**Skin Biopsy Applications in Free Ranging Marine Mammals: A Case Study of Whale Skin Biopsies as a Valuable and Essential Tool for Studying Marine Mammal Toxicology and Conservation**

Catherine F. Wise, John Pierce Wise, Jr., Sandra S. Wise and John Pierce Wise, Sr

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54212


**2. Challenges of recovery, recycling, collection, and disposal of solid wastes by private company**

#### **2.1 Description of the practice**

### *2.1.1 What the practice*

This practice is the result of recent challenges in the recovery, recycling, collection, and disposal of solid wastes by private individuals and businesses. In this instance, the activity is being carried out by a registered solid waste private company in Nigeria's historic city of Kano. It is a limited liability company undertaking collection and disposal of solid wastes on a commercial basis. Its services include on site separation of these waste before transporting the collected waste via a temporary site before final disposal, this company also undertakes in the sweeping of street/market, supply of refuse bins, collection and disposal of refuse. At present its services are available to individuals, private, and governmental organizations within the Kano Metropolitan area. The customers of this company are typically home or company owners who currently have a contract with the refuse collection and disposal company that specifies the anticipated volume of waste to be collected on a regular basis and on the basis of which the company informs the customer of the monthly fee. Currently, a client will pay N2, 000.00 a month on average to have a single 200-liter drum utilized as a waste

can and cleaned out once a week. The refuse is being disposed of through recovery, recycling, and leveling of what is left into a borrow pits in designated areas approved by the Kano State Urban Planning and Development Authority (KNUPDA). They operate a workshop for the maintenance of their machines and equipment. Kano is a developing city in Northern Nigeria that is expected to become economic vibrant from the activity of this stakeholder activity in the coming years, this of course is regarded as a recent advance for a developing nation in this twenty-first century era.


4. Assessing welfare in farmed fish

3.4.5. Other senses

120 Animal Domestication

Figure 2. The sensory worlds of fish.

There are sensory systems in fish that are completely alien to us. The lateral line for example, which serves as a receptor for hydrodynamic stimuli such as those generated by conspecifics, predators or prey. Although the biological processing of hydrodynamic signals has been well studied, not much is known about how fish can discern these from natural occurring events [94]. As all fishes experience night, darkness or turbid waters, there is strong selection for the use of non-visual senses in all fish species. Anatomical diversity suggests that the lateral line is one of the most important senses for fishes. However, research on the function of the lateral line has lagged due to poor understanding of hydrodynamics at small scales and lack of this sense in humans, making it difficult to imagine a fish's hydromechanical world [95]. Electrical sensing is ancestral to fishes and is present in most non-teleosts as well as certain teleost species. The electrosensory world of fishes is rich with electric fields from a multitude of sources including the earth's magnetic field and the bodies of all aquatic organisms including the electrosensing fish itself. The fish's extremely high sensitivity to these fields enables orientation, navigation, communication, and even detection and localization of other fish, both prey

Not only the sensory world of fishes is difficult to relate to, but also the physics of movement underwater in a three-dimensional world can be challenging to understand for humans, who exist roughly in a 2D world. Despite this challenge, it is nonetheless a critical next step for the

and conspecifics [96–98]. Figure 2 summarises the sensory world of fish.

Welfare in aquaculture has been a motive of academic work in the recent past. Several authors have addressed the topic in reviews and research papers [16, 68, 108–110], and the COST action Welfare of fish in European aquaculture has been promoted aiming to (i) improve the knowledge on welfare of fish, (ii) formulate a set of guidelines embodying a common and scientifically sound understanding of the concept of welfare in farmed fish, and (iii) construct a range of targeted operational welfare indicator protocols to be used in the industry [111]. The results of this action were incorporated in many research projects, not only in Europe but also in the USA, Canada, and New Zealand. In addition, major stakeholders in the industry were also involved, including the European Aquaculture Associations, the Fish Farmers Association and the scientific department of EU responsible for fish welfare.

