Peripheral Nerve Injury

### **Chapter 2**

## Nerve Injury Model in Rabbits: Benefits and Pitfalls

*Wesley P. Thayer and Sara C. Chaker*

### **Abstract**

Peripheral nerve injury is widely studied through the sciatic nerve injury model. Although many animal models are used for sciatic nerve injury studies, rabbits are reported as the third most commonly used animal model. However, there is a significant gap in the literature describing common postoperative complications following sciatic nerve injury in rabbits. This chapter analyzed postoperative complications recorded from an original study that tested 40 mm sciatic nerve gap repairs in 56 rabbits. Autophagy of the toes and pressure ulcer development on the injured limb were the two most common and severe postoperative complications seen. These impairments ultimately led to 23.2% of the rabbits requiring euthanasia prior to the study endpoint. This raised the cost of the study by over \$25,000. This chapter outlines the risks and benefits of using this animal model in sciatic nerve injury studies. It also proposes treatment methods for common postoperative complications that can substantially reduce future study costs. To preserve ethical animal care standards in research, we recommend alternative models be used instead of rabbits to study sciatic nerve injuries. However, if rabbits must be used, we encourage using the treatment protocol outlined below.

**Keywords:** peripheral nerve injury, animal model, rabbit animal model, laboratory animal welfare, sciatic nerve injury, ethics and welfare

### **1. Introduction**

Peripheral nerve injury is an exceedingly common injury that comprises nearly 2–3% of all patients admitted to a Level 1 trauma center [1, 2]. Despite this high incidence of patients with this injury, clinically accepted methods for treatment have yet to establish consistent success in restoring nerve function. It is reported that only 20–40% of direct nerve repairs achieve a high level of motor and sensory recovery [3]. In addition, the complex cellular and molecular pathology behind nerve recovery following injury provides an additional barrier to developing an effective treatment for traumatic peripheral neuropathies. Therefore, research endeavors have dedicated extensive efforts to developing treatment methods that address the pathophysiology of this injury to yield more remarkable functional recovery and tissue regeneration.

In pre-clinical research, animal models have massively expanded our knowledge of the biochemical mechanisms of diseases. In peripheral nerve injury research, *in-vitro* techniques are less commonly used due to their inability to mimic endogenous nerve

regeneration complexities [4]. Conversely, *in-vivo* research has shown great translational potential for clinical treatments, making this method more common. The sciatic nerve injury model is often the model of choice for the exploration of this injury. The relatively large size of the sciatic nerve provides easy access for implementing and studying surgical interventions [4]. Although the sciatic nerve is the most common nerve for the study of peripheral nerve injury, there is a great deal of variance in the literature regarding the choice of animal model for experimentation.

Rats are often the animal model of choice for many researchers studying sciatic nerve injury [5–7]. In recent years, there has been a relative increase in nerve injury studies using rabbit models [8–13]. The use of rabbits in the sciatic nerve injury model has shown to be an excellent method due to their size, docility, ease of handling, and short life span [14–16]. However, their sensitivity to surgical implants, although beneficial when determining immunologic responses, may lead to greater costs than anticipated due to experiment-related complications. Additionally, the extensive maintenance required for the welfare of these animals can lead to higher experiment costs. Cages must be made from non-toxic materials so a rabbit would not be harmed if chewed or licked. It also must be designed so that a rabbit cannot hurt itself with sharp edges and be able to be maintained easily and repeatedly [17]. Pain in rabbits can also present difficulties in the use of this animal model. Rabbits do not display explicit behaviors when in pain, which without identification, can cause rabbits to die within 36 hours from distress from the pain [14]. Additional complications such as self-mutilation and the development of pressure ulcers can also result in the early termination of the experiment.

When conducting animal research, it is vital to adhere to the "Three Rs" principle, Replacement, Reduction, and Refinement. This framework allows proper adherence to maintain the welfare and minimize the distress of animals used in experimental studies. More specifically, replacement refers to using alternative methods in an area where animals would have been used, reduction refers to lessening the number of animals used in a study, and refinement refers to improving study protocols to minimize the amount of distress experienced by the animal. Given this universal standard for conducting proper animal research and the commonality of rabbit models for the study of peripheral nerve injury, it is essential to analyze the general benefits and drawbacks of the use of rabbits in these experiments. This chapter will provide data on the unforeseen complications and proposed interventions we have documented during an original experiment testing 40 mm sciatic nerve gap repairs in rabbits [18]. We hope this chapter provides greater information on this animal model to reduce pain in rabbits in future experiments and potentially persuade others to opt for an alternative animal model when studying nerve injury.

### **2. Sciatic nerve injury in rabbits**

Although relatively common, the use of the rabbit model in sciatic nerve injury studies still presents many gaps in the literature outlining specific techniques for success. A recent study by Merolli et al. investigated the sciatic nerve gap-injury model in rabbits and provided greater detail for nerve-gap repairs in this animal model [19]. In the clinical setting, peripheral nerve injuries may require an artificial device called a "nerve conduit" placed over the nerve to aid in the regeneration of the nerve. These devices have been most commonly studied in the rat animal model for sciatic nerve injury. However, this model provides minimal translational potential due to the *Nerve Injury Model in Rabbits: Benefits and Pitfalls DOI: http://dx.doi.org/10.5772/intechopen.107094*

relatively small size of the rat sciatic nerve restricting the length of the devices being studied. In this case, the rabbit model can be instrumental given their relatively larger sciatic nerve and general ease of handling compared to other higher species animal models. Merolli et al. successfully introduced a novel method for transecting the nerve in a uniform and timely manner [19]. Despite the higher regulatory standards for the use of rabbits in experimental studies, the authors still believe that the higher translational potential of this animal model outweighs the higher standard of care.

