor-endo-epoxide, Aldrin, Dieldrin, Endrin, o,p-DDD, p,p-DDD, o,p-DDE, p,p-DDE, o,p-DDT, p,p-DDT, alpha-endosulfan, beta- endosulfan, endosulfan-sulfate, cischlordane, trans-chlordane, methoxychlor), 44 organophosphate (Acephate, Azinphos ethyl, Azinphos methyl, Bromophos ethyl, Bromophos methyl, Carbophenothion, Chlorfevinphos, Chlorpyriphos methyl, Chlorthiophos, Chlorpyriphos ethyl, Coumaphos, Diazinon, Dichlorvos, Dimefox, Dimethoate, Ethion, Fenchlorphos, Fenchlorphos-oxon, Fenitrothion, Fenthion, Fenthion sulfone, Fonofos, Isofenphos, Malaoxon, Malathion, Methacriphos, Methamidophos, Methidathion, Mevinphos, Omethoate, Paraoxon methyl, Parathion ethyl, Parathion methyl, Phorate, Phosalone, Phosmet, Pirimiphos methyl, Profenofos, Pyrazophos, Quinalphos, Terbufos, Tetrachlorvinphos, Thionazin), 11 carbamate pesticides (Propham, Metolcarb, Oxamyl, Methomyl, Propoxur, Chlorpropham, Bendiocarb, Carbofuran, Terbucarb, Carbaryl, Methiocarb) and 10 pyrethroid insecticides Tetramethrin, Cyhalothrin-lambda, Permethrin, Cyfluthrin, Flucythrinate, Fenvalerate, Bifenthrin, Cypermethrin, Fluvalinate, Deltamethtrin), triazine herbicides and metaldehyde was performed through a semiquantitative GC-MS method (Brown et al., 2005; Jones and Charlton, 1999). The LOQ for all compounds is 0,1 mg/kg except for Methaldeyde (50 mg/kg). The sample was extracted with acetone and injected (1 µL) into an ion trap GC-MS system (Varian 240-MS ion-trap, equipped with a Varian 450-GC). A capillary column DB 5MS (30 m, 0.25 mm i.d., 0.25 µm film) was used as stationary phase, the gas carrier was helium as mobile phase. The chromatographic separation was carried out by means of a thermal gradient. The detection was performed by the ion-trap mass analyzer; full mass spectrum of each compound was acquired, providing the best identification (Brown et al., 2005). 2.3. Statistical analysis Descriptive statistic was performed using IBM SPSS Statistics for Mac, Version 25.0 (Armonk, NY: IBM Corp.). Graphs were created using Prism for Mac, Version 8.2.0 (GraphPad Software Inc., La Jolla, CA, USA). 3. Results A total of 4606 samples were analyzed between 2005 and 2014. Of these, 41 (0.89%) were collected in Campania, 2852 (61.92%) in Emilia Romagna, 1530 (33.22%) in Lombardy, 4 (0.09%) in Tuscany and 2 (0.04%) in Veneto, whereas for 177 (3.84%) specimens the geographical origin was unknown. The analyzed samples were: animals/carcasses (n=3864, 83.90%), intestinal content (n=155, 3.37%), honeycomb (n=2, 0.04%), feces (n=14, 0.30%), liver (n=17, 0.37%), intestine (n=2, 0.04%), unidentified material (n=12, 0.26%), various materials (n=86, 1.87%), proventriculus/gizzard (n=2, 0.04%), gastric juice (n=130, 2.82%) and viscera (n=322, 6.99%). Two thousand and six (43.55%) of the analyzed samples were found positive for pesticides (Fig. 1), with the following proportions: carbamates 17.55% (n=352), cyanide 0.10% (n=2), chloralose 0.95% (n=19), metaldehyde 6.63% (n=133), organochlorines 29.21% (n=586) (Fig. 2), organophosphates 15.15% (n=304), pyrethroids 0.35% (n=7), anticoagulant rodenticides 21.09% (n=423), strychnine 6.23% (n=125), thallium 0.15% (n=3) and zinc phosphide 2.59% (n=52). In Table 1 classes of pesticides and compounds detected in positive samples are illustrated. The geographical distribution in the study area of the detected pesticides is showed in Fig. 3.

