Forensic Potential of Entomotoxicology

Acknowledgements

The author would sincerely like to thank Dr S from the University of Central Lancashire for supervising throughout the writing of this dissertation. He would also like to thank his family and his support mentor for their guidance throughout the writing of this paper and the rest of his degree.

Abstract

The following study uses global research to examine the forensic potential of entomotoxicology. A variety of existing research studies have been examined to determine how xenobiotics affects insect development and an evaluation has been carried out on the range of analytical techniques used. The purpose of this paper is to assess whether entomotoxicology can be used to determine a post-mortem interval and to work towards the standardisation and validation of a methodology for entomotoxicological research methods and processes.

A major contemporary aspect considered within this paper is the lack of a definitive correlation between the concentration of xenobiotics inside the insect and the chosen substrate. This has led to lengthy discussion around the type of substrate used and sites from which samples are taken. Consequently, a need to consider the standardisation of entomotoxicological research processes is apparent.

Some definitive conclusions can be drawn from the global research considered to date. For example, xenobiotics do influence insect development and can affect time of death estimations. Current research agrees that entomotoxicology does have forensic potential. However, currently there is insufficient evidence to suggest a correlation between the insect and the concentration of xenobiotics found inside the substrate. This apparent lack of correlation could perhaps be remedied if standard procedures were followed. Therefore, the analysis of contemporary research and findings have inspired the author’s determination to develop such a standardisation. A standard protocol has been proposed and developed within this paper.

Keywords: Forensic science, Entomotoxicology, Post-mortem interval (PMI), Xenobiotic detection, Standardisation, Protocol

Introduction

The field of forensic science is rapidly changing due to both the ever-increasing complexity of and advancements in, technology and human enquiry. Entomotoxicology as an aspect of forensic science, has untapped potential for further enquiry.

Because entomotoxicology is still in its research and development phase there is currently no agreed definition. Goff and Lord (1994 p.2) first defined entomotoxicology ‘as the use of insect specimens for toxicological analysis in the absence of tissues and fluids normally taken for purposes of death investigations.’

However, da Silva et al., (2017, p.1399) defines entomotoxicology as ‘the use of insects as evidence of whether a toxicant is present in an environment such as a corpse, river or landscape’.

Hodecek (2020, p.20) combined both of the above definitions and defined entomotoxicology ‘as the analyses of the effects of xenobiotics on insects and uses xenobiotics present in insect bodies as evidence of environmental pollution. The environment can be pictured as carrion, soil, river, landscape, or even the body of a patient (according to the clinical applications)’. However, without a replicable approach to this discipline, its importance and contribution could be overlooked.

This study will use current global research to determine which areas of entomotoxicology have previously been investigated and where there is the potential for further enquiry. The study will consider methods where insects can be used to recover samples from badly decomposed bodies and the pros and cons of using insects as toxicological samples. This dissertation will propose the use of a standard protocol for entomotoxicological research in the UK.

According to Gennard (2012, P.1) forensic entomology is the “use of information about insect lifecycles and behaviour to help interpret evidence in a legal context relating to both humans and wildlife”. The main application of forensic entomology in a legal context is estimating the amount of time that someone has been dead. This is known as the post-mortem interval (PMI) (Tomberlin and Byrd, 2019). After death, a human body goes through a scientific process known as decomposition. Decomposition is ‘the process by which organic matter is broken down into simpler forms of matter’ (French and Jacques, 2020). The process of decomposition can be split into five different stages. These are known as: ‘fresh, bloat, active/advanced decay, dry, and remains’ (Tomberlin and Byrd, 2019, p.143). During the process of decomposition, the body goes through multiple biological, physical and chemical changes (Tomberlin and Byrd, 2019). Many different saprophagous arthropods appear at the body, to feed on both the dying tissue and the other insects found in the body. Forensic entomology is mainly used on a cadaver that has been decomposing longer than 72 hours. This is because there is very little medical data left to be used by a pathologist to determine a PMI (Gennard, 2012).

