Review of Predator Emitted Volatile Organic Compounds and Their Potential for Predator Detection in New Zealand Forests

Abstract

This review covers the volatile organic compounds (VOCs) emitted by the bodies and secretions of introduced mammalian predators in New Zealand forests, with a specific focus on mice, rats, ferrets, stoats, and possums. The primary aim is to compile information on these VOCs and assess the potential of exploiting these VOCs as unique biomarkers for predator detection in the forest. While a universal VOC has yet to be identified, the review discusses various VOCs associated with predators and their potential as unique biomarkers for detecting the presence of predators, including 2-heptanone, 4-heptanone, 2-octanone, and benzaldehyde. Furthermore, a brief overview of different VOC detection techniques is provided, connecting the selection of biosensing techniques with the detection of volatile biomarkers. Further research and advancement in the biosensing field hold substantial potential to enhance biomarker detection technologies and elevate predator management strategies within New Zealand forests.

1. Introduction

The native ecosystems of New Zealand are unique in many ways, one being that it boasts a remarkable absence of native mammalian predators with the exception of two species of bat. Fostering a favourable environment for the thriving evolutionary diversity of endemic species, New Zealand boasts a wide range of both extinct and extant ground-dwelling birds (e.g., moa, kiwi, and kakapo), and an ancient lizard, the tuatara [1,2,3,4]. Unfortunately, the introduction of mammalian predators to New Zealand, including rodents, possums, and mustelids, has had a detrimental impact on the local ecosystems, posing an ongoing threat to native prey species (Figure 1). This ecological disturbance has disrupted the delicate balance that once allowed for the flourishing diversity of New Zealand’s unique flora and fauna [5]. It was reported that the invasion of mammalian predators in New Zealand, such as stoats, possums, and rats, has been directly linked to the decline or even complete extinction of numerous native species within New Zealand forests [3,5,6]. This alarming trend highlights the devastating impact these animals have had on the delicate ecological balance, resulting in a loss of biodiversity and the gradual decline of native species’ populations [5,6,7,8]. For instance, the invasion of brushtail possums, mustelids, and rodents is largely responsible for the extinction of many native bird species in New Zealand, including the brown kiwi, blue duck, weka, kaka, parakeet, and cuckoo [6,9]. O’Donnell and co-workers estimate that native birds in New Zealand lose nearly 27 million eggs and nestlings each year due to introduced mammalian predators [5]. In addition, rats and mice pose a significant threat to the ecological systems within forests and islands, as they actively hunt nestling birds, eggs, and lizards [10,11,12]. Another study indicates that brushtail possums not only inflict severe damage on bird reproduction and the overall ecological environment of the forest, but they also act as disease vectors, including the transmission of bovine tuberculosis [13].

The task of reducing or eradicating mammalian predators has emerged as a formidable challenge for New Zealanders, culminating more recently in the national ‘Predator Free 2050′ conservation mission [15,16,17]. To date, New Zealand has employed various strategies to reduce predator populations, including traps, poison and lure baits, dog tracking, and hunting. All these methods necessitate human involvement, which carries potential risks and costs [18]. For example, automated trapping systems and camera monitoring have emerged as a popular method for predator eradication efforts. These traps use mechanical triggers, pressure pads, or infrared light to capture images or videos of animals. However, the subsequent identification of captured species usually requires manual assessment by experienced professionals [19,20]. Alternatively, employing an advanced AI-driven framework for image recognition could reduce labour requirements but it might come at the cost of increased power consumption [21,22,23]. There are also techniques developed for tracking and detecting predators, such as faecal sample collections followed by deoxyribonucleic acid (DNA) or messenger ribonucleic acid (mRNA) extractions and identifications [24]. Although the method demonstrates impressive accuracy based on gene alignment, the rapid degradation of DNA and RNA samples in the environment poses significant challenges to the sample collection process. Moreover, human intervention is often necessary during sample collection and the unpredictable environmental conditions in the wild could amplify potential risks for operators. Additionally, further identifications, which are based on specific genes of different species, demand not only a high-cost instrument and database but also professionally trained operators [25,26,27,28]. The challenge of exploiting these current methods includes the real-time monitoring and the sample collection processes within intricate forest environments, thereby resulting in potential risks and costs associated with human involvement, such as the risk of accidental injuries during tracking and sample collection procedures, along with the consideration of the associated time and cost.

Volatile organic compounds (VOCs) are organic molecules with low boiling points at room temperature, and they contribute to the characteristic scents of various objects [29]. It is suggested that general body odours or specific odours from glands or secretions (such as urine or faeces), can persist in the environment and continue to disseminate olfactory information after the predator has left the area [30,31]. Certain VOCs are emitted by animals and plants, serving as semiochemicals that facilitate information exchange between conspecific or different species [32]. These VOCs play crucial roles in processes such as sex attraction and species identification, and their potential applications have been discussed and studied in lure and bait formulations [18,33]. These odours emitted from predators and their secretions consist of a diverse array of unique VOCs that could theoretically serve as unique biomarkers for detecting the presence of mammalian predators in the forest.

The VOCs of insects and plants have been comprehensively studied and exploited for insect pest detection and plant disease monitoring [34,35,36,37,38,39,40]. However, based on our current knowledge, there is a scarcity of studies in the literature focused on investigating the potential of utilizing VOCs and pheromones emitted by mammalian pests as unique biomarkers for predator detection. VOCs assume a pivotal role in the realm of forest biosecurity and pest control, acting as fundamental chemical cues for detection and management. Their utility extends to the development of sophisticated biosensors and monitoring systems, thereby enabling the early identification of invasive species and pests. Here, VOCs associated with the most destructive and detrimental invasive mammalian predators in New Zealand forests, including mice, rats, mustelids, and possums, are comprehensively reviewed. The sources of these VOCs encompass not only the overall body but also specific glands and secretions. Subsequently, the review provides a brief comparison concerning various techniques employed in VOC detections and a detailed exploration of the potential benefits and challenges in exploiting these VOCs as volatile biomarkers for the detection of targeted predators.