(IV) Freedom to express normal behaviour—by providing sufficient space, proper facilities

Domestication and Welfare in Farmed Fish http://dx.doi.org/10.5772/intechopen.77251 123

(V) Freedom from fear and distress—by ensuring conditions and treatment that avoid

This concept forms the basis of recommendations and legislations worldwide and, despite their age, are extensively employed for academic, educational and veterinary purposes with great practical utility [113]. Also, they paved the way so that animals could be considered by

Research on animal sentience, situated between ethology and psychology, was initiated by ethologists such as Dawkins [115, 116] and cognitive psychologists such as Toates [117], around the early 1980's. These studies allowed a deeper understanding of animal minds and depicted how animals perceive the world and how environmental stimuli may affect their welfare level. Most importantly, they opened the door to the understanding of animal's subjective experiences. These experiences may be positive and negative, and the latter include suffering, which is fundamentally a wide range of unpleasant emotional (or emotion-like) states. Negative experiences occur when unpleasant subjective feelings are acute or continue for a long time when an animal is unable to carry out the actions that would normally reduce risks to life and reproduction in those circum-

The five freedoms concept is nonetheless criticised. Some authors claim that this framework is overly guided by anthropocentric thinking about how animals ought to be handled, neglects the concept of allostasis (stability through change) and generally reflects a more ethical view than a science-based approach [119]. The concept may be misleading on, for example, stress and stress indicators such as cortisol [120], because it is well known that stress hormones are also involved in healthy adaptation [121–123]. Capacity to change, allostasis and biologically relevant challenges are crucial for good health and welfare, therefore stable conditions and homeostasis should not be considered optimal [119, 124]. As discussed in Section 3, phenotypes that are selected exclusively for production traits often show signs of structural and morphological imbalance. This occurs because symmorphosis (i.e. a match between structural design and functional demand) is disrupted [125]. In many farm animals (e.g. broiler chickens or heavily selected strains of trout), the structural design of internal organs does not match functional demand [48, 126]. This imbalance is responsible for many health problems in farm

The Concept of Animal Welfare based on Allostasis aims to be an alternative to the Five Freedoms. It incorporates recent scientific developments in behavioural physiology and neu-

• Stability through change (allostasis) and capacity to change are crucial to good health and good animal welfare. Health in this concept has the same meaning as defined in the World Health Organisation's (WHO) constitution as 'a state of complete physical, mental and

social well-being and not merely the absence of disease or infirmity' [127].

robiology and can be summarised as follows [119]:

and company of the animal's own kind;

European law as sentient beings in the Lisbon Treaty of 2007 [114].

mental suffering.

stances [115, 118].

animals.

In recent years, the FishEthoBase project (http://fishethobase.net) has been working in order to bridge the gap even further between the scientific community and the fish farming industry. This open-access database on fish ethology and welfare provides a platform where scientific knowledge is scrutinised and summarised in order to answer relevant criteria regarding welfare in aquatic animal farming. The aim is to cover all fishes farmed nowadays as well as other aquatic species, delivering concrete solutions for fish farmers, pointing to knowledge gaps for researchers and providing awareness for the general public and other stakeholders. This is accomplished in two ways: (1) full profiles of farmed species, where over 40 criteria and sub-criteria on ethology, but also ecology, physiology and general biology are reviewed in-depth according to the scientific literature. Based on these findings, a series of recommendations are proposed to address identified welfare issues in aquaculture; (2) short profiles of farmed species, where a sharp evaluation of 10 critical criteria is performed, covering ecological, behavioural and physiological traits transversal to all fish species: home and depth range, migration, reproduction, aggregation, aggression, habitat type, stress, malformations and slaughter. In addition, sustainable feeding and domestication are also addressed. These 10 criteria are answered using a welfare assessment protocol based on standardised risk analysis methods. For each species, this protocol provides (i) a comparison between the wild behaviour and the fish welfare state under conventional farming conditions, (ii) the overall welfare potential and (iii) the certainty of our findings. The sum of high scores of each species in these three measures throughout all 10 criteria results in the FishEtho-Score, an index that summarises the general welfare state of the species. As occurs with all indexes, the FishEthoScore incurs the risk of oversimplifying a complex array of data concerning welfare. However, by asking the same questions to (ultimately) all farmed fish species, it offers a unique possibility not only for a comparative approach, but also for a global perspective on which species may be farmed most humanely. This is apparently the first effort to create such a welfare assessment scheme for aquaculture, and it may constitute a decisive step for a near-future welfare certification in the industry.