Effects of entomotoxicology on a minimum post-mortem interval (mPMI)

There are two pieces of evidence that entomologists can use to estimate a post-mortem interval. These are insect development and ecological succession (Tomberlin and Byrd, 2019). Different types of arthropods are categorised into different families by forensic entomologists. Each family of insects appears at different stages of decomposition of the body. The measurement of the patterns that different families of insects appear is known as ecological succession (Tomberlin and Byrd, 2019).

The effects that different types of xenobiotics have on insect succession and insect development is still under-researched, however, it is recognised that drugs can cause significant changes in the development rate of necrophagous insects (Lord, 1990). It is also known that these changes can alter PMI estimates, leading to substantial errors in calculations if they are not accounted for during the investigation (Lord, 1990). This dissertation provides some information as to how certain drugs affect the growth rate of insects and how this affects the post-mortem interval (PMI).

Size based developmental estimates

Entomologists can also estimate a PMI by referring to regression models. These are used to determine the relationship between the size of an insect and its age (Villet et al.,2010). GOFF et al., (1992) found that when larvae of Sarcophaga Ruficornis Fabricius were fed on tissues contaminated with Methamphetamine, they matured at a smaller size than the control group. Similar effects were observed when larvae of Calliphora vomitoria were fed on liver spiked with nicotine (Magni et al., 2016). Another study found that the body lengths of the larvae of Lucilia sericata and Lucilia cuprina reared on liver spiked with a median lethal dosage of Ethylene Glycol, were smaller than the larvae that had been reared on a control substrate (Essarras et al., 2018). Diazepam was also found to stunt the growth size and shape of larvae and puparia of Chrysomya albiceps (Carvalho et al., 2001; Carvalho, L. M. L., 2010). The opposite was seen when larvae of Sarcophaga peregrina were found to grow to a larger size than the control, when they were exposed to Cocaine (Goff et al., 1989).

Event based developmental estimates

Another way that entomologists can estimate a PMI is by measuring the time taken to reach various developmental milestones (Villet et al., 2010). GOFF et al., (1992) found that larvae of Sternocera ruficornis grew at a faster rate than the control group, when fed on tissues containing Methamphetamine. This caused estimates to be inaccurate in the post-mortem interval by up to 18 and 48 hours, based on the larval and puparial development.

Unfortunately, the effect of a particular toxicant does not always have the same effect on two flies that are closely related. One example of this is Ketamine. Ketamine was found to substantially prolong both the larval and pre-pupal stages of C. megacephala (Lü et al., 2014), but had the opposite effect on the larvae of L. sericata (Zou et al., 2013). No reports currently exist that show a toxicant’s effect on cadavers’ tissues, in relation to timing or patterns of succession. Furthermore, any changes to the development rates of necrophagous insects may impact on ecological succession (AbouZied, 2016).

The above research shows that drugs can influence the evidence that entomologists use to determine a minimum post-mortem interval. If the entomologist is not familiar with this, it will lead to a misinterpretation of the minimum post-mortem interval. Unfortunately, this area of entomotoxicology has not been researched enough for entomologists and toxicologists to be able to confidently use these results. Clearly It will never be able to be used as an autonomous piece of evidence, due to there being so many variables that can affect the results. However, if research has been carried out properly using standard protocols, entomologists will be able to use the findings to assist with a more reliable time of death estimation.

What are some of the applications of forensic entomotoxicology?

The main application of forensic entomotoxicology is the detection of different types of xenobiotics in insects of forensic importance (Hodecek, 2020). However, Hodecek (2020) suggests four other potential applications of forensic entomotoxicology, which are: the detection of particular xenobiotics; determining the relationship between the concentration of xenobiotics detected in the recovered insect samples and the environment that they were found in; the influence that certain drugs have on the growth rate and morphology of the recovered insects; and the testing of different methods for xenobiotic detection.

Methods of detection for xenobiotics

Several analytical techniques have been used in entomotoxicological research for xenobiotic detection. These include: Gas Chromatography Mass Spectrometry (GC/MS), Liquid Chromatography Mass Spectrometry (LC/MS), High Performance Liquid Chromatography (HPLC), and immunoassay techniques.

Chophi et al., (2019) analysed the trends in analytical techniques across 66 papers between 2001-2018 and confirmed that all the above methods provided positive detections for xenobiotics.