2. Volatile Organic Compounds (VOCs) Emitted by Predators

2.1. Mice

Mice exhibit a strong sense of territory and mark their individual territories using scent, typically through urine and faeces [41]. These scent marks contain specific volatile compounds that play a crucial role in information transfer among conspecifics. For example, several studies indicated that mice can assess the age of other individuals based on the volatile compounds emitted in their urine [42,43,44]. Mice also possess the ability to discern individuals that have been afflicted by parasites or diseases [45]. Female mouse urine has been found to contain volatile compounds that serve as sex-attractive pheromones to male mice [46,47]. In an investigation reported by Varner and co-workers, gas chromatograph-mass spectrometry (GC-MS) was employed to analyze bedding materials contaminated with mouse secretions. Their study revealed the presence of 28 VOCs in these samples. Among them, four specific VOCs, namely butyric acid, 2-methyl butyric acid, 3-methyl-butyric acid, and 4-heptanone, were exclusively detected in samples collected from female mice [48].

In recent decades, there has been a gradual increase in research focused on exploring volatile pheromones and unravelling the intricate functions of these compounds [46,47,48]. In a more recent investigation reported by Tang and co-workers, the volatile profile of female mouse urine was examined, resulting in the identification of 77 unique VOCs. The identified compounds encompass a wide range of chemical classes, including hydrocarbons, alcohols, aldehydes, ketones, and some aromatic compounds, which can also be found in the urine of other rodent species [49].

Among these compounds, six specific volatiles in relatively high concentrations may function as prospective biomarkers for mice (Table 1) [50]. 2-sec-butyl-4,5-dihydrothiazole, previously believed to function as an alarm pheromone in mice, shares a structural similarity with heterocyclic sulfur-containing compounds present in stoat and ferret anal sac secretions [51]. This volatile compound, along with 2,3-dehydro-exo-brevicomin, can be found in the urine of all adult male mice, which can elicit aggression in other males and possess properties that are attractive to females [52,53,54,55]. 2,5-dimethylpyrazine is a female-specific compound known for its role in suppressing estrus. It is utilized by females during mate selection processes [56].

Additionally, distinct volatile profiles could also be distinguished among various mouse species. Soini and co-workers identified 47 VOCs and eight unknown volatile compounds in the urine of a species of mouse, Mus Spicilegus, and suggested five kinds of main VOCs emitted from Mus Spicilegus that are distinct to that of Mus Domesticus (listed in Table 2) [57].

Table 1. Six specific VOCs identified from mouse urine or body.

Volatiles	Source	Conc	Gender	Feature	Reference
2-sec-butyl-4,5-dihydrothiazole	Urine	Low	Male and Female	Age determination; sex-attractive signal; territory mark; warning	[50,51,58,59]
2,3-Dehydro-exo-brevicomin	Urine; body	High	Male and Female	Estrus-related; accelerating puberty	[50,60]
2-heptanone	Urine	Low	Male and Female	Estrus prolongation	[50,60]
6-hydroxy-6-methyl-3-heptanone	Urine; body	Low	Male	Accelerating puberty	[50,60]
EE-α- and E-β-farnesenes	Body	High	Male	Estrus signal; sex-attractive signal; aggressive signal; postponed puberty	[50,60,61]
2,5-dimethylpyrazine	Urine	High	Female	Sex-attractive signal; aggressive signal; postponed puberty; estrus signal	[50,56,62,63]

Table 1. Six specific VOCs identified from mouse urine or body.

Volatiles	Source	Conc	Gender	Feature	Reference
2-sec-butyl-4,5-dihydrothiazole	Urine	Low	Male and Female	Age determination; sex-attractive signal; territory mark; warning	[50,51,58,59]
2,3-Dehydro-exo-brevicomin	Urine; body	High	Male and Female	Estrus-related; accelerating puberty	[50,60]
2-heptanone	Urine	Low	Male and Female	Estrus prolongation	[50,60]
6-hydroxy-6-methyl-3-heptanone	Urine; body	Low	Male	Accelerating puberty	[50,60]
EE-α- and E-β-farnesenes	Body	High	Male	Estrus signal; sex-attractive signal; aggressive signal; postponed puberty	[50,60,61]
2,5-dimethylpyrazine	Urine	High	Female	Sex-attractive signal; aggressive signal; postponed puberty; estrus signal	[50,56,62,63]

Table 2. The main difference between the urinary volatile profiles of male Mus spicilegus and Mus domesticus.

Mus spicilegus	Mus domesticus
γ-hexalactone	2-sec-butyl-4,5-dihydrothiazole (SBT)
δ-hexalaactone	2-isopropyl-4,5-dihydrothiazole (IPT)
Coumarin	Dimethyl disulfide
2-coumaranone	Bis(methylthio)methane
γ-octalactone	Methyl (methylthiio)methyl disulfide

Table 2. The main difference between the urinary volatile profiles of male Mus spicilegus and Mus domesticus.

Mus spicilegus	Mus domesticus
γ-hexalactone	2-sec-butyl-4,5-dihydrothiazole (SBT)
δ-hexalaactone	2-isopropyl-4,5-dihydrothiazole (IPT)
Coumarin	Dimethyl disulfide
2-coumaranone	Bis(methylthio)methane
γ-octalactone	Methyl (methylthiio)methyl disulfide

It has been reported that the quantity of volatiles emitted from mouse urine is influenced by the physiological and psychological conditions of mice [64]. Studies indicate variations in the volatiles emitted by mice, distinguishing between healthy and sick individuals [42,45,65]. Notably, male mice have been observed releasing EE-α- and E-β-farnesenes to attract female mice and announce aggressive signals to other male mice; these are not found in female individuals [60]. Another gender-dependent volatile, Trimethylamine, serves as an attractive scent signal to mice of the opposite sex. It can be identified in the urine of a range of mammals and be found in high concentrations in mouse urine. Importantly, male mouse urine is reported to contain a concentration approximately 20-fold higher than that of female mouse urine [66].

Investigations also suggest the presence of volatile compounds in sex-attractant pheromones deposited by female mice. In a field experiment reported by Musso and co-workers, it was observed that corn cob bedding contaminated with secretions from female laboratory mice had a significant impact on their attraction to wild mice of the opposite sex [67]. During the estrus period of female mice, a high concentration of 2,5-dimethylpyrazine can be found in volatiles released by mouse urine to spread the estrus signals and attract male individuals [63]. Furthermore, 1-ido-2-methylundecane has been specifically identified in the urine of female mice during the proestrus and estrus stages [68]. Dehydro-exo-brevicomin, in either its 2,3- or 3,4-isomer form, is a well-known semiochemical found in both male and female mouse urine, and a rise in its concentration in the urine of female mice was identified during the estrus phase [69]. Tang and co-workers reported that several compounds, including 3,4-dehydro-exo-brevicomin, butanoic acid, pent-1-ene/cyclopentane, 1,2,3-/1,2,4-trimethylbenzene, heptadecane, dioctyl ether, dodecane-1-ol, and 2-ethylhexyl salicylate, were identified to be more abundant or exclusively present in samples collected during the fertile phase [49].

