Wildlife poisoning: a novel scoring system and review of analytical methods for anticoagulant rodenticide determination.

Anticoagulant rodenticides (ARs) are commonly used to control rodent populations and frequently involved in wildlife and domestic animal poisoning. These poisoning cases (especially for ARs) are a challenge for forensic toxicologists, and adequate post-mortem examination and toxicological analyses become essential for a proper diagnosis. Publications describing different analytical methods for AR analysis in biological samples are growing, and a clear compilation of the overall picture is needed to standardize methodologies in future research. This review aims to compile and compare the analytical procedures applied for AR determination in the literature. Using this information, a scoring system was developed for those techniques using liver and blood as matrices, and the techniques were ranked considering different criteria (i.e. sample amount required, recoveries, limits of quantification (LOQs), number of ARs analysed, points of the calibration curve and multi-class methods). This review shows an overview of the main methods used for AR analysis in forensic toxicology and will help to elucidate future directions to improve multi-residue techniques to detect the ARs involved in wildlife lethal poisoning.

A schematic sampling protocol for contaminant monitoring in raptors.

Birds of prey, owls and falcons are widely used as sentinel species in raptor biomonitoring programmes. A major current challenge is to facilitate large-scale biomonitoring by coordinating contaminant monitoring activities and by building capacity across countries. This requires sharing, dissemination and adoption of best practices addressed by the Networking Programme Research and Monitoring for and with Raptors in Europe (EURAPMON) and now being advanced by the ongoing international COST Action European Raptor Biomonitoring Facility. The present perspective introduces a schematic sampling protocol for contaminant monitoring in raptors. We provide guidance on sample collection with a view to increasing sampling capacity across countries, ensuring appropriate quality of samples and facilitating harmonization of procedures to maximize the reliability, comparability and interoperability of data. The here presented protocol can be used by professionals and volunteers as a standard guide to ensure harmonised sampling methods for contaminant monitoring in raptors.

Temporal Persistence of Bromadiolone in Decomposing Bodies of Common Kestrel (Falco tinnunculus).

Bromadiolone is a second generation anticoagulant rodenticide (SGAR) used to control pest rodents worldwide. SGARs are frequently involved in secondary poisoning in rodent predators due to their persistence and toxicity. This study aims to evaluate the persistence of bromadiolone in liver at different stages of carcass decomposition in experimentally-dosed common kestrels (Falco tinnunculus) to understand the possibility of detecting bromadiolone in cases of wildlife poisoning and the potential risk of tertiary poisoning. Twelve individuals were divided into the bromadiolone-dose group (dosed with 55 mg/kg b.w) and the control group. Hepatic bromadiolone concentrations found in each stage of decomposition were: 3000, 2891, 4804, 4245, 8848, and 756 ng/g dry weight at 1-2 h (fresh carcass), 24 h (moderate decomposition), 72 h, 96 h (advanced decomposition), seven days (very advanced decomposition), and 15 days (initial skeletal reduction) after death, respectively. Liver bromadiolone concentrations in carcasses remained relatively stable over the first four days and raised on day 7 of decomposition under the specific conditions of this experiment, presenting a risk of causing tertiary poisoning. However, at the initial skeletal reduction stage, liver bromadiolone concentration declined, which should be considered to interpret toxicological analyses and for proper diagnosis. This experimental study provides for the first time some light to better understand the degradation of SGARs in carcasses in the wild.

Mercury Exposure in Birds Linked to Marine Ecosystems in the Western Mediterranean.