The rabbit sciatic nerve injury model has also been implicated in investigating potential new therapies to promote nerve regeneration following injury. Li et al. evaluated the efficacy of nerve growth factor following a sciatic nerve crush injury in rabbits. They found that high-frequency ultrasound-guided injections of nerve growth factor led to more remarkable nerve recovery outcomes [20]. The size of the rabbit sciatic nerve and the greater translational potential of this larger animal compared to smaller animal models makes their results incredibly promising. The study of nuanced treatment methods for peripheral nerve injury is becoming increasingly common in the literature. Therefore, with more studies employing this animal model, it is pertinent to develop and provide a standardized protocol for the adequate care of rabbits used to study sciatic nerve injury.

### **2.1 Common operative technique**

The sciatic nerve injury model was first developed by Wall et al. in an effort to study the behavioral effects caused by the complete transection of this nerve in animals [21]. The sciatic nerve is the largest nerve, therefore handling and repair are comparably easier than other nerves [22]. One of the most common lesions studied is axonotmesis, or compression of the nerve [4, 23]. Compression injuries utilize a crushing mechanism that leads to damage of the axons without disrupting the integrity of the epineurium. This method of injury leads to functional damage to the sciatic nerve without the need for surgical repair. Crush experimental models are mainly used to mimic and subsequently understand the biological mechanisms behind peripheral nerve injuries caused by mechanical compression.

The other common nerve lesion studied is neurotmesis, or complete transection of the nerve. This injury requires complete disruption of the axons and epineurium. Clinically, one-third of peripheral nerve injuries are caused by a laceration from a sharp object [24, 25]. Due to the nature of this injury, lacerations often lead to neurotmesis of a peripheral nerve. Therefore, *in-vivo* studies investigating transection nerve lesions are more prevalent in the literature for their high translational potential into clinical nerve repair methods.

In our original experiment, we measured different repair techniques following complete transection of the sciatic nerve in rabbits. The rabbits were sedated with a mixture of Ketamine (40 mg/kg) and Xylazine (9 mg/kg) given intramuscularly once on the non-experimental leg. Anesthesia was then maintained with Isoflurane 2% at 3 mL/minute. A 5 cm incision was made longitudinally parallel to the femur using standard sterile operating procedures. The left sciatic nerve was exposed through a split-muscle technique, and 40 mm of sciatic nerve graft was harvested. In our original experiment, rabbits were subject to one of three nerve injury cohorts, cut without repair, reverse autograft, and repair with a sterile nerve conduit. For the second cohort, the sciatic nerve was cut using sharp scissors, and the harvested nerve section was reversed 180° and reattached to the proximal nerve at both ends using a 9–0 nylon suture (Ethicon, Somerville, NJ). For the third cohort, the 40 mm deficit was repaired by suturing in a nerve conduit using 9–0 Ethicon sutures (Ethicon).

Once the specific nerve injury was completed, the muscle plane was closed by layers using a 3–0 Vicryl (Ethicon) interrupted suture pattern, and the skin was closed with a 5–0 Monocryl running suture pattern (Ethicon). This operative procedure was conducted in accordance with the Guide for Care and Use of Laboratory Animals and approved by our corresponding Institutional Animal Care and Use Committee (IACUC).

### **2.2 Postoperative care**

Postoperative care is a critical step in maintaining the well-being of the animal and avoiding any potential complications. Despite multiple regulations and rules for the overall reduction of pain and suffering of laboratory animals, complications can be inevitable. Here we outline the specific steps we took following the surgical procedures to minimize distress in the rabbits.

Immediately following surgery, rabbits were monitored by study personnel until they fully recovered and were able to be transported back to our animal facility per our IACUC protocol. For pain management, every 8 hours for the first 24 hours, 0.02–0.05 mg/kg of buprenorphine hydrochloride was administered subcutaneously or intramuscularly. After the initial 24 hours subsided, 0.1–0.3 mg/kg/24 hours of meloxicam was given as needed for 72 hours. Pain assessments were made using the rabbit grimace scale [26]. This scale evaluates facial actions such as orbital tightening, cheek flattening, nose shape, whisker position, and ear position. This scale has shown to be a reliable method for assessing pain in rabbits.

Rabbits were individually housed in Allentown rabbit cage rack systems, and a non-contact Techboard was placed underneath the racks for urine and excrement collection. Cages were changed every 2 weeks. Rabbits were fed through an automatic feeder and had free access to food and water 24 hours a day. A high-fiber diet and water were replaced every day. Additionally, the room in the animal facility that held the rabbits was set to a 12-hour light/dark cycle. Rabbits were monitored weekly by study personnel and daily by animal care staff. During the weekly inspections, any complications were recorded, and rabbits were weighed. At the end of the specified postoperative period (3, 6, or 12 weeks), the rabbits were euthanized by intravenous injection of 125 mg/kg sodium pentobarbital. For the purpose of the original study, the nerves were then harvested for either immunohistochemistry or magnetic resonance imaging [18].