The preputial gland, also known as the clitoral glands in females, releases secretions that can be mixed into the urine. It is another source of odour in mice. Zhang and co-workers conducted a GC-MS analysis to investigate the differences in volatile compounds emitted from the preputial gland secretion and urine of house mice [64]. They identified a total of 42 volatile compounds in the preputial gland secretions, including 32 esters, eight alcohols, and two sesquiterpenes. The result exhibited differences from the identified VOCs in the mouse urine while the urine itself serves as a rich source of additional compounds. Moreover, research by Röck and co-workers demonstrated that aliphatic aldehydes, such as pentanal and decanal, play significant roles in the mouse body scent. Additionally, other compounds, including nitromethane, propanoic acid, dimethyldisulphide, 1-octene, 1-hexanol, hexanoic acid, indole, and α- and β-farnesene, were found in the air surrounding mice while 1-methoxy-2-propane, 6-hydroxy-6-methyl-3-heptanone, phenol and 4-methyl phenol compounds could be found in both the body scent and the urine of mice [60].

In summary, mice establish individual territories through the scent of their secretions, including urine and faeces. Among over 80 VOCs identifiable in mouse urine, six specific VOCs have been found in higher concentrations. Significantly, diverse VOC profiles are identifiable among different mouse species. Moreover, certain VOCs display sex-specific characteristics or manifest variations in concentrations between different genders. These differences can be attributed to the various sexual stages of mice and the secretions from specific preputial glands, which play an essential role in sexual attraction and information exchange between mice of different genders.

2.2. Rats

Rats, like mice, exhibit similar behavioural traits such as territoriality and communication through scent signals. Rat urine contains specific compounds, including squalene, 2-heptanone, and 4-ethylphenol, which play a crucial role in transmitting information among individuals [18]. A study by Zhang and co-workers reported that male rats in the mature stage release a mixture of VOCs in their urine, including squalene, 2-heptanone, 4-ethylphenol, 4-heptanone, and phenol. These compounds are suggested to have sex-attractive functions in female rats [70]. In a similar study, Takács and co-workers collected bedding materials contaminated with rat secretions (urine and faeces) and identified nine male-specific VOCs in these samples [71]. Furthermore, they exploited six of these VOCs, namely 2-heptanone, 4-heptanone, 3-ethyl-2-heptanone, 2-octanone, 2-nonanone, and 4-nonanone, to attract female rats. The results showed a significant increase, approximately ten-fold, in the attraction of female rats compared to the control group.

It is noteworthy that while 2-heptanone and 4-ethylphenol are primarily found in the urine of male rats, they can also be detected in the urine of female rats, though in significantly lower concentrations [70]. Osada and co-workers also reported the attractiveness of 2-heptanone and 4-ethylphenol to female rats and they further identified 4-methyl phenol, which exhibits similar functions in female rats, emitted by adult male rats [72]. Squalene, a compound synthesized by the preputial glands of rats, is naturally present in the nest of female rats and areas where they conduct their activities. However, its concentration significantly increases during the pre-estrus and estrus stages to transmit mating information to the opposite sex [73]. Furthermore, male rat urine has been found to contain compounds such as 2-(octylthio) ethanol, and 1-chlorodecane, known to attract female conspecifics. Female rats in the estrus stage produce compounds like hydroperoxide, 1-nitropentane, and 4-azidoheptane, which are particularly attractive to male rats. Interestingly, 1-nitropentane also elicits attraction in female rats [74].

A scientific study has revealed the presence of distinct volatile compounds in rat urine, including 1-chlorodecane, 2-methyl-N-phenyl-2-propenamide, hexadecane, and 2,6,11-trimethyl decane [75]. Notably, these compounds form complexes with major urinary proteins within rat urine. This binding mechanism serves to prolong the lifespan of the volatiles in the air, allowing for their sustained presence while controlling their overall concentration [3,75]. Another study revealed the presence of specific compounds in the preputial glands of ship rats, including cyclohexene, beta-bisabolene, 1-pentene, hexadecatetraene, 3-cyclohexene, farnesol 1, and farnesol 2. Among these compounds, only the farnesol compounds were found to be bound with major urinary proteins within rat urine [76].

Byrom and co-workers report that over 20 kinds of volatile compounds can be emitted from the body of the rat. Further simulation experiments investigated four VOCs that make the greatest contribution towards creating rat body odour (pyrazine and thiazole-related compounds) [18]. Another study conducted by Schneeberger and co-workers identified a total of 27 biologically relevant VOCs in rat odour, comprising 11 carboxylic acids, 10 aldehydes and ketones, four alkanes, one ester, one alcohol, one sulfone, and one terpene [77]. The investigation also involved a comparison of the mean relative abundance of these compounds, as determined by the ratio of a particular compound’s peak area to the total peak area in the chromatographic profile. It is noteworthy that the study revealed substantial variations in the relative concentrations of seven specific volatiles present in rat odours between individuals in a hungry state and those in a satiated state, as delineated in

Table 3. Seven various VOCs released from rats were reported in different concentrations between hungry and satiated individuals [77].

VOCs	The Difference in Mean Relative Abundance (Hungry/Satiated)
Propanoic acid	1.3 × 106/2.2 × 106
Butyric acid	1.6 × 106/2.5 × 106
Butyl acetate	3.8 × 105/0
3-methyl butyric acid	1.9 × 105/0
Pentanoic acid	0/3.8 × 105
2-heptanone	5.9 × 105/1.3 × 106
Dimethyl sulfone	6.9 × 105/1.7 × 106

. Notably, butyl acetate and 3-methyl butanoic acid were exclusively released by hungry rats, while pentanoic acid was identified solely in the odour of satiated rats.

Carbon disulfide is a typical volatile compound identified in the exhaled breath of rodents. The specialized olfactory sensory neurons of rats can detect the presence of carbon disulfide emitted from their conspecifics and employ this chemical signal to acquire information regarding the safety of food sources [78]. In addition, it is reported that hexanal and 4-methyl pentanal can be identified in the odour released from anxious rats [79]. The identification of these VOCs provides insights into the relationship between VOC components and the emotional state and well-being of rats, which can also serve as potential biomarkers for anxiety in rats and offer a possible non-invasive approach for evaluating the emotional and physiological states of laboratory animals.