Quantitative analyses

Despite providing a positive detection for xenobiotics, some methods are more sensitive and accurate than others at detecting the quantity and concentration of drugs. It is important that the method chosen is as sensitive as possible, to allow for the lowest concentration of drugs to be detected (Gosselin et al., 2011).

HPLC was used on larvae that had been reared on minced meat spiked with morphine concentrations of 500 ng/g and 1000 ng/g. It was found that this technique could not quantify morphine at these levels of concentration. Morphine quantification was only found to be possible in larvae that had been fed on minced meat with a concentration of 2,500ng/g or more.

Chophi et al., (2019) and Gosselin et al., (2011) found that the best method for quantification of drugs at low dose levels is either GC/MS or LC/MS. Pien et al., (2004) found that the capacity to quantify and detect the drug Nordiazepam and its metabolite Oxazepam was possible from a single larva and puparia down to the picogram level. Previous research has not always tested with such precision and this will obviously have implications for any forward going research.

Additionally, researchers have managed to quantify drugs from different areas of the body using GC/MS and LC/MS. The results have shown varied concentrations inside the lungs, liver, heart, blood, brain, urine and skin (Pien et al., 2004; Bushby et al., 2012). The concentration of drugs was found to be higher inside the liver than any other organ (Chophi et al., 2019). It is therefore advised that quantitative results are interpreted with caution (Chophi et al., 2019). This will have implications for standardising any further research protocols.

Correlation analyses

Debate regarding drug concentrations found within substrates and insects is ongoing and provokes much controversy around this topic (Gosselin et al., 2011).

Entomotoxicological research has shown that analyses can be performed on insect samples and their larvae, however, numerous scientists are still sceptical about correlation studies (Gosselin et al., 2011). This problem exists because ‘drug pharmacokinetics, metabolism, drug redistribution, drug accumulation and feeding activity of larvae’ can all have an impact on correlation studies, but it is not known to what extent (Chophi et al., 2019.p33). Tracqui et al., (2004) conducted a study on 29 human cadavers that were suspected to have died from poisoning. The investigation did not find a correlation between the drug concentration in human tissue and larval samples. After analysing the inter-site variation it was found that the difference in drug concentration in the larvae was due to movement around the cadaver.

Trends In analytical techniques

There are two main variables that affect the ability to identify drugs in putrefied or decomposing tissue. These being the effectiveness of the extraction method and the chosen analytical technique (Chophi et al., 2019).

In the early stages of entomotoxicological research, scientists used techniques such as immunoassay and chromatography. However, the inefficiency of these techniques resulted inconclusive analysis with the possibility of false positives.

In recent years research has been carried out to try and find alternative spectroscopy techniques that are non-destructive and can be used in conjunction with statistical tools, such as ‘PCA (Principal Component Analysis), SPA (Successive Projection Algorithm), and GA (Genetic Algorithm)’ (Oliveira et al., 2014, p41).

Fluoxetine was detected by UV spectroscopy at all development stages and in the exuviae of D. Maculatus (Zanetti, Ferrero and Centeno, 2016). Another study showed that NIR spectroscopy in conjunction with statistical tools could provide information regarding the differences in concentration of the drug flunitrazepam (Oliveira et al., 2014). From this It has been concluded that the use of statistical tools can be applied with accuracy and with reliability. However, it was recognised that further testing was required on the methodology and recommended that larger databases on a wider range of insects should be produced (Oliveira et al., 2014).

Factors effecting xenobiotic concentration

Unfortunately, there are many post-mortem, biological and chemical changes that can affect the quantification of entomotoxicological samples (Tomberlin and Byrd, 2019). As a result it is believed that entomotoxicology is of limited quantitative value in forensic toxicology (Tomberlin and Byrd, 2019).

For example changes in concentration of drugs due to enzymatic activity and post-mortem bacterial degradation, leads to the production of ethanol (Tomberlin and Byrd, 2019). This is a problem for toxicologists as the alcohol detected by the toxicological analysis, could be misinterpreted for alcohol that had been consumed before death (O'Neal and Poklis, 1996).

What is my overall aim and what are my objectives?