### **3. Postoperative complications and proposed solutions**

### **3.1 Autophagia**

Injury to the sciatic nerve causes muscle weakness in the affected limb and decreased sensation [4]. This often leads to complications experienced across multiple animal models. Autophagia is often reported among these complications. Initial theories believed self-mutilation was due to the animal's inability to sense the injured limb and consequently attack it as if it was a foreign body [23]. However, the histology behind peripheral nerve injury and known mechanisms following nerve injury support the theory that the onset of this phenomenon is linked to the axonal regeneration and subsequent generation of abnormal sensations from the injured nerve [23, 27].

*Nerve Injury Model in Rabbits: Benefits and Pitfalls DOI: http://dx.doi.org/10.5772/intechopen.107094*

In our original study, self-mutilation occurred in about 36% of the rabbits. The affected rabbits mainly chewed on the digits of the affected limb, however, there was additional chewing to the surgical site and the dorsal and web spaces of the injured foot. Our solution to this complication was to place a plastic Elizabethan collar with soft edges on the rabbits in the hope that it would deter autophagia. After this intervention's implementation, the autophagia incidence dropped by nearly 10% in the affected rabbits (p = 0.0093). In addition, euthanasia related to self-mutilation decreased from 7.5 to 1.04% (p = 0.00164).

Initially, when autophagia was sighted, the rabbits were placed in donut collars (**Figure 1**). These collars were initially chosen due to their softness, lack of toxicity, and ease of application and cleaning. However, this type of collar did not prevent the rabbits from reaching their hind limb. Therefore, we were required to use plastic Elizabethan collars with soft edges. We found that due to the greater range around the rabbit's head, these collars were more successful at preventing the rabbits from reaching and subsequently harming the toes on the injured limb. In addition, the relatively inexpensive cost of these types of collars proved to be incredibly advantageous. We purchased two sets of 10 collars, which made the treatment of autophagy expedited. It also allowed for an immediate exchange of soiled collars for clean ones.

A critical aspect of this treatment to note if used is that often our study personnel would find the collars covered in waste. Therefore, to maintain sanitary conditions and prevent eye infections in the rabbits, the collars had to be changed regularly and checked daily. Additionally, with these collars, if moisture collects where it is attached to the neck of the animal, fungal rashes can occur. Thus, it is also essential to check the necks of the rabbits to ensure there is no excessive moisture. Lastly, we found that the rabbits would often chew at the rims of the collars, so we had to provide non-toxic coverings, such as hypoallergenic surgical tape, to replace the soft sides of the collar. Despite these minor issues, this simple solution was shown to be successful in reducing this complication in our rabbit model.

### **Figure 1.**

*Example of an Elizabethan collar (left) and a Donut collar (right) used as a treatment for autophagy in original study.*

### **3.2 Pressure ulcers**

An additional complication we encountered was pressure ulcers on the plantar aspect of the injured foot. Pressure ulcers can cause excruciating pain and adequate steps must be taken to prevent the development of this ailment in the animals. Ulcers, specifically on animals' heels, are incredibly common in sciatic nerve injury models. Rabbits are often more prone to this type of injury due to the overall lack of sub-dermal padding in the heel of their hind limbs [28]. Additionally, following nerve injury, dysesthesia can occur in the injured limb leading to dragging of the foot, ultimately contributing to the overall poor condition of the injured foot [29].

In our original study, we reported that about 23% of the rabbits developed pressure ulcers. This was the second most frequent postoperative complication experienced. These ulcers often developed into severe morbidities where the base of the ulcer reached the calcaneus. To prevent and lessen animal suffering, we developed an "ulcer cushion" and utilized this device to treat the ulcers. Our "ulcer cushion" consisted of a

### **Figure 2.**

*Illustration of our developed "ulcer cushion" utilized in our original experiment to treat and prevent severe pressure ulcers.*

*Nerve Injury Model in Rabbits: Benefits and Pitfalls DOI: http://dx.doi.org/10.5772/intechopen.107094*

foam sheet custom-fitted to the bottom of the rabbits' foot (**Figure 2**). Two personnel were used to apply the cushion, one to safely hold the rabbit and another to place the foam onto the affected foot. After the foam was placed onto the foot, it was wrapped in cast padding, and the entire foot was wrapped in veterinary bandaging. When the bandaging was placed onto the foot, appropriate pressure was applied so the cushion would stay in place but not affect the circulation of the foot.

This treatment method was based on the clinical standard for treating this condition in humans. Unfortunately, there is yet to be a commercially made device to treat this ailment in animals, which led to the development of our own manufactured device. We believe this method eased pressure from the foot and even acted as a disease prevention mechanism for autophagia. Additionally, besides routine changing of the bandages, there were no other issues found with this treatment compared to the collars mentioned previously. Therefore, applying this cushion to the injured foot following sciatic nerve injury in rabbits is strongly recommended as a preventative tool for pressure ulcers.

### **4. Cost analysis**

Compared to larger animal models, rabbits are a cost-effective option for sciatic nerve injury experimentation [4]. Yet, rabbits can often experience many complications that ultimately lead to increased costs. When the animal experiences complications beyond remedy, euthanasia must occur, and additional rabbits and their corresponding supplies will have to be ordered to replace the lost animals. This goes against the principle of reduction, a pillar in the universal framework to promote humane animal research. Our treatment methods outlined above completely subsided the need and cost for additional rabbits. In our original study, the total cost for the purchase and care of the rabbits required to complete the experiment should have been around \$108,000. The added cost of acquiring additional rabbits to replace the ones that were lost was \$25,414.94. Therefore, complications alone increased the cost of the study by 23.54%.