#### 5. Fish domestication and the Five Freedoms

The concept of the Five Freedoms was coined in 1965 in the Brambell Report [112] concerning husbandry of livestock and revised by the Farm Welfare Council of the UK in 1979 into its present form [113]:

(IV) Freedom to express normal behaviour—by providing sufficient space, proper facilities and company of the animal's own kind;

involved, including the European Aquaculture Associations, the Fish Farmers Association and

In recent years, the FishEthoBase project (http://fishethobase.net) has been working in order to bridge the gap even further between the scientific community and the fish farming industry. This open-access database on fish ethology and welfare provides a platform where scientific knowledge is scrutinised and summarised in order to answer relevant criteria regarding welfare in aquatic animal farming. The aim is to cover all fishes farmed nowadays as well as other aquatic species, delivering concrete solutions for fish farmers, pointing to knowledge gaps for researchers and providing awareness for the general public and other stakeholders. This is accomplished in two ways: (1) full profiles of farmed species, where over 40 criteria and sub-criteria on ethology, but also ecology, physiology and general biology are reviewed in-depth according to the scientific literature. Based on these findings, a series of recommendations are proposed to address identified welfare issues in aquaculture; (2) short profiles of farmed species, where a sharp evaluation of 10 critical criteria is performed, covering ecological, behavioural and physiological traits transversal to all fish species: home and depth range, migration, reproduction, aggregation, aggression, habitat type, stress, malformations and slaughter. In addition, sustainable feeding and domestication are also addressed. These 10 criteria are answered using a welfare assessment protocol based on standardised risk analysis methods. For each species, this protocol provides (i) a comparison between the wild behaviour and the fish welfare state under conventional farming conditions, (ii) the overall welfare potential and (iii) the certainty of our findings. The sum of high scores of each species in these three measures throughout all 10 criteria results in the FishEtho-Score, an index that summarises the general welfare state of the species. As occurs with all indexes, the FishEthoScore incurs the risk of oversimplifying a complex array of data concerning welfare. However, by asking the same questions to (ultimately) all farmed fish species, it offers a unique possibility not only for a comparative approach, but also for a global perspective on which species may be farmed most humanely. This is apparently the first effort to create such a welfare assessment scheme for aquaculture, and it may constitute a decisive step for a near-future

The concept of the Five Freedoms was coined in 1965 in the Brambell Report [112] concerning husbandry of livestock and revised by the Farm Welfare Council of the UK in 1979 into its

(I) Freedom from hunger and thirst—by ready access to fresh water and a diet to maintain

(II) Freedom from discomfort—by providing an appropriate environment including shelter

(III) Freedom from pain, injury or disease—by prevention or rapid diagnosis and treatment;

the scientific department of EU responsible for fish welfare.

122 Animal Domestication

welfare certification in the industry.

full health and vigour;

and a comfortable resting area;

present form [113]:

5. Fish domestication and the Five Freedoms

(V) Freedom from fear and distress—by ensuring conditions and treatment that avoid mental suffering.

This concept forms the basis of recommendations and legislations worldwide and, despite their age, are extensively employed for academic, educational and veterinary purposes with great practical utility [113]. Also, they paved the way so that animals could be considered by European law as sentient beings in the Lisbon Treaty of 2007 [114].

Research on animal sentience, situated between ethology and psychology, was initiated by ethologists such as Dawkins [115, 116] and cognitive psychologists such as Toates [117], around the early 1980's. These studies allowed a deeper understanding of animal minds and depicted how animals perceive the world and how environmental stimuli may affect their welfare level. Most importantly, they opened the door to the understanding of animal's subjective experiences. These experiences may be positive and negative, and the latter include suffering, which is fundamentally a wide range of unpleasant emotional (or emotion-like) states. Negative experiences occur when unpleasant subjective feelings are acute or continue for a long time when an animal is unable to carry out the actions that would normally reduce risks to life and reproduction in those circumstances [115, 118].

The five freedoms concept is nonetheless criticised. Some authors claim that this framework is overly guided by anthropocentric thinking about how animals ought to be handled, neglects the concept of allostasis (stability through change) and generally reflects a more ethical view than a science-based approach [119]. The concept may be misleading on, for example, stress and stress indicators such as cortisol [120], because it is well known that stress hormones are also involved in healthy adaptation [121–123]. Capacity to change, allostasis and biologically relevant challenges are crucial for good health and welfare, therefore stable conditions and homeostasis should not be considered optimal [119, 124]. As discussed in Section 3, phenotypes that are selected exclusively for production traits often show signs of structural and morphological imbalance. This occurs because symmorphosis (i.e. a match between structural design and functional demand) is disrupted [125]. In many farm animals (e.g. broiler chickens or heavily selected strains of trout), the structural design of internal organs does not match functional demand [48, 126]. This imbalance is responsible for many health problems in farm animals.