The overall aim of this project is to determine whether entomotoxicology has forensic potential and if it can be used as an aid in forensic enquiries. The objectives of this project are, to provide an overview of the stages of decomposition and insect succession; use secondary research to assess the significance of forensic entomotoxicology with regards to PMI and determining cause of death; to discuss the weaknesses and strengths of entomotoxicology; to investigate the usefulness of a standard protocol for entomotoxicology research across the UK; to determine whether more research is required to ascertain the value of entomotoxicology

Results

Analysis of techniques used across entomotoxicological research

Chophi et al., (2019) analysed data on the analytical techniques used across 66 papers between 1980 and 2018 (see table 1).

Table 1

Published Papers Showing Techniques used for Analyses of Toxicological Samples
Technique used for analyses of toxicological samples	Number of published papers using technique	References
Immunoassay	17
(LC/GC) Liquid or gas chromatography	6
Spectroscopy techniques	7
(HPLC) High-performance liquid chromatography	10
(GC/LC-MS) Liquid or gas chromatography- mass spectrometry	26

Figure 1: shows that the most used analytical technique across entomotoxicological research is GC/LC-MS at 39%, followed by Immunoassay techniques at 26%, with HPLC at 15%, then spectroscopy techniques at 11% and LC/GC at 9%.

Most used classes of drug in experiments (see Chart 2)

Chart 2

Bhardwaj et al (2020) Collated the data from 66 papers written between 2001-2020. One area that they highlighted was the most common drugs and toxins being used across the 66 studies. They found that 27% of the studies were using stimulants, 23% were using sedatives, 19% were using opioids, 17% were using miscellaneous drug classes and finally 14% used insecticides.

They also found that the most used insect across the 66 studies was C. megacephala (Bhardwaj et al., 2020), due to it being the most abundant forensically relevant fly across the world (Badenhorst and Villet, 2018). C. albiceps and C. putoria were found to be the second and third most used dipteran species in entomotoxicological research (Bhardwaj et al., 2020).

Data analysis of key themes across entomotoxicological research

Ten studies all written between 2010 and 2020 were analysed to identify any key themes within entomotoxicological research and three were identified across the ten studies. The three key themes were: the need for standardisation and validation of methodology (see table 2); the correlation between the amount of toxin detected in the substrate and the insects (see table 3); and whether entomotoxicology has forensic potential (see table 4).

Standardisation and validation of entomotoxicology

Table 2

Comment	Number of Studies	References
Agreed that there is a need for standardisation and validation of research	7	(Bhardwaj et al., 2020; Chophi et al., 2019; da Silva et al., 2017; Gosselin et al., 2011; Hodecek, 2020; Ishak et al., 2019; Magni et al., 2018)
Disagreed that there is a need for standardisation and validation of methodologies	0
No analysis of the need for standardisation and validation of methodology	3	(Basilicata et al., 2019; Magni et al., 2016; Salimi et al., 2018)

Table 2 shows that out of the ten studies analysed, 70% agreed that entomotoxicological methodology needs to be validated and standardised. None of the studies disputed the idea that standardisation and validation is needed. The remaining 30% of the studies did not discuss this key issue.

Correlation between concentration of drug in the insect and substrate

Comment	Number of studies	References
Agrees that there is a correlation	0
Disagrees that there is a correlation	5	(Basilicata et al., 2019; Chophi et al., 2019; Gosselin et al., 2011; Ishak et al., 2019; Salimi et al., 2018;)
No discussion on correlation	5	(Bhardwaj et al., 2020; da Silva et al.,2017; Hodecek, 2020; Magni et al., 2018; Magni et al., 2016)

 

Chart 4

Table 3 shows that out of the ten studies analysed, 50% concluded that there is no correlation between the concentration of drugs found inside the substrate and insects. None of the studies agreed that there is any correlation at all. The remaining 50% of the studies did not cover this theme.

Does Entomotoxicology have Forensic Potential?