The cost of our interventions proved to be substantially inexpensive. When the cost of all the materials used for the treatment of the autophagy and pressure ulcers were summed, our treatments were around 66 times less expensive than ordering more rabbits (\$376.95 vs. \$25,038.44). These intervention methods proved to have the ability to reduce the number of animals required for the completion of the study, which can save thousands of dollars in experimental costs.

### **5. Discussion**

The use of rabbits for sciatic nerve injury models can provide greater acuity for the investigation of peripheral nerve injury repair methods compared to smaller rodent animal models. In addition, their ease of handling and biocompatibility make rabbits an excellent choice for nerve injury experimentation. Although this model is less costly than larger animal models, rabbits can experience many complications that can almost negate this difference. In our original experiment, where we utilized rabbits to test sciatic nerve repairs, we recorded all postoperative complications experienced by the subjects. We found autophagy and ulcers of the heel to be the most common complications experienced by the rabbits. In this chapter, we outline how we treated and

managed these complications in the hope that future studies can use these techniques to prevent these morbidities from occurring.

Autophagia or self-mutilation of the toes of the injured limb is a common complication across multiple animal models. With sciatic nerve injury, dysesthesia is common and is suspected to be the cause of this response in rabbits [22]. Amputation or euthanasia before the intended study endpoint is often required when this complication becomes severe. However, these solutions lead to greater distress for the rabbit and increased costs. Our proposed solution for this complication includes placing an Elizabethan collar on the rabbit, making it more challenging to bother the injured limb. With this inexpensive and simple method, we were able to reduce the number of animals with autophagia by nearly 10% and reduce the number of animals requiring euthanasia due to severe self-mutilation by 6%.

The second most common complication we encountered was the development of ulcers on the heel of the injured foot. Currently, there is no standard method to treat this complication in animal models. These ulcers can become extremely severe if left untreated, often developing so deep into the foot that the bone of the heel can be visible. Therefore, to maintain the well-being of the rabbits, it is vital to develop a standardized method of care when this morbidity occurs. Our solution was the development of an "ulcer cushion." This essentially was a custom-fitted piece of foam placed under the injured foot and then secured with cast padding and veterinary tape. Although we did not perform a statistical analysis on the effectiveness of this method, we believe this solution alleviated the pressure placed on the injured foot and even worked indirectly to prevent autophagia.

These developed treatments drastically decreased the overall cost of the experiment as well. The price of replacing rabbits that had to be euthanized prior to the intended study endpoint can lead to substantial unbudgeted costs for the experiment. In our original study, the price for additional rabbits and the subsequent housing and care required, led to an additional cost of \$25,414.94. This raised the cost of the entire experiment by nearly 24%. However, our proposed treatments dramatically reduce this cost to merely \$377. Therefore, these developed treatment methods can save thousands of dollars, increase the welfare of the animals, and lessen the number of animals needed to complete the experiment.

One aspect of both treatments to note is that they require high levels of maintenance. The collars had to be changed often to maintain sanitation and avoid bacterial infections. Study personnel often returned to see the collars covered in feces. Additionally, moisture can be easily trapped around the neck of the rabbit where the collar is placed, leading to a high risk of developing fungal dermatitis. Similarly, the ulcer cushions had to be replaced and monitored often throughout the study. Another aspect future researchers should consider if utilizing these proposed treatments is how behavioral assessments may be affected. Moreso, with the placement of the cushion on the injured foot, the rabbit's mobility will be disturbed and subsequently interfere with any walking assessments. Ambulatory assessments are the most used postoperative measurements in nerve repair studies [30–33]. Therefore, with a cumbersome cast placed on the rabbit's foot, one can anticipate the animal to drag their foot and even have an exaggerated movement of the protected foot when raising it, compared to the unaffected limb. These details should be strongly considered when implementing these treatments in an experiment that requires postoperative behavioral assessments. However, if these additional features of the treatments cannot be handled accordingly, we encourage using alternative animal models that allow for large sciatic nerve injury modeling but may not be as sensitive as the rabbit model.

*Nerve Injury Model in Rabbits: Benefits and Pitfalls DOI: http://dx.doi.org/10.5772/intechopen.107094*

### **6. Conclusions**

In this chapter, we have outlined our experience working with rabbits to study repair methods for sciatic nerve injury. This animal model can provide great benefits for pre-clinical experimentation of peripheral nerve injuries. However, rabbits tend to have greater sensitivity to this particular impairment. With the lack of literature outlining common post-operative complications experienced in this animal model, it was imperative to discuss our findings and potential solutions for future research. Therefore, if an experiment cannot meet the suggested treatment protocols for maintaining the animals' well-being, we suggest opting for an alternative, more resilient model for studying sciatic nerve injury. If a rabbit animal model must be used, applying the treatment methods outlined in this chapter immediately postoperatively can drastically reduce the amount of animal suffering and allow the experiment to progress successfully.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Wesley P. Thayer\* and Sara C. Chaker Department of Plastic and Reconstructive Surgery, Vanderbilt University Medical Center, Nashville, United States

\*Address all correspondence to: wesley.thayer@vumc.org

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Section 3 Pesticides

### **Chapter 3**

## Effect of Agricultural Pesticides and Land Use Intensification on Amphibian Larval Development

*Claudia Patricia Camacho-Rozo and Jairo Antonio Camacho-Reyes*

### **Abstract**

The increase in human population had to increase the demand for vital resources, including food, generating intensive and extractive exploitation, and impacting natural ecosystems and biodiversity. Land degradation of ecosystems is a serious and widespread problem in the world. The expansion of the agricultural frontier is by direct or indirect human-induced processes, expressed as long-term reduction or loss of biodiversity. The expansion and industrialization of agriculture had been negatively affected by soil fertility, the climate, biogeochemical cycles, bodies of water, and loss of biodiversity on different spatiotemporal scales. Intensive agriculture, in the form of monocultures, is subjected to strict pest controls for the use of highly toxic agrochemicals. Pesticides are used in monocultures by spraying aqueous dilutions. Knowing the toxic effect of pesticides and agrochemicals on amphibians is very important. These animals have special ecophysiological conditions because they have biphasic life cycles composed of an embryonic and larval aquatic development stage and the adult stage in humid terrestrial environments. For these reasons, the amphibians have been observed with increased mortality rates, reduced prey availability, and affected growth rates.