The Concept of Animal Welfare based on Allostasis aims to be an alternative to the Five Freedoms. It incorporates recent scientific developments in behavioural physiology and neurobiology and can be summarised as follows [119]:

• Stability through change (allostasis) and capacity to change are crucial to good health and good animal welfare. Health in this concept has the same meaning as defined in the World Health Organisation's (WHO) constitution as 'a state of complete physical, mental and social well-being and not merely the absence of disease or infirmity' [127].

• Good animal welfare is characterised by a broad predictive physiological and behavioural capacity to anticipate environmental challenges.

6. Ethics vs. welfare in fish domestication

phers and scientists:

points, felt sorry for the other's dog' [131].

The ethical discussion on welfare of animals is controversial. This occurs because often the perspectives of scientists studying welfare science(s) and philosophers debating about ethics lie on very different standpoints. While science uses mostly operational and measurable concepts, such as the ones described throughout this chapter, ethics is focused on experiencing

Domestication and Welfare in Farmed Fish http://dx.doi.org/10.5772/intechopen.77251 125

• The contract theory states that, despite the naturally selfish nature of man, there is a need to waiver rights in exchange for the benefits of a contract (implicit or explicit) [129]; • The utilitarianism theory states that the best action is the one that maximises utility, and

• The deontologist theory states that the ethical value of an action is dependent on the motivation behind it and not on the consequences. It criticises the utilitarianism theory stating that the right of the individual can never be sacrificed for the common good [130].

The following allegory provides a good metaphor for the misunderstanding between philoso-

'Two dog owners met one day to walk their dogs together. One owner had grown up in a small family that valued health, safety, and orderly, disciplined behaviour. The dog of this owner received regular veterinary care, two meals a day of low-fat dog food, and was walked on a leash. The other owner had grown up in a large community that valued conviviality, sharing of resources and close contact with the natural world. This dog (the owner's third - the first two had been killed by cars) had burrs in its coat, was fed generously but sporadically, and had never worn a collar in its life. Each owner, judging quality of life from very different view-

The challenge lies in the different concepts, assumptions and vocabulary that scientists and philosophers use, which function as two distinct cultures with little mutual understanding or communication. Since the early days of the animal welfare debate, the two sides have struggled to communicate with each other, even though both were (and are) working with the common goal of understanding and improving an appropriate relationship between humans and other species of animals [132]. In fact, scientific research on animal welfare began because of ethical concerns over the quality of life of animals, and the public looks to animal welfare research for guidance regarding these concerns. The conception of animal welfare used by scientists must therefore relate to these ethical concerns in order to make sure that the orientation of the research and the interpretation of the findings are to address them successfully [131]. In order to bridge the gap and seek common ground between ethics and welfare science, it is important to recognise three classes of problems that may arise when the adaptations present in an animal do not fully correspond to the challenges posed by its current environment. These problems summarise the ethical concerns about the quality of life of animals [131]: • If animals present adaptations that no longer serve a significant function in the new environment, then unpleasant subjective experiences may arise, yet these may not be

values and critically reflecting on them. Three main ethical theories are followed:

therefore actions are to be evaluated majorly for their consequences [129];

To summarise, the Five Freedoms were primarily derived in relation to the welfare of farm animals, but, with the exception of the fifth freedom, would appear to consider that animals are passive within their environment [128]. Despite its undeniable role in the development of present (and future) welfare standards, this concept would benefit from an update in order to incorporate both ultimate (i.e. adaptive) and proximate (i.e. physiological) mechanisms. Integrating phylogeny and ontogeny in the design and analysis of husbandry practices would result in broader and overall better welfare schemes (Figure 3).

Figure 3. Animal welfare in relation to environmental challenges as shown by the out-dated concept based on homeostasis and the new concept based on allostasis. Adapted from [117].