Mercury (Hg), particularly as methylmercury (MeHg), is a nonessential, persistent, and bioaccumulative toxic element with high biomagnification capacity and is considered a threat to marine environments. We evaluated total Hg concentrations in liver, kidney, and brain in 62 individuals of 9 bird species linked to marine ecosystems from western Mediterranean admitted in a Wildlife Rehabilitation Center (WRC) (Alicante, Spain, 2005-2020). Age- and sex-related differences in Hg levels, as well as the cause of admission to the WRC, were also evaluated in certain species. The species studied were: northern gannet (Morus bassanus), European shag (Phalacrocorax aristotelis), great cormorant (Phalacrocorax carbo), osprey (Pandion haliaetus), Balearic shearwater (Puffinus mauretanicus), yellow-legged gull (Larus michahellis), razorbill (Alca torda), common tern (Sterna hirundo), and black-headed gull (Chroicocephalus ridibundus). Concentrations in feathers of 27 individuals, and concentrations in internal tissues in 7 other individuals of 7 different species were also reported but not statistically evaluated due to the limited number of samples. Results suggest that individuals were chronically exposed to Hg through diet. The differences in Hg concentrations among species may be explained by their diet habits. Mercury concentrations strongly correlated between tissues (r = 0.78-0.94, p < 0.001, n = 61-62). Some individuals of certain species (i.e., European shag, northern gannet, and great cormorant) showed Hg concentrations close to or above those described in the literature as causing reproductive alterations in other avian species. Consequently, certain individuals inhabiting western Mediterranean could be at risk of suffering long-term, Hg-related effects. Some of the species evaluated are listed within different categories of threat according to the International Union for Conservation of Nature (IUCN) and are endangered at a national level, so this study will provide valuable information for assessors and authorities in charge of the management of the environment and pollution.

Mercury and Organochlorine Pesticides in Tissues of Loggerhead Sea Turtles (Caretta caretta) Stranded Along the Southwestern Mediterranean Coastline (Andalusia, Spain).

Nineteen loggerhead sea turtles (Caretta caretta) stranded along the southwestern Mediterranean coastline (Andalusia) were used in this study. A total of 68 samples of fat (n = 18), liver (n = 15), kidney (n = 13), pectoral muscle (n = 19), and brain (n = 3) were analysed for total mercury (Hg) and organochlorine pesticides [OC: ∑Dichlorodiphenyltrichloroethanes (∑DDT), ∑Hexachlorocyclohexane (∑HCH), ∑Heptachlor, ∑Drins and ∑Endosulfan]. These loggerhead sea turtles showed tissue Hg and OC concentrations similar to or lower than those reported in other studies. Few growth-related variations in Hg or OC levels in relation to straight carapace length were found, probably because the specimens were mostly juveniles. This study will help to fill the gap on spatio-temporal exposure data and ascertain the real world-wide picture of the contamination levels in loggerhead sea turtles.

Selection of new chemicals to be used in conditioned aversion for non-lethal predation control.

Globally, native predators and scavengers are threatened through the incidence of illegal poisoning due to increasing human-wildlife conflicts. The use of conditioned taste aversion (CTA) may mitigate such conflicts. CTA is a robust learning paradigm that occurs when animals associate a food with a discomfort induced by a chemical, thereby avoiding that food in subsequent encounters. We reviewed the potential of 167 chemical compounds to be used in CTA, considering effects, margin of safety, accessibility, and detectability. After the review, 15 compounds fulfilled the required characteristics, but only five (thiabendazole, thiram, levamisole, fluconazole and fluralaner) were finally selected to be tested in CTA assays with dogs. Of the tested compounds, thiabendazole, thiram and levamisole caused target food rejection by dogs and reduced the time spent eating during post-conditioning. However, despite being microencapsulated, levamisole appeared to be detectable by dogs, whereas thiram and thiabendazole were not. Fluconazole and fluralaner did not produce any CTA effect. Thiabendazole, thiram and levamisole can therefore induce CTA, and thus are potential candidates as aversive compounds for wildlife management. Thiram is an undetectable, relatively safe and accessible compound that can induce CTA in canids, and opens new possibilities to develop methods of non-lethal predation control.

Spatiotemporal variations of organochlorine pesticides in an apex predator: Influence of government regulations and farming practices.

BACKGROUND: Intensification of agricultural practices has caused several negative effects to the environment. The use of fertilizers and pesticides may alter geochemical cycles or cause direct wildlife intoxication. Detrimental effects of organochlorine pesticides (OCPs) have forced the authorities to ban or restrict its use. This study evaluates the variation in levels of OCPs in a sentinel species in relation to changes in government regulations and the spatial configuration of agricultural practices around the nests. METHODS: Between 2003 and 2007, we analysed OCP levels in 256 blood samples of Eurasian Eagle-owl (Bubo bubo) chicks nesting in area of intensive commercial agriculture with historical frequent use of pesticides, in South-eastern Spain. We studied year-to-year variations in OCP concentrations and their relation with land use configuration around raptor nests by Generalized Linear Mixed Models (GLMM). RESULTS: OCPs were detected in 100% samples surveyed in 2003 and 2004, while dropped to 27% in 2005, 6.8% in 2006 and 6.3% in 2007, coinciding with the ban of OCPs. The presence of the main OCPs was related to agricultural practices. In particular, endosulfan and lindane were related to irrigated crops and urban areas, while DDT-related compounds and dieldrin were associated with dry land farming. CONCLUSIONS: OCP concentrations in blood samples of Eurasian Eagle-owls may respond quickly to the implementations of new regulations about the use of agricultural products. This raptor was confirmed as a good sentinel species allowing rapid detection of changes in pesticides use.