**Keywords:** agriculture, growth, human land use, malformation, pesticides use, sublethal effects, survival

### **1. Introduction**

Ecosystem transformation may be an inevitable outcome of the combined impacts of multiple drivers [1–3], including the increase in the human population, their activities, and the demand for resources [4, 5]. The conversion of natural habitat to other land covers through changes in human land use is a principal cause of deforestation, the loss of biodiversity, and local/global extinction of species in natural terrestrial ecosystems [1, 3, 6]. In the world, ecosystem transformation is associated with the rise of agricultural systems, these new ecosystems increase human appropriation of the Earth's net primary production, reducing the amount of energy available for all other species, and influencing a range of ecosystem processes and services [3, 4, 7].

To provide for human needs, over 50% of the global usable land is already for pastoral or intensive agricultural uses [3, 5]. The increase in the agricultural frontier and overexploitation lead to the use of a high number of chemical products that affect both the communities present in bodies of water and the soil [8–10]. Among the communities most affected by these agents are amphibians, which have limited spatial mobility [11–13], physiological and ecological specificities, that restrict their distribution and habitat use [14–16].

Amphibians present a biphasic life cycle in their development, an aquatic larval stage, adapted for rapid growth, and a terrestrial adapted for dispersal and reproduction [17–19]. The aquatic environment is the first habitat faced by anurans with complex life cycles [16, 18], because different restrictions imposed by the environmental gradient may be present during larval development [16, 17].

In the terrestrial environment, adult anurans select and colonize different habitats for oviposition and development of their young [20–22]. Tadpole uses different microhabitats, such as semi-permanent pools, permanent pools, temporary pools, phytotelmata seasonal and permanent streams or rivers, and under leaf litter [16, 18]. These bodies of water can be mesotrophic or oligotrophic, and present different biotic and abiotic characteristics, which could affect their adaptation [20, 23].

The larval stages of anurans in disturbed environments and recovery processes are crucial for the persistence of the species in a specific locality [20, 24, 25]. Their survival depends directly or indirectly on the requirements of these organisms for conditioning and the presence of suitable microhabitats for the species throughout their life cycle [24, 26, 27].

Environmental and spatial processes present in anuran assemblages with complex life cycles respond differently within water bodies [22]. These organisms play an important role in aquatic ecosystems, especially in the absence of higher trophic levels [28]. These organisms present different traits in the oral disc and diets that allow them to occupy more functional space. The tadpole's filter-feeding can change the composition and abundance of algal species, thus affecting the amount of chlorophyll and primary productivity [25, 28]. Scraper larvae can affect other primary consumers as epiphytes, present on the bottoms of water bodies. The changes in the structure and function of freshwater ecosystems provide insight into the mechanisms of interaction between anuran assemblages and their relationship with the ecosystem [25, 28]. However, numerous environmental variables can directly or indirectly affect anuran larval growth, survival, and mortality rates [20, 29, 30]. Tadpoles can inhabit water bodies that present a gradient of permanence over time [20]. For the tadpoles, the opportunity to remain longer in a water body would imply increasing size and decreasing the probability of mortality during metamorphosis [19, 31]. The larger size would be related to an earlier onset of reproductive maturity, which is advantageous in terms of egg production and sexual attraction, among other variables [21, 31, 32].

Agrosystems use fertilizers and pesticides, these affect the amphibians populations of the terrestrial and aquatic ecosystem. Pesticides affect amphibian populations with lethal, as well as sublethal effects. The sublethal effects occur in the medium and long, decreasing the availability of prey for amphibians, which together with ecological and physiological stressors could restrict the transmission of matter and energy and growth rates [33, 34]. We reviewed the literature on dermal pesticide absorption and toxicity studies for aquatic life stages of amphibians.

*Effect of Agricultural Pesticides and Land Use Intensification on Amphibian Larval Development DOI: http://dx.doi.org/10.5772/intechopen.106268*

### **2. Transformation of the territory and its impact on biodiversity**

The transformation of ecosystems and ecological processes in the world is associated with the establishment and needs of human populations and their socio-cultural processes [5, 35]. The demographic increase has been progressive in the world. Humans have transformed between 40–50% of the natural ecosystems of savannas, forests, and wetlands into agricultural and urban systems [5, 36]. Over 50% of the global usable land is already in pastoral or intensive agricultural uses and urban systems, reducing the potential for the sustainable provision of many goods and services from natural ecosystems. These new systems have been causing negative effects on climate, soil fertility, biogeochemical cycles, land use, and diversity at different spatial scales [5, 37, 38].