In addition to other sources, the glands of rats contribute significantly to overall rat odours. The scent glands of rats contain a mixture of alcohols, aldehydes, and acids derived from both saturated and unsaturated aliphatic or aromatic compounds. These compounds found in the scent glands function as pheromones in rats [80]. Specific volatile compounds released from rat bodies (including the general body and various scent glands) and rat secretions are summarized in

Table 4. Some specific VOCs identified in the odour of rats and rat secretions.

Volatiles	Source	Gender	Feature	Reference
Squalene	Urine; preputial glands	Male and Female	Sex-attractive functions; mating signals released by females during pre-estrus and estrus stages;	[18,73]
2-heptanone	Urine; Faeces	Male and Female	Sex-attractive functions; scent marks	[18,70,71]
4-ethylphemol	Urine	Male and Female	Sex-attractive functions	[18,70]
4-heptanone	Urine; Faeces	Male and Female	Sex-attractive functions in female rats; scent marks	[70,71]
Phenol	Urine	Male	Sex-attractive functions in female rats;	[70]
3-ethyl-2-heptanone	Urine and Faeces	Male and Female	Sex-attractive signal; scent marks	[71]
2-octanone	Urine and Faeces	Male and Female	Sex-attractive signal; scent marks	[71]
2-nonanone	Urine and Faeces	Male and Female	Sex-attractive signal; scent marks	[71]
4-nonanone	Urine and Faeces	Male and Female	Sex-attractive signal; scent marks	[71]
4-methyl phenol	Urine	Male	Sex-attractive signals, estrus-related	[72]
Ethanol	Urine	Male	Attract female conspecifics	[74]
2-octylthio	Urine	Male	Attract female conspecifics	[74]
1-chlorodecane	Urine	Male and Female	Attract female conspecifics; bound with major urinary proteins	[74,75]
hydroperoxide	Urine	Female	Estrus-related; attract males	[74]
1-nitropentane	Urine	Female	Estrus-related; attract conspecifics	[74]
4-azidoheptane	Urine	Female	Estrus-related; attract males	[74]
2-methyl-N-phenyl-2-propenamide	Urine	Male and Female	Bound with major urinary proteins; longer lifespan	[75]
Hexadecane	Urine	Male and Female	Bound with major urinary proteins; longer lifespan	[75]
2,6,11-trimethyl decane	Urine	Male and Female	Bound with major urinary proteins; longer lifespan	[75]
Farnesol 1 and 2	Preputial glands	Male and Female	Bound with major urinary proteins; longer lifespan	[76]
Pyrazine	Body	Male and Female	Contribute to the body odour	[18]
2-ethyl-5-methyl pyrazine	Body	Male and Female	Contribute to the body odour	[18]
Butyl acetate	Body	Male and Female	Hungry signals	[77]
3-methyl butyric acid	Body	Male and Female	Hungry signals	[77]
Carbon disulfide	Exhaled breath	Male and Female	Food safety-related signals	[78]
Hexanal	Body	Male and Female	Emotionally related; releases anxiety signals	[79]
4-methylpentanal	Body	Male and Female	Emotionally related; releases anxiety signals	[79]
di-n-octyl phthalate	Cheek glands; preputial glands	Male	Elicit attraction from females	[81,82]
1,2-benzene dicarboxylic acid (2-methylpropyl) ester	Cheek glands	Female	Elicit attraction from conspecifics	[81]
2,6,10 dedecatrien-1-ol, 3,7,11-trimethyl-(Z, E)	Cheek glands; preputial glands	Female; Male	Elicit attraction from conspecifics	[80,81,82]
E-E-α-farnesene and E-β-farnesene	Clitoral glands; preputial glands	Female; Male	Estrus-related; attract the opposite sex conspecifics	[70,80]

. The cheek glands of rats play a significant role as a source of odour-producing secretions. A diverse range of compounds, including alkanes, aliphatic acids, esters, and alcohols, were discovered in the cheek gland secretions of laboratory rats [81]. Male rats’ cheek glands were found to contain di-n-octyl phthalate to elicit attraction solely from females. Furthermore, the study identified two key components in the cheek gland secretions of female rats: 1,2-benzene dicarboxylic acid (2-methylpropyl) ester and 2,6,10 dedecatrien-1-ol, 3,7,11-trimethyl-(Z, E). Notably, these compounds demonstrated attractive properties for both male and female rats [81]. The study reported by Kannan and co-workers has indicated that the clitoral gland of female rats secretes specific compounds, including 6,11-dihydro-dibenzo-b,e-oxepin-11-one, 2,6,10-dodecatrien-1-ol-3,7,11-trimethyl(Z), and 1,2-benzene dicarboxylic acid butyl(2-methylpropyl) ester. These compounds are believed to serve as signals of attraction, playing a role in communication between conspecifics [80]. Early research also investigated that the preputial glands of male rats release volatile compounds, including 2,6,10-dodecatrien-1-ol-3,7,11-trimethyl, and di-n-octyl phthalate, to attract female rats [82]. Similarly, a higher concentration of E-E-α-farnesene and E-β-farnesene can be emitted from male rat glands compared to female rat glands, which plays a role in attracting opposite-sex conspecifics [70,80]. In another study investigating testosterone-dependent volatile compounds, researchers identified a total of 34 different volatiles in the preputial gland of rats, including 15 alkanes, six sterols and steroids, four terpenes and terpenoids, four fatty acid esters, one chlorinated compound, and four other compounds [83].

In comparison to mice, rats exhibit similar territorial instincts and behavioural characteristics. Three specific VOCs, namely squalene, 2-heptanone, and 4-ethylphenol, play a crucial role in delineating individual territories and facilitating inter-individual communications. As observed in mice, rats also exhibit sex-specific variations in the concentrations of certain VOCs. These differences are primarily attributed to specific preputial glands responsible for signalling sexual attraction. Moreover, the scent glands in rats contribute to their overall body odour, resulting in a more complex blend of VOCs. Notably, the concentrations of these specific VOCs can be different based on various physiological states, including different estrus states and satiety levels.