Lead exposure in common shelduck (Tadorna tadorna): Tracking the success of the Pb shot ban for hunting in Spanish wetlands.

Levels of lead (Pb) were analyzed in tissues of 25 Common shelduck (Tadorna tadorna) from La Albufera Natural Park (NP) in Valencia (Eastern Spain), which were found dead due to a botulism outbreak. The objective was to assess Pb exposure in waterfowl, fourteen years after the implementation of the Real Decreto 581/2001, which banned the use of Pb ammunition for hunting activities in internationally relevant wetlands. For this purpose, this study measured the actual ingestion of lead pellets in Common shelduck from La Albufera de Valencia and provide data on Pb residue concentrations in different tissues of this species breeding in this NP. The Pb distribution pattern in tissues was bone > kidney > liver > brain > muscle, with Pb concentrations of 778.96 ±â€¯998.64, 122.40 ±â€¯89.18, 92.08 ±â€¯57.65, 18.90 ±â€¯16.30 and 15.13 ±â€¯11.85 ng/g (wet weight), respectively. This work presents, as far as we are aware, the first published data on Pb concentrations in Common shelduck. The Pb levels detected were lower than those reported in other studies on Anatid species from Spanish wetlands, which may be explained by this ban. Moreover, lead concentrations found were below those levels for which any observable effect has been described for acute or chronic lead exposure.

Interspecific differences in the antioxidant capacity of two Laridae species exposed to metals.

The main aim of the present study was to assess the concentration of metals (Pb, Cd, Hg, Cu and Zn) in blood, and elucidate their potential effects on oxidative stress biomarkers in red blood cells of Audouin's gull (Ichthyaetus audouinii) and Slender-billed gull (Chroicocephalus genei) chicks in Southeastern Spain. For this purpose, total glutathione (GSH) content, antioxidant enzymes activities (glutathione peroxidase, GPx; superoxide dismutase, SOD; catalase, CAT and glutathione-S-transferase, GST), and lipid peroxidation (TBARS) were analyzed. In general, metal exposure in both species can be considered low, except for Hg in Audouin's gull. Our findings show higher antioxidant levels in Audouin's gull than in Slender-billed gull; probably due to a combination of different basal antioxidant capacity between species, and to an up-regulation of the antioxidant system in Audouin's gull as a response to the higher Hg, Cu and Zn concentrations. This could reduce the production of TBARS keeping them at lower levels than those found in Slender-billed gull. In spite of this, the significantly higher Hg levels found in Audouin's gull (13.6µg/dl wet weight) in comparison to Slender-billed gull (2.7µg/dl), likely related to their different diet and the former consuming discarded fish, were able to produce lipid peroxidation in this species. The positive effect of Hg on SOD activity in Slender-billed gull, and of Pb on GSH levels in Audouin's gull, could reflect the necessity of the organism to upregulate these antioxidants to balance the increased oxidative stress caused by metals. The degree of metal exposure seems to be essential in the response of the antioxidant system, which may suffer up or down-regulations depending on metal concentrations. This study supports the interactive effects of metals on oxidative stress biomarkers, the complexity of the antioxidant system and the close cooperation between antioxidants, which requires the study of several metals and biomarkers to evaluate oxidative stress and damage in wild birds.

Delta-aminolevulinic acid dehydratase (δALAD) activity in four free-living bird species exposed to different levels of lead under natural conditions.