Currently, more than half of the world's population lives in cities, occupying 3% of the earth's surface [3, 35, 39]. Humans appropriate one-third of the net primary productivity of the land and 8% of the ocean to meet their food, energy, and production and consumption needs for goods and services [39, 40]. Natural terrestrial ecosystems are a major source of timber, fuels, fuelwood, resins, and fibers, which provide provisioning services for humans [2, 3]. However, their extension and coverage have been replaced by new crop areas (12%) and pastures for livestock (25%) to produce the necessary food for a constantly increasing population; being the main drivers of the transformation of the structure and functionality of the landscape at different spatial and temporal scales [1, 5, 37]. The intensification of agricultural systems transforms natural terrestrial ecosystems by changing land cover and land use, resulting in deforestation, defaunation, and land-use change [1, 5, 9, 36].

Forest clearing processes have been permanent and at different intensities around the world, configuring different patterns of land use and, therefore, different deforestation dynamics [5, 41]. In the case of tropical rainforests, deforestation has increased, giving way to agricultural, mining, and silvopastoral uses [5, 36]. These changes in the landscape present differences in cover types, being heterogeneous matrices where the radiation balance and temperature have extreme fluctuations day and night [42, 43]. The matrices are characterized by having greater exposure and change in abiotic variables, such as wind intensity and frequency, temperature, solar radiation, relative humidity, greater water flow, and high water or saline erosion of soils [5, 36, 43]. In this sense, depending on the use, anthropogenic systems affect the quality and quantity of habitat for both vegetation and fauna found on the edges of the remaining forest fragments [1, 5, 44].

These matrices are surrounded by small fragmented patches of forest, which have different soil types and vegetation, and vary in size, shape, and isolation [45, 46]. The biological and physical interaction of these two transition zones generates an effect of changing environmental and biotic conditions at the edges [45, 47]. Thus, the edge effect is mostly associated with fragmentation and habitat loss [6, 46]. As habitat fragments, the geographic extent of the anthropogenic matrix increases, and the edge effect on remnant native forests increases, which increases their isolation as habitat loss is accentuated [20, 48]. As landscapes are transformed, the original continuity of native cover is broken, decreasing the reproductive success of native species and the genetic exchange between populations [1, 7, 49].

Fragmentation in tropical ecosystems directly affects population densities and the number of species, especially endemic species [50, 51]. Likewise, the edge effect and fragmentation generate an alteration of the habitat of native species [1, 7, 49]

and favor the appearance of new exotic/invasive or disturbance-adapted species that compete with the rest of the species for resources [50, 52]. Habitat degradation and loss caused by ecosystem transformation processes [44, 52] result in the extinction of species of the taxonomic groups most sensitive to disturbance, such as amphibians and some reptiles [49, 52].

In 2000, it was considered that approximately 60% of the world's tropical forests were degraded due to anthropogenic disturbances [2]. Worldwide 70% of native forests are located less than 1000 m from a productive system [53]. In the productive system the activities, such as burning, spraying, logging, unmanaged soil fertilization, use of pesticides, and herbicides, are traditionally carried out [5]. These activities generate strong impacts not only locally, but also lead to ecological footprints on a regional scale. Elements, such as the edge effect, alterations in the composition of biota, presence of water bodies, and soil permeability, negatively affect biodiversity and ecosystem services [37, 54].

### **3. Response of amphibian assemblages to ecosystem transformation**

Amphibians are essential components of many natural ecosystems. They are indicators of ecosystem health [55] and have important roles in natural food webs [56]. These animals are sensitive to environmental changes and require moist habitats with relatively low and constant temperatures [55]. In addition, the distribution of amphibians in forests may be determined by the heterogeneity of ground cover [57, 58] and the availability of microhabitats [56, 59]. The stages of larval of anurans (eggs, embryology, and tadpoles) are subject to different biotic and abiotic conditions that affect survival, development, and size during development [31, 60, 61]. Many anuran larvae exhibit phenotypic, physiological, and ethological adaptations in response to changes in the environment that will directly influence premetamorphic growth and development, developmental speed, growth rates, body size, weight at the end of metamorphosis and the length of the larval cycle [17, 62, 63].

The choice of the aquatic environment, by adult amphibians for oviposition, influences the rates of fertilization, embryonic development, larval growth, survival, and mortality of these organisms (**Figure 1**) [64–66]. Likewise, the structural and physiognomic characteristics of the aquatic habitat are determined by: 1) the temperature of the medium, 2) the amount of food available, 3) the amount of radiant energy through sunlight, 4) the amount of oxygen available, 5) the amount of accumulated excreted waste, 6) bacterial growth, 7) predation, 8) the space available per organism (density), 9) presence of growth-inhibiting substances and 10) variation in habitat size [16, 67]. In anurans, changes in environmental selective pressures accompany and determine development, body size, weight, and the timing of metamorphosis [18, 68], affecting the distribution of species and their use of different habitats [66].

The amphibian's different life-history stages may occur in disjunct habitats and function at different spatiotemporal scales (**Figure 1**) [22, 23, 69, 70]. Changes in the physical and biotic environment primarily affect physiological, ecological, and biological processes [18].

The interactions of these factors in anuran larvae (**Figure 1**) depend on the characteristics of the habitats selected. The parents select the site to lay eggs, as well as the characteristics of the microhabitats they use as a mechanism to mitigate competition and predation, due to different relationships over time [18, 70]. Tadpole survival, growth, and development are secondarily affected by the behavior of each species in predator-prey relationships and their phenotypic plasticity (**Figure 1**) [18, 70].