Table 4

Comment	Numbers of studies	References
Agree that it has potential	6	(Bhardwaj et al., 2020; Chophi et al., 2019; Gosselin et al., 2011; Hodecek, 2020; Ishak et al., 2019; Magni et al., 2016)
Does not agree that it has potential	0
Does not discuss whether it has potential	4	(Basilicata et al., 2019; da Silva et al., 2017; Magni et al., 2018; Salimi et al., 2018)

Table 4 shows that out of the 10 studies analysed, 60% of them agreed that entomotoxicology has potential for use in forensic science, none thought that it had no potential at all, while 40% of the studies analysed, did not discuss its potential.

Discussion

Key themes across entomotoxicological research

The idea of using insects as toxicological samples was first proposed in the latter half of the 20^th century, however, it was not until the early 2000’s that increased research into this area of entomology was seen. Since the idea of entomotoxicology was first proposed there have not been many papers written on this subject. Nevertheless, multiple key themes have arisen from these articles, with most focussing on entomotoxicology’s main application, which is the use of insects as toxicological samples.

The most common theme identified is the need for standardisation of practice and the creation of protocols for entomotoxicological research. Da Silva et al (2017) concluded that the creation of a standard protocol would be ideal for scientists around the world and this would make the results more comparable and therefore valid. Hodecek also identified this key theme in his research and stated that entomotoxicology is inconsistent and needs to have its methodology standardised (Hodecek, 2020). Da Silva et al (2017) also pointed out that the standard protocol should be both easy to use and cost efficient. they state that it should make replication and hormesis, i.e., effective low dose usage, observable and valid.

Another key theme found across entomotoxicological research is the issue of whether there is a correlation between the concentration of drugs found in both the substrate and insects. This notion is discussed by Chophi et al (2019) in which they stated that this is an area of high controversy. When considering several lines of enquiry, Chophi et al., found that there was hardly any evidence to suggest that there is a correlation between the concentration of the drug detected inside a human tissue and the concentration found in larval samples and that hardly any quantitative relationship exists (Chophi et al., 2019). Additionally, the type of analytical techniques used to extract and detect drugs in insect samples is a key area of discussion. Chophi et al. (2019) analysed the trends of analytical techniques used across entomotoxicology and concluded that both chromatographic and mass spectrometry techniques had been used most and were the preferred choice of technique. They also point out that in previous years immunoassay techniques were widely used, but as time passed by, they were superseded by spectroscopy techniques. However, they do conclude that immunoassay techniques are good for detecting drug classes and identifying whether further analytical tests are needed.

Both da Silva et al., (2019) and Tomberlin and Byrd (2019) expand on these weaknesses. Tomberlin and Byrd concluded that the primary weakness of entomotoxicological research was the myriad of methodological approaches used in the research (Tomberlin and Byrd, 2019, p.299). Da Silva et al., also highlighted some key weaknesses in entomotoxicological research. These included no standardisation, insufficient replication and expense (da Silva et al., 2017, p.1409).

The final key theme identified concerned insect sampling sites. Both Gosselin et al (2011) and Chophi et al (2019) argue in favour of the importance of collecting multiple insect samples from different parts of the body and both concluded that drug concentration is found to be at its highest inside the liver. Gosselin et al., further expanded on this theory and concluded that areas such as the head and muscles are the best places to recover an insect sample when the liver has fully decomposed (Gosselin et al., 2011).

One unexpected finding of this research was that very few studies investigated alcohol and its effect on insect development (Tomberlin and Byrd, 2019). This was surprising as alcohol is the drug that is most detected by forensic laboratories and is one of the leading causes of drug related deaths (Tomberlin and Byrd, 2019). Alcohol detection can provide vital information as to the circumstances in which someone died (Tomberlin and Byrd, 2019). A possible explanation for the lack of studies, is that toxicology results involving alcohol are very difficult to interpret even in routine casework (Tomberlin and Byrd, 2019). This is because the body naturally goes through a process known as post-mortem fermentation during decomposition, when the bacteria in the body breaks down and produces ethanol (O'Neal and Poklis, 1996). This is a problem for toxicologists as the alcohol detected by the toxicological analysis, could be misinterpreted for alcohol that had been consumed before death (O'Neal and Poklis, 1996).