The purposes of this study were: (1) to determine the δALAD activity and δALAD ratio in blood of four free-living bird species (Griffon vulture, Eagle owl, Slender-billed gull and Audouin's gull); (2) and to investigate the correlations between δALAD activity/ratio and Pb concentrations in blood samples. A decrease was observed in δALAD activity in Griffon vultures and Eagle owls exposed to Pb. In addition, negative relationships were found between δALAD ratio or δALAD activity and Log blood Pb levels in Griffon vultures and Eagle owls, and these relationships were stronger in areas with the highest Pb exposure. We provide equations that may be helpful to estimate δALAD activity and δALAD ratio using blood Pb concentrations. Regarding gull species, δALAD activity found in the present study may be considered the normal activity in Slender-billed gull and Audouin's gull species, since very low blood Pb concentrations and no correlations were found in these species. Although both δALAD activity and δALAD ratio are sensitive biomarkers of Pb exposure and effect in birds, the use of δALAD ratio may improve the results. Besides, this study provides blood threshold concentrations at which Pb bears effects on δALAD enzyme (5µg/dl in Eagle owl; 8µg/dl in Griffon vulture; and probably >2µg/dl in Slender-billed gull and Audouin's gull). Our findings show that Eagle owl seems to be more sensitive to δALAD enzymatic inhibition by Pb than Griffon vultures. Eagle owls and Griffon vultures exhibited up to 79% and 94% decrease in δALAD activity when blood Pb concentrations exceeded 19 and 30µg/dl, respectively. Regarding the effects related with δALAD inhibition, significant negative correlations were found between δALAD activity and hematocrit in Eagle owls and Griffon vultures, which may be related to compensatory response associated with a decrease in δALAD activity. In addition, an effect on creatine kinase activity and total proteins in plasma was found in Griffon vultures. The significant negative correlations found between δALAD activity and tGSH in Griffon vulture, and between δALAD activity and Log CAT activity in Eagle owls, may be related to a protective response of antioxidant system against reactive oxygen species (ROS). The negative relationship found between δALAD activity and TBARS levels in Griffon vulture is probably related to an induction of lipid peroxidation by ROS that may be generated by δALA accumulation when δALAD activity is depressed. δALAD activity and δALAD ratio in blood are suggested as important nondestructive biomarkers for Pb exposure and effect for future biomonitoring studies in Griffon vulture and Eagle owl. Further studies are recommended to provide new data on Pb concentrations at which δALAD activity is affected in different wild bird species and to elucidate why different species tolerate Pb in different ways.

Effects of heavy metals on biomarkers for oxidative stress in Griffon vulture (Gyps fulvus).

Metals are involved in the formation of reactive oxygen species (ROS) which may result in metal-related oxidative stress that can lead to oxidative damage to lipids, DNA and proteins. It is necessary to understand the mechanisms of metal toxicity in wild birds, and the concentrations that cause effects on oxidative stress biomarkers. The aim of this study is to assess the concentrations of lead (Pb), cadmium (Cd), mercury (Hg), copper (Cu) and zinc (Zn) with regards to oxidative stress in blood samples of 66 Griffon vultures (Gyps fulvus) from two areas of the Autonomous Community of Valencia (East of Spain). The two study areas (Alcoy n=36 and Cinctorres n=30) were selected as random locations of interest that had not yet been studied, and are feeding stations where supplementary food, mainly of pork origin, is provided for vultures. Given that the two study areas are not considered polluted sites, we expected to find low metal concentrations. However, there are no known threshold concentrations at which metals can affect antioxidant systems, and low metal levels may have an effect on antioxidant biomolecules. In this study, since sampling was done at the beginning of the hunting season, the low Pb levels found in most Griffon vultures from Alcoy and Cinctorres (median=12.37 and 16.26µg/dl, respectively) are suggestive of background levels usually found in vultures that feed on pork carcasses all year round. The ingestion of game meat with bullet fragments in carcasses or with Pb shots embedded in the flesh could be the cause of the high blood Pb concentrations found in three vultures from Cinctorres (83, 290 and 362µg/dl). Griffon vultures feeding in Cinctorres had enhanced CAT and GST activities and tGSH concentrations, which may be interpreted as protective response against the higher TBARS levels. This study provides threshold concentrations at which metals affect antioxidant system derived from 66 samples of Griffon vulture. Blood Cd concentrations greater than 0.05µg/dl produced an induction of 33% in GPx and of 44% in CAT activity in erythrocytes of vultures from Alcoy. Hg concentrations in blood higher than 3µg/dl produced an induction of 10% in SOD activity. Concentrations of Pb above 15µg/dl in blood produced an inhibition of 12.5% in GPx and 11.3% in CAT activity, and a TBARS induction of 10.7% in erythrocytes of Griffon vultures.