*Effect of Agricultural Pesticides and Land Use Intensification on Amphibian Larval Development DOI: http://dx.doi.org/10.5772/intechopen.106268*

### **Figure 1.**

*Summary of factors affecting the biology and ecology of the anurans (adults, eggs, hatchlings, and tadpoles). These processes are influenced by primary factors in or of the biotic environment. The secondary factors are reflected in the reproductive ecology and behavior of adults; they can affect the biotic environment of a larva (microhabitat selection). The arrows indicate interactions and their directions, which need not be directly causal. Data from [18].*

Biotic factors include but are not limited to food availability and quantity, population density (crowding), and predation [62]. Abiotic factors, such as environment, ambient temperature, photoperiod, and water body (water level, quality) directly influence amphibian larvae and physiological mechanisms related to growth and differentiation [62, 71–73]. The anthropogenic transformation of the landscape tends to be heterogeneous, thus imposing new adaptation challenges to amphibians, presenting in some cases high biodiversity and only those species that have greater phenotypic plasticity to abiotic variations and ecosystem disturbance can adapt [16, 17]. However, the patchy distribution of aquatic habitats together with the reduced dispersal capacity of some amphibian species could impede the colonization of new habitats by adults, so the selection of breeding sites could determine the occurrence of tadpoles in each habitat [22]. In the water body,

tadpoles may select occurrence sites according to microhabitat characteristics based on food availability and predation risk [16, 22].

Intensification of agricultural practices is observed on a global scale, generating habitat loss, reduced landscape heterogeneity, and connectivity [10, 12, 74]. Amphibians depend on the quality of aquatic habitats for reproduction and development, as well as the surrounding landscape for the terrestrial phase [34]. In agrosystems the composition and abundance of amphibian species are low. Presenting generalist species that have different traits that allow these organisms to survive. Amphibians that lay their eggs in lentic water bodies and whose larvae develop there (without parental care) tend to be more tolerant to the anthropogenic transformation of the landscape [20, 75]. An example of this is foam nests which can be a successful strategy to colonize highly dynamic and ephemeral anthropogenic bodies [20].

### **4. Effect of pesticides on the development and growth of amphibian larvae**

In the world, with the expansion of the agricultural frontier, the use of fertilizers and pesticides has become widespread, with negative effects on amphibian populations [76], and the use of herbicides and pesticides contaminate the air, soil, surface water, and subsoil, generating serious problems for community dynamics and biodiversity [76–78]. Chemical pesticides have been one of the main resources used in intensive and conventional agrosystems for the control of some pathogenic fungi, insect pests, and weeds [7, 77]. The chemical composition of pesticides varies according to the degree of toxicity and persistence associated with their origin (natural and synthetic). Natural pesticides originate from pyrethrin, nicotine, and rotenone [7, 79].

Synthetic pesticides have hydrocarbons, chlorinated pesticides, organophosphates, and carbamates as their groups of origin. However, cyclodienes, carbamates, and organophosphates were eliminated from 50% of the world commercial market [77, 79], due to their high toxicity and collateral effects on other nontarget species [77]. In wild populations (amphibians, reptiles, birds, and mammals) [79], some effects are caused by residues of organic insecticides and organophosphates, which are abundant in the environment, are known [77, 79].

Pesticides in amphibians, not only reduce prey availability, but also reduce the transmission of matter and energy and, ultimately, growth rates [20, 34]. Within agroecosystems malformations in adult anurans and tadpoles are very common, being one of the main causes of high amphibian mortality rates [34, 80].

Among the different adverse effects of organophosphates on anurans are changes in survival and growth rates, morphological malformations, and some behavioral problems [33]. Organophosphate pesticides, such as endosulfan, are highly toxic in the environment [77, 79] and degrade slowly, leading to accumulations in food chains [79]. The impact of endosulfan on wildlife is associated with lethal effects on some fish and on the larval stage of amphibians present in water bodies near the areas where the pesticide is applied. Among the nonlethal effects, there are delays in the growth and development rates of amphibian larvae [31, 81].

Chlorpyrifos remains in the water for only a few days or weeks [33]. The tadpoles to high concentrations of chlorpyrifos have significant negative effects on growth and metamorphosis development time. On the other hand, the use of pyrethroids are pest controllers due to their low toxicity in birds and mammals, nevertheless showing that they are highly toxic to aquatic organisms [82]. The tadpoles both lethal and sublethal *Effect of Agricultural Pesticides and Land Use Intensification on Amphibian Larval Development DOI: http://dx.doi.org/10.5772/intechopen.106268*

effects have been recorded, which are associated with behavioral changes, affecting larval cohorts and gregarious behavior patterns that favor the search for food and cause greater predation in intoxicated larvae [78, 82].

Among the pyrethroids is cypermethrin (Cy), which is highly active and effective against a wide range of pests that affect agriculture, public health, and domestic animals [33, 78], but it also reduces the biodiversity of aquatic organisms, such as crustaceans, aquatic insects, fish, and anuran larvae, which are not part of the target species for control [78, 82]. The cypermethrin in amphibian larvae causes the death of nerve cells in anurans, and have also been determined that, when exposed to this pesticide during the early stages, developmental inhibitions are observed [78]. However, the different concentrations of cipermectrin that produce mortality and those required to produce malformations are different.

Pesticides are a group of substances with varying degrees of toxicity and very diverse characteristics, among which two large groups can be distinguished: (1) elements that are defined by the type of use of the pesticide, according to the organism on which they act, such as insecticides, herbicides, acaricides, fungicides, and rodenticides. (2) according to the chemical structure of the substances with pesticidal activity, which are divided into organophosphates, organochlorines, carbamates, carboxylic acids, pyrethroids, amides, anilines, alkyl derivatives of urea, heterocyclic compounds with nitrogen, phenols, imides, inorganic compounds [77].