Journal Pre-proof Positive and negative samples per species are reported in Fig. 4, 5 and 6, whereas Table 2 and 3 illustrate the pesticides involved in animal and in wildlife species poisoning, respectively. The temporal trend of the classes of pesticides detected in positive sample per year is shown in Fig. 7.

lP

re

-p

ro of

3.1 Classes of pesticides detected in positive samples 3.1.1 Insecticides The insecticides found in positive samples are reported in Table 1. Organochlorines (n=586, 46.92%) were the most frequently detected class, followed by carbamates (n=352, 28.18%), organophosphates (n=304, 24.34%) and pyrethroids (n=7, 0.56%). The most frequently isolated organochlorines were α- (n=289, 49.32%) and β-endosulfan (n=284, 48.46%). Methomyl was the most commonly detected carbamates (n=216, 61.36%), whereas among organophosphates, methamidophos was the most frequent compound (n=190, 62.50%). Regarding the pyrethroids, permethrin was the most frequently found (n=4, 57.14%). Fig. 8 shows the temporal trends of αand β-endosulfan during the study period. 3.1.2 Rodenticides All the rodenticides identified are reported in Table 1. Anticoagulant rodenticides (n=423, 68.01%) were the most commonly isolated compounds followed by strychnine (n=125, 20.10%), zinc phosphide (n=52, 8.36%), chloralose (n=19, 3.05%) and thallium (n=3, 0.48%). Brodifacoum (n=130, 30.73%), coumatetralyl (n=110, 26.00%) and bromadiolone (n=81, 19.15%) were the most frequently detected anticoagulant rodenticides. 3.1.3 Other pesticides Other isolated pesticides were cyanide (n=2, 0.10% of the total positive samples) and metaldehyde (n=133, 6.63% of the total positive samples).

Jo

ur

na

4. Discussion Pesticides, as a result of inappropriate and careless use (Caloni et al., 2018), and as intentional baits, represent the first class of compounds responsible of intoxications in all animal species: companion animals, horses, ruminants and wildlife (Berny et al., 2010; Caloni et al., 2018, 2012; Guitart et al., 2010a, 2010b). In this study, 4606 samples have been analyzed, allowing to outline a general trend for animal poisoning, with concern to both domestic and wild species, in Italy, over a period of ten years. The majority of the samples were from the northern regions Emilia Romagna (n=2852, 61.92%) and Lombardy (n=1530, 33.22%). Concerning the geographical distribution (Fig. 3) of the different classes of pesticides, organochlorines represented the main cause of intoxication in Emilia Romagna and Campania, whereas in Lombardy the main culprit was found in the anticoagulant rodenticides. Lombardy is located in the north of Italy and represents one of the major industrial areas of the country (mainly service sector), moreover its territory is characterised by the presence of a large plain that covers approximately half of the region’s surface (Pianura Padana) with intensive agricultural and animal husbandry activities. The Emilia Romagna borders with Lombardy to the north and it is also characterised by a large amount of farmland, thus the agricultural sector represents a very important part of the region’s economy, together with the industrial sector. Campania is located in the south of Italy and this region also has a strong agricultural vocation. In this context, the presence of pesticides in the environment could be related to the production activities but it is interesting to underline that most of the organochlorines (the main cause of intoxication in Emilia Romagna and Campania) have been banned, thus the identification of these substances can be related to their high environmental persistence but also to illegal practices,

