Going wild: Pesticide screening method for poisoned wildlife and pets

Wildlife and pets threatened by pesticides

Image: National Park Service

The widespread use of pesticides inevitably leads to incidents of accidental poisoning of other creatures which were not the intended targets, be they wild animals, livestock, pets, birds, or important insects like bees. Unfortunately, these are not the only wildlife poisoning events that occur, with unscrupulous criminals targeting wildlife and birds, often using pesticides.

In the UK, the Wildlife Incident Investigation Scheme (WIIS) investigates pesticide poisonings with two key objectives. Firstly, they aim to enforce the correct use of pesticides while identifying and penalising those who misuse and abuse them. Secondly, they provide information to the regulator on pesticide hazards to wildlife and companion animals and beneficial invertebrates.

In Scotland, this operation is carried out by Science and Advice for Scottish Agriculture (SASA) which is based in the outskirts of Edinburgh. Here, they investigate cases of suspected animal poisoning, carrying out all of the sample preparation and analysis work. Apart from the victims of poisoning, the scientific team also undertake the analysis of suspicious substances and baits that have been laced with pesticides.

It came as a surprise to me that the number of suspected poisonings referred to SASA in 2010 and 2011 totalled 233 and 237, respectively, and these incidents are on the increase. For 2011, the major mammalian victim was the fox (60 incidents), with far fewer occurrences for other species such as badger (2) and red squirrel (2). The most reported incidents for birds affected the buzzard (60), red kite (17) and sparrowhawk (15) but many common birds like sparrows, finches, crows and swans were also investigated. Poisoned pet cats and dogs were also brought in for examination.

Until 2010, the scientists at SASA used GC/MS/MS methods almost exclusively for monitoring multiple pesticide residues in the biological specimens but this was limited to some extent because not all pesticides are amenable to this technique. Now, they have added LC/MS/MS to their inventory of analytical techniques to expand the range of pesticides for which they can test. Michael Taylor, Head of Pesticides & Wildlife at SASA, and colleagues, noted the dearth of reported methods for measuring multiple pesticides in pets and wildlife, so they developed their own.

More than 100 pesticides

One key aspect of method development was the desire to streamline the sample preparation procedure, so the researchers based the extraction around ethyl acetate, which was in place for the GC/MS method. This way, a single extract can be divided up for analysis by GC/MS and LC/MS.

A total of 102 target pesticides, metabolites and degradation products were included, some being chosen to extend the WIIS-Scotland inventory, reflecting past and present pesticide usage patterns in Scotland. Other pesticides that had been detected in non-target animals in the UK, but were hardly detected in Scotland, were also included, such as fenthion and methomyl. Finally, banned pesticides like carbofuran and aldicarb were added as they are known to have been used in illegal Scottish poisoning incidents.

Samples of chicken muscle and liver tissue that were described as organic were bought from local outlets to prepare blank matrices using the standard extraction procedure. Solutions of the pesticides were also added to the tissues at 0.1 or 1.0 mg/kg, these concentrations being consistent with the lower levels found in accidentally or deliberately contaminated animals.

The extracts were analysed by LC/MS/MS with electrospray ionisation in positive ion mode. In the vast majority of cases, the protonated molecule was selected for multiple reaction monitoring and the compounds were screened following HPLC separation using a solvent gradient.

Due to limitations of the data system, it was not possible to screen and confirm all 102 analytes in one run. Instead, when the preliminary screen suggested a positive result, the sample was rerun while monitoring a second MRM transition for confirmation, using a more rapid isocratic HPLC separation. The recovery figures for the spiked tissues were all deemed to be acceptable for screening purposes and the pesticides were measured by comparison with external calibration curves.

Eagle eyed technique

The new LC/MS/MS technique was used at SASA throughout 2011, detecting pesticide levels from 0.01 mg/kg to several thousand mg/kg in dead creatures. Exposure to pesticides was confirmed as the cause of death, supported by post mortem examinations which ruled out other causes.

Apart from the species listed in the 2011 Annual Report on Pesticide Poisoning of Animals, Taylor told spectroscopyNOW.com that "the method has been applied to the investigation of the suspected poisoning/contamination of many other species. The range is diverse, as we have also tested such species as Scottish wildcat, hare, hen harrier, honeybee, pine marten, tree creeper, osprey and meerkat!"

In one example, the crude liver extract of a dead golden eagle recovered in Scotland was found to contain carbofuran, which was banned from 2001 in the UK. This pesticide is permitted in other countries but is known to be highly toxic to birds and mammals.

The team did offer one note of caution. If some samples need to be re-examined at any stage, then pesticide stability becomes a factor. In one test, the peak area response of carbosulfan was reduced by 46% during seven days of storage, so measurement would be affected.

New pesticides can be added to the target list easily and the method can be used to prove or disprove the presence of pesticides in many species. It can also be applied to blood, vomit and faeces in cases where animals have become sick but not died, providing a wide ranging method for pesticide poisoning studies.