Razorbills (Alca torda) as bioindicators of mercury pollution in the southwestern Mediterranean.

Levels of mercury (Hg) were analyzed in the tissues of 50 Razorbills (Alca torda), from the Mediterranean area, which had drowned in fishing nets. The mercury distribution pattern in tissues was similar to those of other studies (liver > feather vane > kidney > muscle > brain > feather shaft), with mercury concentrations of 2.85 ± 0.90, 2.66 ± 1.60, 2.23 ± 0.87, 1.54 ± 0.54, 1.48 ± 0.54 and 1.30 ± 0.76 mg/kg (dry weight), respectively. It could be considered that Razorbills in the southwestern Mediterranean were chronically exposed to relatively low levels of MeHg, probably below 0.5 ppm, via dietary intake. We have proposed prediction equations for brain and kidney Hg concentrations using feather shafts as non-invasive samples. This work provides a solid understanding of Razorbill Hg exposure both in their wintering and breeding grounds, and shows that this species can be useful for assessing marine environmental health in the Mediterranean area.

Razorbill (Alca torda) feathers as an alternative tool for evaluating exposure to organochlorine pesticides.

The aim of this study was to explore the usefulness of feathers as a biomonitoring tool for organochlorine pesticides (OC) in a razorbill population (Alca torda). Fifteen OC were analyzed in feathers, including α-, ß- and δ-hexachlorocyclohexane, lindane, aldrin, dieldrin, endrin, endosulfan I and II, endosulfan sulfate, p,p'-DDT, DDD, DDE, heptachlor and its epoxide. The geometric mean concentrations observed in this study were ∑DDT 67.40 ng/g, ∑HCH 62.88 ng/g, ∑Heptachlor 61.75 ng/g, ∑Endosulfan 19.70 ng/g, and ∑Drins 10.17 ng/g. The higher OC levels found in this study compared with other studies are probably affected by the razorbill diet and migration status. However, levels found in the feathers of the present study are related to concentrations in internal tissues below those which cause adverse reproductive and behavioral effects or other signs of organochlorine-pesticide poisoning in birds. Age does affect the concentration of OC pesticides in feathers. Thus, feathers would appear to be a promising tool for OC biomonitoring in seabirds, since it is possible to quantify OC compounds.

Assessment of organochlorine pesticide exposure in a wintering population of razorbills (Alca torda) from the southwestern Mediterranean.

Sixteen organochlorine pesticides (OC) were analyzed in several tissue types (abdominal and subcutaneous fat, liver and brain) from juvenile (n=14), immature (n=9), subadult (n=7) and adult (n=20) razorbill (Alca torda) collected from the southwestern Mediterranean coastline, in the East of Spain (La Marina, Elche, Alicante, Spain). These razorbills had drowned in fishing nets (most probably) while searching for food. The objective was to assess the exposure to organochlorine pesticide residues in this wintering population of marine birds. This paper presents, as far as we are aware, the first published data on OC concentrations in razorbills. The highest levels were found in abdominal fat followed by subcutaneous fat, liver and brain. A significant positive relationship was found between age and OC levels in tissues, and with the highest levels in adults. The group of sigma Drins had the highest concentrations, followed by sigma DDT, sigma Endosulfan, sigma HCH and sigma Heptachlor, with endrin aldehyde being the compound which reached the highest levels. The p,p'-DDE/p,p'-DDT ratio in fatty tissues suggests exposure to non-degraded DDT and thus is present in the environment despite its prohibition. The OC levels detected were higher than those found in other studies on Alcidae, which may be explained by the Mediterranean habitat in which the birds were found. However, these levels are below concentrations for which any observable effect has been described.

Heavy metals in tissues from loggerhead turtles (Caretta caretta) from the southwestern Mediterranean (Spain).