Journal Pre-proof

Jo

ur

na

lP

re

-p

ro of

whereas anticoagulant rodenticides are largely used for rodent control in these areas, in the cities and in the countryside. It is interesting to note that, even if strychnine has been banned many years ago, all the regions involved in this study still presented positives related to this alkaloid. This is an issue, probably related to the presence of a black market and old stocks, that needs to be addressed and that it is also been reported in other European countries (Berny et al., 2010; Guitart et al., 2010b). Pesticide trends (Fig. 7) during the decade of this study: with regard to the above-mentioned strychnine, the number of positives has remained fairly constant over the years, whereas a decreasing trend can be observed after 2009 for the banned α- and β-endosulfan, even if they are still present (Fig. 8). A declining trend was also recorded for acetylcholinesterase inhibitors from 2013 and for organochlorines and anticoagulant rodenticides from 2012, while metaldehyde poisoning cases always increased (Fig. 7). With regard to the old substances, while cyanide has no longer been found responsible of intoxications since 2009 and thallium has been identified just in three samples in 2006, chloralose has shown a different trend: it has been isolated in one sample in 2011, but since then its use has slightly increased, with just a small decline in 2013 (Fig. 7). Insecticides, mainly represented by acetylcholinesterase inhibitors, carbamates and organophosphates, are the first category of pesticides involved in intoxications, in domestic as well as in wild animals (Table 2 and 3), followed by rodenticides and molluscicides. Most samples were positive to the potent carbamate methomyl, often fatal, followed by carbofuran, the most common insecticide involved in wildlife poisoning (Caloni et al., 2018; Modrä and Svobodová, 2009; Vandenbroucke et al., 2010) whereas in the group of organophosphates, methamidophos accounts the highest number (Table 1). Organochlorines, well-known persistent pollutants that the EU started to ban in the 1980s (EC, 1979), are paradoxically still present. If DDT has still been found in poisoned baits in a research performed in Italy in 2017 (Chiari et al., 2017), most positives (n = 573) (Table 1) concern endosulfan (α and β) (Caloni et al., 2018; Chiari et al., 2017; De Roma et al., 2016) and even if the number of cases had declined from 2009, the amount remains high (Fig. 8). Pyrethroids, that have commonly been found responsible of accidental intoxications of domestic carnivores in Italy, according to a study performed on data related to suspected cases of domestic animal poisoning reported to the Milan Poison Control Centre (Caloni et al., 2018, 2012), were detected only in one cat and two dogs (Table 2), and have not been related to wild species (mammals or birds) intoxications, which however have shown a huge number of positives to organochlorine, both birds and mammals species (Table 3). In the class of rodenticides, second and third generation anticoagulants, bromadiolone and brodifacoum, continue to be the main culprit of poisoning (n=211) (Table 1), even if coumatetralyl and warfarin, first generation compounds, are still present (n=147 samples) (Table 1). Strychnine, also taken off the market many years ago (2006)(EC, 2006), remains largely used, confirming data reported in previous epidemiological studies (Berny et al., 2010; Caloni et al., 2018, 2012), where dog results the principal species involved (n=100) (Table 2), while only sporadically red fox, wild boar, wolf and wild birds (Table 3). Old products like cyanide (n=2) (Table 2), only detected in the first period of our investigation (Fig. 7) and the rodenticides chloralose (n=19) (Table 2), and thallium (n=3) (Table 2), even if intoxications are decreasing, are sometimes detected in laboratories receiving suspected baits (R. Gupta, 2018), as it happened in our investigation (Chiari et al., 2017). Anticoagulants, followed by strychnine and zinc phosphide, are the rodenticides mainly detected in wild mammals (Table 3).

Journal Pre-proof

ro of

Metaldehyde poisoning in dogs and cats is a big issue in Europe (Caloni et al., 2018), and data revealed carnivores as the target species, while only one case is confirmed in a wildlife mammal (European badger)(Table 3). Thus, comparing domestic and wild animals, it can be observed that insecticides are firstly implicated in intoxications in both categories but, though in wild animals acetylcholinesterase inhibitors are most frequently implicated, in domestic species (mainly dog) organochlorines are the predominant insecticide class involved. Moreover, while exposure to anticoagulant rodenticides is a concern for wild as well as domestic animals, some substances that frequently cause intoxications in domestic animals like metaldehyde and strychnine do not have the same numerical impact in wild animals, with just few cases reported. Furthermore, unlike domestic animals, no positives in wild animals concerning cyanide and pyrethroids have been detected. Finally, there is an aspect concerning the negative samples that is worthy of remark: these samples had been classified as negative with reference to the limited toxicants investigated and considered the main culprits in animal poisoning (Berny et al., 2010; Bille et al., 2016; Caloni et al., 2018; Chiari et al., 2017; Guitart et al., 2010b; Mcfarland et al., 2017).

ur

na

lP

re

-p

5. Conclusions The data collected in this study provide a comprehensive and up-to-date overview on domestic and wild animal pesticide poisoning, information that is crucial to develop adequate prevention measures and also represents a valuable tool to help veterinarians during diagnosis and treatment. Pesticides remain the class of compounds that is responsible for most of the intoxications involving animal species and, among them, insecticides followed by rodenticides are the categories most frequently involved. Even if a slight trend towards a decrease has been observed for some toxicants, the persistent abuse of already banned substances (i.e. strychnine, aldicarb, carbofuran, methamidophos) is still a very critical issue. However, it should be noted that a great number of intoxications are caused by the exposure to commercially available products, such as metaldehyde and anticoagulant rodenticides, due to their widespread use, also in a domestic context, thus adequate education and sensibilization of people would be essential. Furthermore, in the future, it would be interesting to have a wider range of compounds to detect in order to comprehend the emerging toxicants.