Having identified some of the weaknesses of entomotoxicological research, it is important to note that researchers have also identified some strengths. For example, Hodecek (2020) concluded that despite entomotoxicology having flaws, it does show a great amount of potential in both environmental and forensic sciences. Tomberlin and Byrd (2019) stated that once entomotoxicology is standardised and protocols have been created, the importance of entomotoxicology will increase. Chophi et al., (2019) concluded that when conventional toxicological samples are not available for analysis, insects and larvae can be used reliably as toxicological specimens for xenobiotic detection.

What do the results show?

Overall, it can be argued that these studies highlight the need for the standardisation of entomotoxicology and the need for the creation of protocols. When considering all the above evidence, it seems that entomotoxicology does have some forensic potential, however such studies remain narrow and only really focus on using insects of forensic importance to detect xenobiotics.

Why are standardisation and protocols important in the field of forensic science?

According to Wilson-Wilde (2018) the term standard is used scientifically to describe multiple types of documents developed by different Standards Development Organisations (SDOs).

Historically, the forensic science industry has been largely unregulated. However more recently, attempts at developing standards and guidelines have resulted in the development of an International Organisation for Standardisation (ISO). These standards are now used as a reference point in forensic science across the world.

The International Standards Organisation is an independent organisation that works alongside experts to create ‘voluntary, consensus- based, market relevant international standards’ (ISO, 2021).

The ISO provides guidelines for providers and users to follow in respect of the quality standards of their products and services. These standards can be applied using three different methods. These are: self- regulation, certification and accreditation (Wilson-Wilde, 2018). Forensic science is accredited under the competency-based standards, which are developed by the ISO Committee on COnformity ASsessment (CASCO) (ISO, 2021). In the UK, most forensic science providers (FSP) are now either accredited or awaiting accreditation under the United Kingdom Accreditation Service (UKAS) (UKAS, 2019). Although it is still not compulsory for FSP’s to be accredited under UKAS, it is highly recommended as it reassures the courts and the criminal justice system that the FSP’s are following ISO standards and ensures that there are protocols put into place and that everything is validated (UKAS, 2019).

It is this kind of standard that needs to be applied to entomotoxicology if it is to gain recognition as a reliable tool in forensic science.

Creation of a standard protocol for experimental work

Whilst considering a standard protocol for entomotoxicological experimental research there are three specific areas that need to be controlled and monitored. These are: environmental conditions; storage and collection of samples; analyses of samples. Controlling and monitoring these three specific areas will not only make the experiment more realistic and reliable, but it will also make it easier for other researchers to follow, therefore making the experiment more replicable. The collection and storage of biological samples will follow the guidelines published by Dinis-Oliveira et al (2016) and the collection and storage of entomological samples will follow the guidelines set by (Amendt et al., 2015). The protocol will be based on these two sets of guidelines as they are approved by the European Council of Legal Medicine (Tomberlin and Byrd, 2019).

Whilst conducting the research it is very important that the insects are exposed to a realistic environment (Gosselin et al., 2011). Conditions such as humidity, temperature and climate need to be controlled in accordance with the region where the insects originate from (Gosselin et al., 2011). The experiment will also need to have a realistic photoperiod e.g., 12 hours light and 12 hours darkness (Gosselin et al., 2011).

Another issue that the protocol will address is the collection of biological and entomological specimens. Dinis-Oliveira et al., (2016) recommend using plastic containers for the storage of specimens, as they are less likely to break when exposed to cold conditions inside a freezer. However, when volatile compounds are being stored such as solvents or gasses, they recommend that glass containers are used and that they are sealed using Teflon or an aluminium foiled lid. Each lid should have a self-adhesive tamper-proof sticker placed across it (Dinis-Oliveira et al., 2016). It is highly recommended that new containers are used where possible and that all equipment is washed with distilled water and sterilised before usage (Dinis-Oliveira et al., 2016). When collecting larval samples, it is recommended that they are recovered using pliable forceps or a spoon (Amendt et al., 2015). A minimum of 10 specimens (preferably closer to 30) should be collected from multiple sites on the cadaver (Dinis-Oliveira et al., 2016; Gosselin et al., 2011; Tracqui et al., 2004). Tomberlin and Byrd (2019) recommend that live feeding larvae should be collected from the cadaver, as they are at the most desirable life stage to perform analysis on. Once all the samples have been collected, they should be washed with distilled water to avoid any surface contamination (Sadler et al., 1997). Each sample should then be packaged separately and labelled with the anatomical area from which they were recovered and their larval feeding stage (Tomberlin and Byrd, 2019). Due to toxin tropism the feeding substrate from which the insects were collected, must be noted on either waterproof paper or in pencil and placed into the storage container. Each container should then be logged on an appropriate recording system (Amendt et al., 2015). After the larvae have been packaged and recorded correctly, they should be placed into a freezer as soon as possible to stop the xenobiotic concentration reducing (Dinis-Oliveira et al., 2016). Any samples that have been collected for toxicological analysis should be stored at a minimum of -20degrees centigrade and no preservatives should be added to the storage container, as this will contaminate the samples (Dinis-Oliveira et al., 2016).