Cadmium, lead, copper, and zinc were measured in tissues of 21 loggerhead turtles (Caretta caretta) from the southwestern Mediterranean coastline. Mean concentrations (dry weight) of essential elements (Zn and Cu) were 107 and 21.6 microg/g in liver, 27.9 and 3.8 microg/g in kidney, 65.4 and 5.0 microg/g in pectoral muscle, 11.1 and 3.45 microg/g in brain, and finally 19.2 microg/g and undetected in bone, respectively. Mean concentrations of heavy metals (Cd and Pb) were 23.4 and 2.8 microg/g in liver, 31.5 and 0.5 microg/g in kidney, 0.2 and 0.3 microg/g in pectoral muscle, 0.2 and 0.7 microg/g in brain, and undetected and 1.2 microg/g in bone, respectively. Metal concentrations were similar to other studies conducted on Mediterranean turtles. However, cadmium concentrations varied widely among individuals, which has been associated with potential sources of cadmium in Mediterranean Sea. This is the first study into metal accumulation in tissues of loggerhead turtle from Spanish Mediterranean coastline.

Changes in blood pesticide levels in booted eagle (Hieraaetus pennatus) associated with agricultural land practices.

We estimated the degree of exposure to lindane and endosulfan in the blood of booted eagle nestlings (Hieraaetus pennatus) (1999-2003), in order to assess the usefulness of these samples as a unit for monitoring changes in exposure as a result of shifts in agricultural practices and the implementation of legal measures. The highest blood lindane concentrations were obtained 1 year prior to its prohibition by the European Union. Subsequent to that year, the drop in blood concentrations was dramatic. Furthermore, endosulfan blood concentrations follow a progression coinciding with an increase in olive, grape and plum-tree crops. We conclude that concentrations of organochlorine pesticides in the blood of booted eagle nestlings may be used to monitor the use of those pesticides over a particular agricultural region and alert the authorities of possible environmental or health risks.

Raptor ecotoxicology in Spain: a review on persistent environmental contaminants.

Initial studies on the pressure from environmental contaminants on raptor populations in Spain date back to the 1980s, and they have been carried out from a range of viewpoints using a range of sentinel raptor species. However, there is no national monitoring scheme, and therefore the research carried out has been sporadic both spatially and temporally. The exposure to metals has not varied over time, except in the case of lead, whose concentration in eggs and tissues has diminished. In general, the concentrations of metals detected in raptor samples from Spain are generally low and not sufficient to produce toxic effects. Excepting DDT and DDE, most organochlorine-based pesticides in raptors from Spain have diminished over the last 2 decades. The concentrations of DDE found in the eggs of various species could in part explain problems in the reproductive success of raptors in Spain.

Relationship of the toxicity of pesticide formulations and their commercial restrictions with the frequency of animal poisonings.

Deliberate poisoning of domestic animals and wildlife with commercial formulations of pesticides has been documented worldwide. We have compiled the analytical results obtained by four Spanish Laboratories of Veterinary Toxicology since 1990. The frequency of poisonings and the intentional use of pesticides were compared between formulations with restricted and unrestricted use, including the toxicity of the commercial formulations as a covariant in the model. The frequency of poisoning was inversely related with the lethal dose of specific formulations, but not with the amounts consumed in agriculture in Spain. The intentional illegal use of some pesticides as poisons was not affected by the commercial restriction of their formulations, but was inversely correlated with their LD(50). The examination of the data permits us to detect three highly toxic compounds (aldicarb, carbofuran, and strychnine), more widely implicated in animal poisonings than other compounds of similar toxicity.

Organochlorine residues in booted eagle (Hieraaetus pennatus) and goshawk (Accipiter gentilis) eggs from southeastern Spain.

Most organochlorine (OC) use has been banned in Spain, but these compounds are persistent and may still adversely affect predatory birds. Data generally are lacking, however. Residues of hexachlorobenzene, alpha-hexachlorocyclohexane, lindane, hexachloro-octahydro-epoxy-dimethanonaphthalene, DDT, dichlorodiphenyldichloroethane, dichlorodiphenyldichloroethylene (DDE), and polychlorinated biphenyls (PCBs) were measured in 22 failed eggs of booted eagles (Hieraaetus pennatus) and goshawks (Accipiter gentilis) from southeastern Spain; both species are declining in this region. Hexachloro-octahydro-epoxy-dimethanonaphthalene, DDE, and sum PCB congener concentrations were significantly higher in booted eagle than in goshawk eggs, and an inverse relationship was found between shell thickness and DDE concentrations in booted eagles. Organochlorides may have been associated with the failure of some booted eagle eggs, but concentrations in booted eagle and goshawk eggs decreased over the period during which populations have dwindled. Thus, although OCs may be a contributory factor, they are unlikely to be the primary cause of the recent population declines in southeastern Spain.