Jo

Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References Armentano, A., Iammarino, M., Magro, S. Lo, Muscarella, M., 2012. Validation and application of multi-residue analysis of eight anticoagulant rodenticides by high-performance liquid chromatography with fluorimetric detection. J. Vet. Diagnostic Investig. 24, 307–311. https://doi.org/10.1177/1040638711433354 Bark, L.S., Higson, H.G., 1963. A review of the methods available for the detection and determination of small amounts of cyanide. Analyst 88, 751–760. https://doi.org/10.1039/AN9638800751 Berny, P., 2007. Pesticides and the intoxication of wild animals. J. Vet. Pharmacol. Ther. https://doi.org/10.1111/j.1365-2885.2007.00836.x Berny, P., Caloni, F., Croubels, S., Sachana, M., Vandenbroucke, V., Davanzo, F., Guitart, R., 2010. Animal poisoning in Europe. Part 2: Companion animals. Vet. J. https://doi.org/10.1016/j.tvjl.2009.03.034

Journal Pre-proof

Jo

ur

na

lP

re

-p

ro of

Bille, L., Toson, M., Mulatti, P., Dalla Pozza, M., Capolongo, F., Casarotto, C., Ferrè, N., Angeletti, R., Gallocchio, F., Binato, G., 2016. Epidemiology of animal poisoning: An overview on the features and spatio-temporal distribution of the phenomenon in the north-eastern Italian regions. Forensic Sci. Int. 266, 440–448. https://doi.org/10.1016/j.forsciint.2016.07.002 Bogan, J., Rentoul, E., Smith, H., Weir, W.P., 1966. Homicidal Poisoning by Strychnine. J. Forensic Sci. Soc. 6, 166–169. https://doi.org/10.1016/S0015-7368(66)70329-6 Boucaud-Maitre, D., Rambourg, M.O., Sinno-Tellier, S., Puskarczyk, E., Pineau, X., Kammerer, M., Bloch, J., Langrand, J., 2019. Human exposure to banned pesticides reported to the French Poison Control Centers: 2012–2016. Environ. Toxicol. Pharmacol. https://doi.org/10.1016/j.etap.2019.03.017 Brown, P., Turnbull, G., Charman, S., Charlton, A., Jones, A., 2005. Analytical methods used in the United Kingdom Wildlife Incident Investigation Scheme for the detection of animal poisoning by pesticides. J AOAC Int 88, 204–20. Caloni, F., Berny, P., Croubels, S., Sachana, M., Guitart, R., 2018. Epidemiology of Animal Poisonings in Europe, in: Gupta, R.C.B.T.-V.T. (Third E. (Ed.), Veterinary Toxicology. Elsevier, pp. 45–56. https://doi.org/10.1016/B978-0-12-811410-0.00003-9 Caloni, F., Cortinovis, C., Rivolta, M., Davanzo, F., 2016. Suspected poisoning of domestic animals by pesticides. Sci. Total Environ. 539, 331–336. https://doi.org/10.1016/j.scitotenv.2015.09.005 Caloni, F., Cortinovis, C., Rivolta, M., Davanzo, F., 2012. Animal poisoning in Italy: 10 years of epidemiological data from the Poison Control Centre of Milan. Vet. Rec. 170, 415. https://doi.org/10.1136/vr.100210 Chiari, M., Cortinovis, C., Vitale, N., Zanoni, M., Faggionato, E., Biancardi, A., Caloni, F., 2017. Pesticide incidence in poisoned baits: A 10-year report. Sci. Total Environ. 601–602, 285–292. https://doi.org/10.1016/j.scitotenv.2017.05.158 De Roma, A., Rossini, C., Riverso, C., Galiero, G., Esposito, M., 2016. Endosulfan poisoning in canids and felids in the Calabria region of southern Italy. J. Vet. Diagnostic Investig. 29, 122–125. https://doi.org/10.1177/1040638716681389 EC, 2007. Commission Decision of 13 June 2007 concerning the non-inclusion of carbofuran in Annex I to Council Directive 91/414/EEC and the withdrawal of authorisations for plant protection products containing that substance. Off. J. Eur. Union L 156, 0030–1. EC, 2006. Commission Regulation (EC) No 777/2006 of 23 May 2006 amending Annex I to Regulation (EC) No 304/2003 of the European Parliament and of the Council concerning the export and import of dangerous chemical. Off. J. Eur. Union L136, 9. EC, 2003. Council Decision of 18 March 2003 concerning the non-inclusion of aldicarb in Annex I to Council Directive 91/414/EEC and the withdrawal of authorisations for plant protection products containing this active substance. Off. J. Eur. Union L 076, 0021–4. EC, 1979. Council Directive 79/117/EEC of 21 December 1978 prohibiting the placing on the market and use of plant protection products containing certain active substances. Off. J. Eur. Union L33, 36. Gocan, S., Cimpan, G., 2004. Review of the Analysis of Medicinal Plants by TLC: Modern Approaches. J. Liq. Chromatogr. Relat. Technol. 27, 1377–1411. https://doi.org/10.1081/JLC120030607 Guitart, R., Croubels, S., Caloni, F., Sachana, M., Davanzo, F., Vandenbroucke, V., Berny, P., 2010a. Animal poisoning in Europe. Part 1: Farm livestock and poultry. Vet. J. 183, 249–54. https://doi.org/10.1016/j.tvjl.2009.03.002 Guitart, R., Sachana, M., Caloni, F., Croubels, S., Vandenbroucke, V., Berny, P., 2010b. Animal poisoning in Europe. Part 3: Wildlife. Vet. J. https://doi.org/10.1016/j.tvjl.2009.03.033