2.3. Mustelids

Before the 1990s, studies on mustelids, including stoats and ferrets, primarily focused on analyzing the odorous components of their anal secretions, which serve as strong sources of odours [84,85,86,87,88,89,90,91]. For example, Crump’s study identified 2-propylthietane as the major component emitted from the anal gland secretions of stoats [86]. Subsequent studies in mustelid odours have revealed the presence of new thietanes in stoat anal glands [88]; and identified 11 volatile compounds, including 2,2-dimethylthietane, 2-propylthietane, 2-pentylthietane, quinoline, and indole, in ferret anal glands [87]. Indeed, a large number of various sulfur-related VOCs could be found in anal secretions of stoats and ferrets; and the abundance of these VOCs shows an association with the gender of mustelids [87,88]. For instance, higher concentrations of 2,3-dimethylthietane and 3,4-dimethyl-1,2-dithiolane can be found in anal secretions of female mustelids while 2-propylthietane is in higher abundance in that of male mustelids [70,86]. In addition, another study revealed the presence of two aldehydes, five ketones, benzothiazole, 2-methylquinoline, and 4-methyl quinazoline in secretions from both male and female mustelids, with a male-specific compound, o-aminoacetophenone, discovered in the secretions. Female secretions, on the other hand, contained 3-ethyl-1,2-dimethyl-1,2-dithiolane [92].

In subsequent studies, the focus on mustelids has shifted towards investigating the pheromones emitted from their fur and urine, given the abundance of preputial and sebaceous glands beneath the skin. The secretions from these scent glands can contaminate the mustelid urine, contributing to its overall odour [93,94,95]. Similar to rodents, male mustelids have larger preputial glands compared to females [96]. A detailed urinary profile for ferrets has been established by Zhang and co-workers, which includes 41 identified compounds and seven unidentified compounds [92]. It is important to highlight the major compounds found in anal secretions, which consist of sulfur-related compounds, differ from the constituents identified in urine. The urine contains higher concentrations of eight identified nonsulfur components, which are highlighted with stars in

Table 5. A summary of volatile compounds identified in the anal secretions and urine of mustelids. The star symbol (★) indicates VOCs found in higher concentrations in urine [92].

Volatile	Species	Source	Gender
Acetic acid	Ferret	Urine	Unisex
Acetophenone	Ferret	Anal secretions; urine	Unisex
Benzaldehyde	Ferret	Anal secretions; urine	Unisex
Benzothiazole	Ferret	Anal secretions; urine	Unisex
Decanal	Ferret	Urine	Unisex
2-Decanone	Ferret	Urine	Unisex
3,3-Dimethyl-1,2-dithiolane	Ferret	Anal secretions	Unisex
2,2-Dimethylthietane	Ferret, stoat	Anal secretions	Unisex
2,3-Dimethylthietane	Ferret, stoat	Anal secretions	Female-specific
2,4-Dimethylthietane	Ferret	Anal secretions	Unisex
3,3-Dimethyl-1,2-dithiolane	Ferret	Anal secretions	Unisex
3,4-Dimethyl-1,2-dithiolane	Ferret	Anal secretions	Female-specific
2,5-Dimethylpyrazine ★	Ferret	Urine	Unisex; higher conc in males
Dimethoxyacetophenone ★	Ferret	Urine	Unisex
3-Ethylcyclopentanone	Ferret	Urine	Unisex
3-Ethyl-1,2-dimethyl-1,2-dithiolane	Ferret, stoat	Anal secretions	Female-specific
2-Ethyl-3-methylthietane	Ferret	Anal secretions	Unisex
2-Ethylthietane	Ferret, stoat	Anal secretions	Unisex for ferrets; female-specific for stoat
(E)-6,10-Dimethyl-5,9-undecadien-2-one	Ferret	Anal secretions; urine	Unisex
Heptanal	Ferret	Urine	Unisex
2-Heptanone ★	Ferret	Urine	Unisex; higher conc in males
4-Heptanone ★	Ferret	Urine	Unisex; higher conc in males
Hexanal	Ferret	Urine	Unisex
Indole	Ferret, stoat	Anal secretions	Unisex
2-Isopropylthietane	Ferret	Anal secretions	Unisex
6-Methyl-5-hepten-2-one	Ferret	Anal secretions; urine	Unisex
6-Methyl-6-hepten-2-one	Ferret	Urine	Unisex
2-Methylquinoline ★	Ferret	Anal secretions; urine	Unisex
4-Methylquinazoline ★	Ferret	Anal secretions; urine	Unisex; higher conc in males
Nonanal	Ferret	Anal secretions; urine	Unisex
Octanal	Ferret	Urine	Unisex
2-Octanone	Ferret	Anal secretions	Unisex
Ortho-aminoacetophenone ★	Ferret, stoat	Anal secretions; urine	Higher conc in male ferrets (unisex); male-specific for stoats
2-Pentylthietane	Ferret, stoat	Anal secretions	Unisex
2-Propylthietane	Ferret, stoat	Anal secretions	Unisex
3-Propyl-1,2-dithiolane	Ferret, stoat	Anal secretions	Unisex
Quinoline ★	Ferret	Anal secretions; urine	Unisex
7-tridecanone	Ferret	Urine	Unisex
1-Undecanol	Ferret	Urine	Unisex

. Among these volatile compounds, 2-methylquinoline is exclusively found in male ferret urine, potentially contributing to sex attraction and scent marking of male territories [92,97]. For a comprehensive overview of identified volatiles in mustelid urines, please refer to Table 5, summarizing VOCs in the anal secretions and urine of ferrets and stoats [92].

In contrast to rodents, mustelids heavily rely on their anal glands and secretions as prominent sources of odours. These emissions encompass a wide array of VOCs, including numerous sulfur-related compounds, along with certain VOCs based on different genders such as o-aminoacetophenone and 3-ethyl-1,2-dimethyl-1,2-dithiolane. These specific compounds play a crucial role in conveying scent signals related to sexual information exchange between different genders. Furthermore, anal glands and other plentiful subcutaneous glands contribute to the overall body odour and can contaminate mustelid urine, resulting in the identification of nearly 50 different VOCs in mustelid urine. Some of these specific VOCs exhibit distinctions between different species and genders.

2.4. Possums

Trichosurus vulpecula, commonly known as the brushtail possum, is a browsing marsupial species that was introduced into New Zealand in the 19th century for the fur industry [3,98]. However, the introduction of brushtail possums had significant ecological consequences, as they became a major pest in New Zealand, posing a threat to forest ecosystems [99].

Compared to rodents, scent communication among marsupials, such as the brushtail possum, is believed to be more complex. It involves the release of sophisticated pheromones from various secretions and numerous glands, suggesting a potential for intricate pheromonal communication at the neurological level [100,101].