Sediment entrainment by drainage and irrigation systems could generate eutrophication processes in aquatic systems and greenhouse gas emanations [83]. Contamination of water sources has negative effects on populations, for example, the amphibians do not have shelled eggs and their skin is a permeable organ. These animals are more sensitive to pollution and deterioration of the environment [33, 78, 82]. Tadpoles could be directly or indirectly in agricultural areas, being exposed to contaminants present in both their aquatic and terrestrial habitats [20, 84, 85]. The sublethal effects of cypermethrin are abnormalities, changes in behavior, the acceleration or delay of metamorphosis due to chemical stress, loss of appetite, mutations, death of amphibian nerve cells, low rates of the embryo, and tadpole development [78, 82] and lethal effects (high mortality rates).

The incidence of agrochemical ecotoxicity in anuran larvae has shown different responses about the species, the concentration of the contaminant, its degradation rate, exposure times, and to predator pressures [86]. Some studies have shown that in crops, the use of agricultural inputs at different concentrations is often not high enough to cause immediate mortality but generates sublethal consequences, such as depressions in the immune system of amphibians, which makes them more susceptible to parasites and malformations in their morphology [33, 78, 82] or the decline of the different species and populations of the impacted habitat, where alterations in the food chain may have greater consequences than direct chemical effects [86].

### **5. Case study**

Under experimental conditions, we evaluated the effects of the biocide cypermethrin (Cy: the substance of the pyrethroid chemical group), the active compound of the commercial product ®Fuminate in 80 larvae of Mannophryne vulcano, at larval development stages 25 and 26 according to Gosner's table [87]. Three treatments with ®Fuminate and control were designed, each with 20 replicates.

In the experimental subjects exposed to different concentrations of cypermethrin and the control, the following variables were studied: (1) stage according to Gosner [87]; (2) body weight (g), and (3) total length (TL). Follow-up of the test for each treatment and larva was carried out until the completion of the metamorphosis process, observing the sublethal effects on its development.

In each of the experiments, the following records were obtained: with treatment level 1) in concentrations of one (1) ml of cypermethrin, diluted in one (1) liter of water (concentration suggested by the manufacturer for the bathing of cattle, horses, and domestic dogs) obtained 100% mortality in less than 12 h. The high degree of toxicity of this pyrethroid to aquatic life organisms is demonstrated, especially to anuran larvae, which cause lethal and sublethal effects [78, 82]. In the treatments of cypermethrin, level 2) at 0.4125 ml/l, level 3) at 0.206 ml/l, and the control treatment (no insecticide), no deaths were recorded during the first 24 h.

The variables studied indicate that *Mannophryne volcano* tadpoles exposed to sublethal concentrations of cypermethrin were affected in the rate of weight gain (**Figure 2**), weight (**Figure 3**), and growth (**Figure 4**), until the end of the metamorphosis. However, no increase in length during the larval period or malformations were observed as suggested by Carr et al. [88] for similar cases. This could be related to the time of exposure to the xenobiotic substance and the embryonic stage in which the organisms were found, being more likely to observe these effects in organisms exposed since the laying of eggs by the female.

Tadpole growth and weight gain showed similar behavior in the three levels evaluated. In the larvae of *M*. *vulcano* there were no significant differences between the different levels in both development and growth, which indicates that the first and final stages are the most critical in larval development [33].

Survival in the two treatments with cypermethrin concentrations registered the lowest values. The highest pesticide concentration had the lowest survival of metamorphs compared to those recorded for the other levels. It is important to keep in mind that tadpoles are more susceptible to contaminants when they are in the transition from an aquatic to a terrestrial phase, where sublethal effects could be greater during this critical phase.

### **Figure 2.**

*Weight gain of Mannophryne vulcano larvae from the average values, during the evaluation of the three treatments.*

*Effect of Agricultural Pesticides and Land Use Intensification on Amphibian Larval Development DOI: http://dx.doi.org/10.5772/intechopen.106268*

**Figure 3.** *Average size at the end of the metamorphosis of Mannophryne vulcano, during the experiment with three types of pesticides.*

### **Figure 4.**

*Average weight at the end of the metamorphosis of Mannophryne vulcano, during the experiment with three types of pesticides.*

It is also important to highlight that the growth, development, and timing of the larval period depend on environmental factors, such as temperature, quality, quantity of food, and density, which were previously evaluated. According to Izaguirre et al. [78] larvae from temporary water bodies tend to be more sensitive to cypermethrin concentrations compared to larvae from lotic habitats.

### **Acknowledgements**

We acknowledged the anonymous reviewers and the associate editor Dr. Mohammad Manjur Shah who provided insightful comments on an earlier

version of the manuscripts. Our case study was financially supported by the Chicago Zoological Society.

### **Competing interests**

We declare that we have no competing interests.

### **Author details**

Claudia Patricia Camacho-Rozo1 \* and Jairo Antonio Camacho-Reyes2

1 Department of Ecology and Territory, School of Rural and Environmental Studies, Pontificia Universidad Javeriana, Bogotá, Colombia

2 School of Biology, Universidad Pedagógica y Tecnológica de Colombia, Tunja, Colombia

\*Address all correspondence to: claudiap.camachor@javeriana.edu.co

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Effect of Agricultural Pesticides and Land Use Intensification on Amphibian Larval Development DOI: http://dx.doi.org/10.5772/intechopen.106268*

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