Journal Pre-proof

Jo

ur

na

lP

re

-p

ro of

Gupta, P.K., 2018. Epidemiology of Animal Poisonings in Asia, in: Gupta, R.C.B.T.-V.T. (Third E. (Ed.), Veterinary Toxicology. Elsevier, pp. 57–69. https://doi.org/10.1016/B978-0-12-8114100.00004-0 Gupta, R., 2018. Non-Anticoagulant Rodenticides, in: Veterinary Toxicology: Basic and Clinical Principles: Third Edition. pp. 613–626. https://doi.org/10.1016/B978-0-12-811410-0.00047-7 Gwaltney-Brant, S.M., 2018. Epidemiology of Animal Poisonings in the United States, in: Gupta, R.C.B.T.-V.T. (Third E. (Ed.), Veterinary Toxicology. Elsevier, pp. 37–44. https://doi.org/10.1016/B978-0-12-811410-0.00002-7 Jones, A., Charlton, A., 1999. Determination of Metaldehyde in Suspected Cases of Animal Poisoning Using Gas Chromatography−Ion Trap Mass Spectrometry. J. Agric. Food Chem. 47, 4675–4677. https://doi.org/10.1021/jf990026d Mcfarland, S.E., Mischke, R.H., Hopster-Iversen, C., Von Krueger, X., Ammer, H., Potschka, H., Stürer, A., Begemann, K., Desel, H., Greiner, M., 2017. Systematic account of animal poisonings in Germany, 2012-2015. Vet. Rec. 180, 327. https://doi.org/10.1136/vr.103973 Modrä, H., Svobodová, Z., 2009. Incidence of animal poisoning cases in the Czech Republic: Current situation. Interdiscip. Toxicol. 2, 48–51. https://doi.org/10.2478/v10102-009-0009-z Poppenga, R.H., Ziegler, A.F., Habecker, P.L., Singletary, D.L., Walter, M.K., Miller, P.G., 2005. Zinc Phosphide Intoxication of Wild Turkeys (Meleagris gallopavo). J. Wildl. Dis. 41, 218–223. https://doi.org/10.7589/0090-3558-41.1.218 Robertson, A., Campbell, J.G., Graves, D.N., 1945. Experimental zinc phosphide poisoning in fowls. J. Comp. Pathol. Ther. 55, 290–300. https://doi.org/10.1016/S0368-1742(45)80027-9 Ruiz-Suárez, N., Boada, L.D., Henríquez-Hernández, L.A., González-Moreo, F., Suárez-Pérez, A., Camacho, M., Zumbado, M., Almeida-González, M., Del Mar Travieso-Aja, M., Luzardo, O.P., 2015. Continued implication of the banned pesticides carbofuran and aldicarb in the poisoning of domestic and wild animals of the Canary Islands (Spain). Sci. Total Environ. 505, 1093–9. https://doi.org/10.1016/j.scitotenv.2014.10.093 VANDENBROUCKE, V., DESMET, N., DEBACKER, P., CROUBELS, S., 2008. Multi-residue analysis of eight anticoagulant rodenticides in animal plasma and liver using liquid chromatography combined with heated electrospray ionization tandem mass spectrometry. J. Chromatogr. B 869, 101–110. https://doi.org/10.1016/j.jchromb.2008.05.011 Vandenbroucke, V., van Pelt, H., De Backer, P., Croubels, S., 2010. Animal poisonings in Belgium: A review of the past decade. Vlaams Diergeneeskd. Tijdschr. 79, 259–268. Figure captions Figure 1. Number of positive and negative samples. The analyzed specimens were animals/carcasses, intestinal content, honeycomb, feces, liver, intestine, unidentified material, various materials, proventriculus/gizzard, gastric juice and viscera. Figure 2. Chromatogram from a carcass, analytes are α- and β-endosulfan. Figure 3. Geographical distribution in Italy of pesticides isolated from samples. The analyzed specimens were animals/carcasses, intestinal content, honeycomb, feces, liver, intestine, unidentified material, various materials, proventriculus/gizzard, gastric juice and viscera. Figure 4. Positive and negative samples in domestic animals. Figure 5. Positive and negative samples in wildlife mammals, insects and unknown species. Figure 6. Positive and negative samples in wildlife birds. Figure 7. Pesticides detected in samples each year from 2005 to 2014. Figure 8. Frequencies of α- and β-endosulfan detected in samples each year from 2005 to 2014.