The brushtail possum is equipped with various specialised glands dedicated to producing scent marks and olfactory signals [102,103]. For example, paracloacal glands of brushtail possums secrete an oily white liquid with onion or garlic odours, which consists of tetradecanyl hexadecanoate, octadecenoate, C₅–C₃₀ fatty acids, and alcohols [104,105,106]. A study by McLean and co-workers identified nearly 150 different VOCs in the cloaca secretion of brushtail possums, comprising 81 acids and alcohols, 27 esters (2,6- and 2,7-dimethyloctanol related), and 39 species of sulfur-related compounds; especially, a relatively high concentration of 2-Methyl-3-pentanol (over 20% relative abundance in the chromatographic profile) and hexadecanoate (over 16%) can be found within the cloacal secretions of possums [98]. Woolhouse and co-workers reported that esters of C₁₆ and C₁₈ fatty acids could be found in the sternal glands of brushtail possums, and Salamon refined the finding that more complex components (23 compounds) can be identified in male possums than in female possums (4 compounds) [106,107]. Zabaras and co-workers reported that acetic acid, 1,1-bis-(p-tolyl)-ethane, C₆–C₁₀ aldehydes, and long alkyl-chain compounds could be considered as general components of sternal gland secretions [108]. The other glands of the brushtail possum, including labial glands, apocrine glands, and sebaceous glands, also contribute to the production of odours [103,109,110].

Urine is considered to be another significant source of marsupial odours, containing various pheromones from glands. In the case of the brushtail possum, urine is believed to facilitate the dispersion of cloacal secretions, enabling the incorporation of pheromones from the paracloacal glands into the urine [111,112]. Analysis of possum urine reveals interesting differences between male and female possums. In male possum urine, pyrazine and methyl ketone derivatives are present, while aldehydes are exclusively found in female possum urine. Notably, both male and female possum urine contain methyl ketones [112]. Additionally, olfactory communication among marsupials may exhibit similarities, prompting researchers to actively pursue the identification of key semiochemicals for the optimal formulations of lures [18,113,114]. A prior investigation conducted by Toftegaard and colleagues on another marsupial (Brown Antechinus) may provide valuable insights. The study identified 16 VOCs in marsupial urine, including two pyrazine derivatives, six ketones, three aldehydes, and five miscellaneous compounds. These compounds play a crucial role in olfactory communication among marsupials and may serve as the most effective formulations for influencing marsupial behaviour [115]. Further details are listed in

Table 6. The urinary analysis of the marsupial (Brown Antechinus) [115].

Pyrazines	Methyl Ketones	Aldehydes	Others
2,6-Dimethyl pyrazine b	2-Heptanone d	Nonanal a	Benzaldehyde c
2-Ethenyl-6-methyl pyrazine b	2-Octanone b	Decanal a	Decanol c
	2-Nonanone b	Undecanal a	2,4-Dithiapentane b
	2-Decanone b		N-Butyl benzene sulfonamide
	2-Hexanone b		Limonene d
	2-Undecanone c

^a: identified in male urine only; ^b: identified in female urine only; ^c: identified in both male and female urine; ^d: identified in the urine of castrated marsupials only.

below.

In conclusion, the presence of a diverse array of glands leads to a more complex volatile profile found in possums’ odours. The secretions from these glands contain a multitude of compounds and potentially contaminate urine and faeces. Furthermore, specific preputial glands are implicated in the production of sex-specific VOCs, such as aldehyde derivatives detected in female urine.

3. Detection of VOCs as Biomarkers in Forests

3.1. VOCs as Unique Biomarker for Detection

The review of VOCs emitted by predators suggests that identifying a single VOC as a unique biomarker applicable to all the targeted forest predators is challenging.

Table 7. A summary of some volatile biomarkers for the target predators: (√) VOCs can be found in the volatile profile of the relevant predator; (X) VOCs cannot be found in the volatile profile of the relevant predator.

Volatiles	Possum	Stoat	Ferret	Rat	Mouse
2-Heptanone	√	X	√	√	√
2-Octanone	√	X	√	√	X
4-Heptanone	X	X	√	√	√
Benzaldehyde	√	X	√	X	√
Decanal	√	X	√	X	√
Indole	X	√	√	X	√
Nonanal	√	X	√	X	√

outlines specific VOCs that are present in the volatile profiles of multiple predators, thereby indicating potential candidates for the selection of VOC biomarkers, which are relevant to three or more species of our target predators. Additionally, some VOCs are linked to two distinct species. For instance, thietane-related compounds like 2,2-dimethylthietane and 2-pentythietane are present in the volatile profiles of mustelids, including stoats and ferrets. Similarly, phenol and 2-methyl phenol are associated with rodents such as mice and rats. Therefore, our study emphasizes the challenges of identifying a single, universally applicable VOC biomarker for all targeted predators. A variety of volatile compounds have the potential to serve as unique biomarkers for detecting two or more groups of predators.

VOCs released in relatively high concentrations are preferred candidates for volatile biomarker selection. For example, 2-heptanone and 2,5-dimethylpyrazine were identified in comparatively elevated levels in the urine of mice [50]. 2-heptanone is also associated with the volatile profiles of four different predator groups (excluding stoats), while 2,5-dimethylpyrazine is associated with ferrets and mice. These VOCs emerge as leading candidates for the volatile biomarkers used in the detection of target predators. However, practical considerations, such as chemical availability and cost, also play a crucial role in laboratory experiments and biosensor development for VOC detection. Some chemicals may be difficult to acquire from the market, and the high cost of chemicals like undecanol and 2,3-dehydro-exo-brevicomin may be challenging to obtain from the market, and the high cost of chemicals like 2,2-dimethylthietane makes them unfavourable options for VOC sensing experiments.

3.2. Techniques of Detection of VOC Biomarkers

The current approaches to identifying biomarkers in pest management involve using laboratory-based analytical instruments, particularly gas chromatography-mass spectrometry (GC-MS), alongside techniques rooted in biosensing technologies. GC-MS is a powerful analytical technique used to identify and quantify the components of complex gaseous samples [116]. It is frequently employed to study gaseous samples in VOC-related investigations. In practice, the gaseous samples released from predators or their secretions are typically procured using manual headspace methods (HS) or concentrated via solid phase microextraction (SPME) before being injected into the GCMS system. Within the gas chromatograph, diverse VOCs exhibit distinct interactions with the column material, leading to different elution times; and the resulting mass spectra generated for each eluted VOC are compared to a commercially available database of mass spectrum libraries containing VOCs to identify the compounds present in the gaseous sample [116]. An alternative widely used method is thermo desorption-gas chromatography-mass spectrometry (TD-GC/MS), employing a two-step sample collection process before injection for GCMS analysis. In this method, VOCs are initially concentrated by adsorbent materials, followed by a heating process to gradually release the collected sample for GCMS identification and quantification [117]. In comparison to SPME-GCMS, TD-GCMS typically offers a larger adsorbing capacity, resulting in higher sensitivity and a lower limit of detection to VOC samples [118].