When samples are being collected for the purpose of DNA analysis, they must be retrieved and stored separately to any samples that have been collected for toxicological analysis (Tomberlin and Byrd, 2019). Once the samples have been collected, the insects should be drowned in twice their volume of >80% ethanol and placed inside a refrigerator (Amendt et al., 2015). The ethanol should be drained and replaced each day to ensure a suitable storage environment is maintained (Amendt et al., 2015).

All xenobiotics will be extracted using the Solid-phase extraction technique and then will be analysed with either of the Gas Chromatography-Mass Spectrometry or Liquid Chromatography-Mass Spectrometry methods, as these have been concluded to be the best analytical techniques by multiple authors (Tomberlin and Byrd, 2019)

Additionally, to assist with standardisation, the scientist will fill in a protocol form whilst conducting the entomotoxicological research (see table 5).

Table 5

Suggested Protocol Form for Experimental Entomotoxicological Research
What were the environmental conditions during experiment?	Temperature- Click or tap here to enter text. Humidity- Click or tap here to enter text. Photoperiod- Click or tap here to enter text.
Information about samples	Insect species- Click or tap here to enter text. Sample size- Click or tap here to enter text. Developmental stage- Choose an item. Type of animal substrate- Click or tap here to enter text. Tissue type- Choose an item. What drug/ drugs was the substrate spiked with? - Click or tap here to enter text. Concentration of the drug - Click or tap here to enter text. At what stage was the drug injected? - Choose an item.
Storage / collection of samples	Was all equipment sterilized before use? - Choose an item. Type of storage container - Choose an item. Storage method - Choose an item. Storage temperature - Click or tap here to enter text.
Analyses	Analysis type - Choose an item. Extraction method - Choose an item. Analytical method - Choose an item.

What are the strengths and weaknesses of forensic entomotoxicology?

Strengths

The results obtained in the research studies indicate that entomotoxicology could be an essential tool in medical legal investigations. Specifically, in drug related deaths, when the bodies are at the later stage of decomposition or have been exposed to fire for long periods of time. Conventional samples such as bodily fluids and tissue would no longer be available in these circumstances (Campobasso and Introna, 2001). Additionally, toxicological analysis of carrion insects will not only help with determining the cause of death, but also with providing more accurate PMI estimations. This is the main role of an entomologist in medical legal cases (Tomberlin and Byrd, 2019).

Entomotoxicology’s main strength according to Tomberlin and Byrd (2019) is the reliability of being able to detect toxicants, when examining different types of evidence and how this relates to the estimation of PMI’s.

Weaknesses

Tomberlin and Byrd (2019) state that the main weakness of entomotoxicological research is the lack of standardisation and the lack of correlation between toxicant levels inside the substrate and larvae. According to Edmund et al., (2014) the analysis of entomotoxicological findings does not currently meet the requirements for the standardisation and validation of protocols set by most international courts.