Jo

ur

na

lP

re

-p

ro of

Journal Pre-proof

Journal Pre-proof Table 1. Classes of pesticides and compounds detected in positive samples. Compounds

Number

Anticoagulant rodenticides

Brodifacoum

130

(n=423)

Bromadiolone

81

Chlorophacinone

12

Coumachlor

1

Coumatetralyl

110

Difenacoum

37

Difethialone

5

Bendiocarb

lP

Carbamates (n=352)

-p

Warfarin

10

na

Carbaryl

37

re

Flocoumafen

ro of

Pesticides

3 1

115

Methiocarb

9

Methomyl

216

Metolcarb

1

Oxamyl

5

Propoxur

2

Jo

ur

Carbofuran

Chloralose

19

Cyanide

2

Metaldehyde

133

Organochlorines (n=586)

α-endosulfan

289

Journal Pre-proof

β-endosulfan

284

β-HCH

1

γ-HCH (Lindane)

9

Para-Para DDT

2

Azinphos methyl

3

Chlorpyrifos ethyl

5

Chlorthiophos

1

Coumaphos

8

ro of

1

-p

Organophosphates (n=304)

α-HCH

Dichlorvos

lP

Dimethoate

3

na

Fonofos

9 1

18

Malathion

7

Methamidophos

190

Omethoate

5

Parathion ethyl

17

Phorate

20

Pirimiphos Methyl

3

Terbufos

3

Trichlorfon

3

λ-Cyalothrin

2

ur

Isofenphos

Jo Pyrethroids (n=7)

8

re

Diazinon

Journal Pre-proof Permethrin

4

Tetramethrin

1 125

Thallium

3

Zinc phosphide

52

Jo

ur

na

lP

re

-p

ro of

Strychnine

Journal Pre-proof Table 2. Pesticides involved in domestic animal intoxication, presented by animal species. Pesticides

N.