The relative abundance of VOCs is commonly employed as a measure to quantify their relative concentrations within the volatile profile. This value serves as an indicator of the relative amounts (%), defined as the proportion of a specific VOC’s peak area to the total peak areas for all VOCs in the chromatographic profile, specifically the total ion current (TIC) [98]. The process of quantifying the actual concentrations of VOCs in GC-MS profiles is inherently complicated and involves multiple steps. For the identified VOCs, establishing a calibration curve requires the analysis of known concentrations of corresponding standard compounds [119]. Additionally, an internal standard, with known concentrations, should be chosen and added to the VOC sample to correct for variations in sample injection, column efficiency, and instrument response [120]. The established calibration curve can then be utilized to convert the peak area or peak height in the chromatographic profile of the identified VOC in the sample into its actual concentration [121]. Considering the complexity of analyzing numerous VOCs in samples and the time-consuming and resource-intensive nature of the quantitation process, many studies have been done to evaluate experimental results based on the relative abundance of VOCs in the samples.

While these techniques are powerful for VOC-related studies, they face limitations such as being time-consuming, costly, lacking portability, and requiring skilled operators [122]. Moreover, their considerable size and intricate structure can make them unsuitable for real-time monitoring of predators in wild environments. To achieve real-time determination of predator presence, the advancement of biosensing technologies dedicated to pest detection emerges as a highly promising and viable solution. The volatile biomarkers present in the air within the vicinity of the target predators in forested areas offer a promising option for remote and real-time predator detection. For example, in a recent investigation conducted by Wang and co-workers, a receptor-derived peptide biosensor with the capability for real-time monitoring was developed, which effectively utilizes gaseous sex pheromone compounds to detect the presence of the cotton bollworm [123]. Ma and co-workers fabricated a biosensor derived from odorant receptors. This specialized biosensor was designed to detect the presence of the migratory locust by monitoring the volatile biomarker 4-vinylanisole [124].

Table 8. Advantages and disadvantages of various techniques used for predator tracking and detection.

Techniques	Advantages	Disadvantages
Camera Trapping	Visual evidence of predator presence Capture behavioural patterns, aiding in ecological studies Remote and real-time monitoring Non-Invasive method	Requires skilled labour and high power consumption for data fusion Time-consuming Weather impact
Environmental DNA Detection	High sensitivity Precise identification of species Non-invasion method	Less stable in complex environments The sample can be contaminated Not suitable for field analysis Not real-time monitoring
GCMS Analysis	High-resolution and precise results Quantitative data Wide range of applications	Requires skilled labours for operation and analysis High equipment cost Time-consuming Not real-time monitoring

presents a comparative analysis of techniques potentially employed for predator detection. In contrast to other methodologies utilized for tracking and detecting predators, the identification of volatile biomarkers has notable potential benefits. The biosensing of volatile biomarkers released from predators requires less human involvement and minimal training, resulting in lower costs for both equipment and labour. This method can effectively monitor the presence of predators enabling management to prevent the repopulation of eradicated areas. Additionally, the biosensing device can be portable or operate remotely, allowing for easy deployment and wireless data collection; and the incorporation of bio-based or biodegradable biosensing materials could mitigate adverse effects on the forest environment (Figure 2) [125,126,127]. The remote capabilities may not only reduce labour costs but may also minimize risks to operators, decreasing human involvement in predator eradication efforts while lowering monitoring expenses and potential exposure to hazards. The utilization of bio-based or biodegradable biosensing materials could further reduce the environmental impact on the forestry environment. Implementing biosensors holds significant potential for enhancing the efficiency and safety of predator eradication strategies.

Over recent decades, the utilization of biosensors for volatile biomarker detection has played a critical role in various fields, contributing to advancements in pharmaceutical-related tests [128,129,130,131,132], diagnoses and prognosis of rare diseases [133,134,135,136,137], risk estimation [138,139,140,141,142], food safety [143,144,145,146], environmental assessment [147,148,149,150,151,152,153], and pollution monitoring [154,155,156,157,158,159,160].

Biosensors can detect the unique volatile biomarkers released from predators and convert their responses into measurable signals, as outlined in

Table 9. Several general signal types of VOC biosensing devices.

Resulting Signals	Description	Reference
Electrochemical signals	measure the current resulting from the electrochemical reaction between the analyte and a redox electrode, or the potential difference between two electrodes resulting from ion-selective interactions between the analyte and a sensing membrane	[162,163,164,165,166,167,168]
Surface plasmon resonance (SPR)	measure changes in the refractive index near a metal surface due to the binding of analytes	[169,170,171,172]
Optical signals	measure the emission of fluorescent light by a fluorophore attached to the analyte, the emission of light from excited states of a molecule, or the absorption of light by the analyte at specific wavelengths	[173,174,175]
Electrical signals	measure resistance/conductance changes of the sensing material due to analyte adsorption or interaction	[176,177,178,179]
Colourimetric signals	measure changes in colour due to reactions between the analyte and reagents	[180,181,182]
Acoustic Signals	measure changes in sound frequency, amplitude, or velocity attributed to interactions between VOC and sensor	[183,184,185]

. While biosensing may not possess the same level of potency and functional versatility as laboratory-based analytical instruments, well-developed biosensors offer several advantages including lower device costs, compact size, portability for remote applications, rapid response time, continuous measurement capabilities, no requirement for specialized skills, high sensitivities, and customizable selectivities [161]. These attributes position biosensing technology as a promising candidate for field-based tracking and monitoring of predators within forest environments.

These biosensors can be categorized into distinct types based on the variation in sensing materials and the measuring signals they employ, such as chemiresistors [186] electrochemical biosensors [187,188,189] and optical VOC biosensors [190,191,192]. Diverse VOC biosensors manifest unique sets of advantages and drawbacks. For instance, chemiresistors offer notable advantages such as a short response time, high sensitivity, modulable selectivity, and a robust structure. However, their performance can be affected by humidity, and they exhibit a limited sensing range, coupled with sensitivity to temperature variations. Electrochemical sensors, on the other hand, provide reliable performance with low power requirements, high repeatability, and accuracy, along with sufficient sensitivity and selectivity. Yet, their drawbacks include ultra-sensitivity to temperature fluctuations, and a shortened lifespan due to exposure to gases. Optical sensors stand out for their fast response, simple determination process, sufficient sensitivity, and cost-effectiveness. However, they face challenges such as interference from the surrounding environment, limited selectivity, and difficulty in determining individual components within a mixture. In practical applications, the choice of biosensing techniques and materials could be guided by the consideration of specific requirements for the biosensor properties, including sensitivity, selectivity, stability, response and recovery rates, biodegradation, and energy consumption.