Another key weakness that needs to be discussed is the type of substrate used in entomotoxicological research. Currently the preferred substrate for entomotoxicological research is spiked animal tissue, due to it being low in cost and easy to prepare. However, this could be a major drawback with the research because the drugs are not being broken down by a living organ system (Gosselin et al., 2011). This is important for interpretation, as in real case scenarios the drug availability will be altered and the produced metabolites will also be consumed by the insect (Oliveira et al., 2009; George et al., 2009). Gosselin et al., (2011) suggest that one way to avoid this major drawback would be to use living animal substrates that have been injected with the drugs before death. This would not only be a more realistic approach but would also be a way to counteract this identified weakness as the drugs would be broken down and metabolised by a living organ system. However, this method also has some drawbacks. First it would be very hard to obtain statistically accurate results from simulated research (Gosselin et al., 2011) and second, approval would have to be gained from the ethics committee which can be a very long and tedious process (Mahat et al., 2009). Additionally, the scientist could never guarantee that it would accurately simulate a human drug overdose due to pharmacokinetic differences (George et al., 2009).

The following researchers have used human tissue in entomotoxicological research (Definis-Gojanović et al., 2007; Kintz et al., 1990; Kintz, Tracqui and Mangin, 1990; Manhoff et al., 1991; Miller et al., 1994; Nolte, Pinder and Lord, 1992; Tracqui et al., 2004). For example, muscle tissue was used by (Bourel et al., 2001; Wilson, Hubbard and Pounder, 1993) and liver was used by (Campobasso et al., 2004; Introna et al., 1990). However, Gosselin et al., (2011, p. 7) state that ‘using human tissue in entomotoxicological research is ethically questionable’.

This combination of findings shows that entomotoxicology does have both weaknesses and strengths, with the lack of validated standard protocols being the main weakness. However, Tomberlin and Byrd (2019) argued that entomotoxicology will be used more readily by scientists in the future as soon as it has been validated and standard protocols have been put into place.

Conclusions

What were the main research findings?

The most significant finding of this dissertation is that most of the researchers agree that entomotoxicology does have forensic potential. The research has also found that drugs do influence insect development and can change the time of death estimation. Consequently, the research has shown that entomotoxicology can be used when determining a minimum PMI, when considered alongside other forensic evidence.

However, further research is required into the use of standard protocols before entomotoxicology can be used as a source of evidence. In particular, the need for the standardisation and validation of entomotoxicological methodology and the need for a standard protocol for its use in forensic science, has been identified.

Additionally, there is very little evidence to suggest that there is a correlation between the concentration of drugs found in the substrate and the insects during analysis, however the creation of standard protocols that ensure samples are collected from similar areas of the body could change this.

The results of this study also indicate that once procedures have been properly validated and protocols have been put into place, entomologists should be able to consider entomotoxicological results when determining a minimum PMI for drug related deaths.

Limitations of entomotoxicological research

The main limitation of entomotoxicological research is the lack of standardisation. The lack of standard protocols for researchers to follow, means that the majority of studies are not using the same methods, which means that data is not scientifically comparable.

The ability to interpret drug concentration results is also a limitation. More research needs to be done into quantitative analysis of insect samples and to determine whether there is a correlation between the concentration of drugs inside a substrate and the insect.

Another limitation of entomotoxicological research is that the majority of studies are using animal models and tissues and not humans. This is a problem as animals will metabolise drugs in different ways to humans, therefore the studies will not be as accurate as using human organs and tissues.

Recommendations for further research

Entomotoxicological research is still in its early developmental phase and several questions remain unanswered. Research using standard protocols is needed to further determine how certain types of drugs affect the growth rate and morphology of the most abundant forensically relevant flies.

Research using animals that have been injected with drugs at the antemortem stage rather than post-mortem stage, is a potential area for further investigation, ethical considerations allowing. This may allow researchers to determine how living organ systems break down xenobiotics and whether this has any effect on toxicology results.

Further research using spectroscopy techniques coupled with statistical analysis software, will not only make interpretation of data easier, but it also makes interpretation more accurate and reliable and cost effective and will allow for spectral databases to be produced.

Finally, research needs to be conducted into the changes that take place inside the human body after death. The extent to which this affects toxicant quantification needs to be determined and also whether there is a correlation between the concentration of drugs in the substrate and insects. The effect of factors such as drug pharmacokinetics, metabolism, drug redistribution, drug accumulation and feeding activity of larvae have on the correlation, all need to be investigated further.