Cat (Felis catus)

carbamates

86

chloralose

16

metaldehyde

36

organochlorines

51

organophosphates

65

pyrethroids

ro of

Species

strychnine

12

carbamates

4

organochlorines

2

re

21

lP

ur Jo Dog (Canis lupus familiaris)

120

zinc phosphide

na

Chicken (Gallus gallus domesticus)

-p

anticoagulant rodenticides

1

organophosphates

5

anticoagulant rodenticides

3

strychnine

1

zinc phosphide

1

carbamates

112

cyanide

2

chloralose

2

metaldehyde

90

organochlorines

489

organophosphates

165

Journal Pre-proof

anticoagulant rodenticides

251

strychnine

100

thallium

2

zinc phosphide

22

organochlorines

2

anticoagulant rodenticides

4

pyrethroids

Jo

ur

na

lP

re

-p

Sheep (Ovis aries)

2

ro of

Pig (Sus scrofa domesticus)

pyrethroids

1

Journal Pre-proof Table 3. Pesticides involved in wildlife animal intoxication, presented by animal species. Species (Mammals)

Pesticides

N.

Beech marten (Martes foina)

organochlorines

2

organophosphates

1

anticoagulant

2

rodenticides

metaldehyde

1

thallium

1

organophosphates

1

anticoagulant

re

European hare (Lepus europaeus)

1

ro of

carbamates

-p

European badger (Meles meles)

1

carbamates

1

organophosphates

1

anticoagulant

2

lP

rodenticides

na

European hedgehog (Erinaceus

Jo

ur

europaeus)

rodenticides

European rabbit (Oryctolagus cuniculus)

carbamates

1

European roe deer (Capreolus capreolus)

zinc phosphide

1

Ferret (Mustela putorius furo)

organophosphates

1

Rat (Gen. Rattus)

anticoagulant

1

rodenticides Red fox (Vulpes vulpes)

carbamates

11

organochlorines

20

organophosphates

15

Journal Pre-proof anticoagulant

26

rodenticides

carbamates

1

organochlorines

2

organochlorines

2

ro of

3

organophosphates

1

strychnine

4

zinc phosphide

1

carbamates

2

organochlorines

2

organophosphates

3

strychnine

1

zinc phosphide

1

Pesticides

N.

carbamates

9

organophosphates

8

strychnine

1

carbamates

5

organochlorines

2

organophosphates

1

strychnine

1

Jo

Species (Birds)

ur

na

lP

Wolf (Canis lupus)

zinc phosphide

-p

Wild boar (Sus scrofa)

3

re

Squirrel (Gen. Sciurus)

strychnine

Common buzzard (Buteo buteo)

Common magpie (Pica pica)

Journal Pre-proof Common pheasant (Phasianus colchicus)

carbamates

1

organophosphates

2

anticoagulant

1

rodenticides organophosphates

1

Eurasian sparrowhawk (Accipiter nisus)

organophosphates

1

Falcon (Gen. Falco)

carbamates

1

House sparrow (Passer domesticus)

carbamates

1

Moorhen (Fam. Rallidae)

anticoagulant

1

-p

ro of

Eurasian jay (Garrulus glandarius)

carbamates

107

chloralose

1

organochlorines

6

organophosphates

16

anticoagulant

6

Jo

ur

na

lP

Pigeon (Fam. Columbidae)

re

rodenticides

Rook (Corvus frugilegus)

rodenticides strychnine

1

zinc phosphide

1

anticoagulant

1

rodenticides Seagull (Fam. Laridae)

carbamates

4

Turtle dove (Fam. Columbidae)

carbamates

4

metaldehyde

1

organophosphates

3

Journal Pre-proof anticoagulant

1

rodenticides Western marsh harrier (Circus

organophosphates

1

Species (Insects)

Pesticides

N.

Honeybee (Apis mellifera)

pyrethroids

3

Other species

Pesticides

N.

Unknown

carbamates

1

metaldehyde

5

-p

ro of

aeruginosus)

6

organophosphates

13

anticoagulant

3

lP

re

organochlorines

Jo

ur

na

rodenticides strychnine

1

zinc phosphide

1

Journal Pre-proof Graphical abstract

Jo

ur

na

lP

re

-p

ro of

Highlights - Insecticides are the pesticides firstly involved in animal poisoning - 50% of the samples resulted positive to already banned products - Anticoagulant rodenticides and strychnine are frequently implicated - Acetylcholinesterase inhibitors are the main culprits in wildlife poisoning - Organochlorines are still detected

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8