Table 10. Comparisons between different material-based chemiresistors used as VOC biosensors. The symbols attached to each qualitative estimation remark the degree, major (↑) or minor (↓), of each characteristic concerning the other groups of materials (Adapted from reference [193]).

Properties	Metal Oxide-Based	Polymer-Based	Carbon-Based
Sensitivity	High ↑↑	High ↑	High
Selectivity	Poor ↓	Poor	Poor
Stability	High	Medium ↓	Medium
Response and recovery rate	High	Low	Low
Biodegradation	Low	High ↑	High
Temperature requirement	High	Low	Low
Energy consumption	High	Low	Low
Miniaturization potential	High	High	High

provides a comparison of different material-based chemiresistors employed as VOC biosensors.

3.3. Challenges of VOC Detection in Forests

In the detection of target VOC biomarkers in forests, various factors need to be considered, including VOC concentrations or abundances, the commonality of VOC biomarkers between species, and the possibility of cross-sensitivities. As mentioned in Section 3, although not all predators share common volatile biomarkers, certain VOCs can be identified in the volatile profiles of multiple species. For example, Table 7 illustrates that 2-heptanone is present in the volatile profiles of mice, rats, ferrets, and possums, and 4-heptanone can be identified in rat and ferret urines; EE-α, and E-β-farnesenes are emitted from both mice and rats, while benzaldehyde is emitted by both mustelids and possums. Trimethylamine, a compound found in the urine of various mammals, also shows promise as a potential biomarker for these mammalian predators.

As a VOC biosensor aims to detect the presence of mammalian predators in forests, it is crucial to consider potential cross-sensitivities arising from volatiles emitted from other species in forests, including plants, insects, fungi, and birds. For instance, decanal is one of the volatile semiochemicals emitted from possums but it has also been identified in VOCs released from brown marmorated stink bugs, H. halys, which potentially established themselves in New Zealand [194,195,196]. This shared presence makes decanal an unfavourable candidate for possum biomarkers. In addition, plants emit numerous VOCs essential for growth, communication, defence, and survival, with over 1700 different VOCs identified in various plant species [197,198,199]. Terpenoid chemicals are the largest class of plant VOCs, which comprise more than 550 volatile organic compounds. It is followed by the second large class of phenylpropanoids and benzenoids which are based on aromatic amino acids [199,200]. While the range of these plant VOCs may not completely overlap with the targeted predators’ VOCs, potential cross-sensitivities of the biosensors to these plant VOCs should be acknowledged as a challenge. New Zealand is known for its extraordinary diversity and exclusivity of plant species, including endemics such as matai (Prumnopitys taxifolia), kauri (Agathis australis), miro (Prumnopitys ferruginea), totara (Podocarpus totara), kahikatea (Dacrycarpus dacrydioides), and rimu (Dacrydium cupressinum), which presents a unique context [201]. VOCs emitted by these native plants in New Zealand have the potential to induce cross-sensitivity in the biosensors. For instance, Effah and co-workers identified 46 VOCs emitted from Dracophyllum subulatum foliage, an endemic plant exclusive to New Zealand [202]. As these VOCs exhibit a slight overlap with VOCs released by rodents (EE-α-farnesene), marsupials (decanal and limonene), and ferrets (heptanal, nonanal, and octanal), recognizing and addressing these potential cross-sensitivities is essential in the development of a suitable biosensor. Enhancing the selectivity of developed sensing devices to specific volatile biomarkers can mitigate the cross-sensitivities, achieved through methods such as functionalized surfaces with recognition groups (e.g., insect odorant receptors [203,204,205] and odorant binding protein [206,207,208]).

The performance of VOC sensing can also be influenced by the moisture content in the environment. Water vapour may act as an electron donor, reacting with the surface of sensing materials, leading to significant changes in the conductivity of chemiresistor-based sensors [209]. While this phenomenon could be utilised to develop humidity sensors, it poses a risk of cross-sensitivity and may negatively influence the performance of the sensors.

Moreover, it is important to recognize that potential volatile biomarkers emitted by target predators might be present in low concentrations. For example, volatile semiochemicals released from rat urine exhibit relatively low concentrations due to their binding with major urinary proteins [210]. This binding mechanism extends the longevity of volatiles in the air but limits their overall concentration [210,211]. Consequently, an effective VOC biosensor for predator detection in forest environments must possess certain properties. These include a rapid response time, high selectivity, and sensitivity to the target volatile biomarker even at low-level concentrations (parts per billion, or ppb). Achieving these characteristics is critical for the reliable and accurate monitoring of predator presence in forest ecosystems using VOC-based biosensors.

4. Conclusions

The diverse range of VOCs emitted by predators carries valuable information about their presence, behaviour, and reproductive status. This review provides insights into potential VOCs associated with the most destructive and detrimental invasive mammalian predators in New Zealand, including rats, mice, mustelids, and possums. It not only highlights specific VOCs that play a crucial role in scent communication among mammalian predators but it also emphasizes the potential of VOCs as valuable biomarkers for predator detection and management. The detection of unique VOCs emitted from target predators, such as sex-specific volatile compounds or variations in concentrations of specific VOCs based on different physiological states, presents both opportunities and challenges for effective predator detection and pest management.

It is noteworthy that although a universal volatile biomarker has not yet been established across all targeted predators, a selection of volatile compounds shows the potential to serve as unique biomarkers for the detection of two or more predator groups. For practical considerations in testing, some of these VOCs are easier to prepare or purchase. Examples include benzaldehyde (linked to mustelids and possums), 2-heptanone and 2-octanone (associated with rodents, ferrets, and possums), 4-heptanone (linked to rodents and ferrets), and trimethylamine (found in mammals’ urine). These compounds hold substantial promise for use as volatile biomarkers in biosensor development and sensing experiments. By detecting and monitoring specific VOCs in the environment, non-invasive methods can be developed to assess predator populations, evaluate the effectiveness of control measures, and determine the impact of predators on native wildlife. This approach has the potential to reduce risks to operators and contribute to advancements in biosecurity and pest control in forests.