**2. Materials and Methods**

#### *2.1. Soil Characteristics*

An incubation study was conducted on soils (0–15 cm depth) that were collected from six locations in Manitoba, Canada. The locations were Carman (Soil 1; 49◦29 6"N, 98◦02 2"W), Carberry (Soil 2; 49◦53 7"N, 99◦22 29"W), Deerwood (Soil 3; 49◦22 1"N, 98◦23 34"W), High Bluff (Soil 4; 50◦01 2"N, 98◦08 9"W), Beausejour (Soil 5; 50◦05 13"N, 96◦29 58"W), and Portage la prairie (Soil 6; 49◦57 9"N, 98◦16 0"W). These were the same six soils used in two previous studies [14,15]. In the Canadian soil classification system, all soils are classified as Chernozems (an equivalent of Chernozem in the FAO classification system) except Soil 4, which is classified as a Regosol (an equivalent of Regosol in the FAO classification system) [18]. The soils were air-dried and ground to pass through a 2-mm sieve. A subsample of each soil was analyzed (Table 1) for organic matter by the wet oxidation method [19], cation exchange capacity by ammonium acetate method [20], urease activity [21], soil texture by pipette method [22], field capacity [23], and pH (soil/water, 1:2) and electrical conductivity with a combined conductivity and pH meter (Orion versaStar, ThermoFisher Scientific Inc., Waltham, MA, USA).

## *2.2. Experimental Design and Treatment Applications*

The experimental setup was a completely randomized design containing two inhibitor treatments of six soils, a factorial layout for eight sampling periods, and was replicated three times for a total of 288 experimental units. The inhibitor treatments were NBPT (10 mg NBPT kg−1 soil) and NBPT plus NI (DI; 10 mg NBPT + 2.5 mg NI kg−1 soil). We used analytical grades of NBPT (CAS: 94317-64-3) and NI (3,4-dimethyl pyrazole phosphate; CAS: 202842-98-6) in this study.

Ten grams of each soil was weighed in 50 mL centrifuge tubes. The soil was wetted to 75% field capacity based on soil mass, capped, and left to equilibrate for 24 h at room temperature. Twenty-four hours after wetting, the soils in the centrifuge tubes were spiked with 0.5 mL of a solution containing either 200 mg NBPT L−1 (NBPT inhibitor treatment) or 200 mg NBPT + 50 mg NI L−1 (DI inhibitor treatment). The ratio of NBPT to NI in the DI inhibitor treatment was the same as the ratio of NBPT to NI in the double inhibitor formulation used in our previous studies [14,15]. However, the current study did not include urea with the inhibitors, as we discovered that the presence of urea interfered with the analytical procedure for NBPT. The tubes were recapped and placed in an incubator (Isotope Incubator, Model 304, Fisher Scientific, Hampton, NH, USA) set at 21 ◦C. On days

0, 0.5, 1, 2, 4, 7, 10, and 14 after treatment application, three replicates or samples of each soil by inhibitor treatment (i.e., six soils × two inhibitor treatments × three replicates for a total of 36 samples) were removed (destructive sampling) from the incubator for NBPT extraction and analysis. Day 0 was immediately after the soil was spiked with the inhibitor treatments.

**Table 1.** Selected soil (0–15 cm) properties.

a Canadian soil classification system.

#### *2.3. Extraction and Analysis of NBPT*

On each sampling day, 25 mL of deionized water was dispensed on the sampled centrifuge tubes and shaken on a reciprocating shaker for 30 min at 120 excursions per minute. After 30 min of shaking, the samples were centrifuged for 5 min at 10,000× *g* to allow soil residues to settle to the bottom. Immediately after centrifugation, about 4 mL aliquot was transferred using a 0.2 μm syringe filter (Basix™ Syringe Filters, ThermoFisher Scientific, Waltham, MA, USA) into a 20 mL vial. This was followed by transferring 1 mL of the filtered aliquot into a 2 mL high-performance liquid chromatography (HPLC) vial (9 mm surestop screw vial, ThermoFisher Scientific, Waltham, MA, USA) containing 0.1 mL dimethyl sulfoxide for NBPT analysis with HPLC-mass spectrometry (HPLC-MS), as described by Engel et al. [17].

The HPLC-MS (Bruker Compact QqTOF, Billerica, MA, USA) used was equipped with an electrospray source that operated in the positive ionization mode. The nebulizer pressure of the source was 0.3 bar with 5 L min−1 of N2 drying gas at 200 ◦C. The capillary voltage was 3500 V, and the capillary exit voltage was 70 V. Reverse-phase chromatography was used to separate NBPT using an Intensity Solo C18 (100 × 2.1 mm, 2 μm) HPLC column (Bruker Daltonik, Billerica, MA, USA). The column was maintained at 35 ◦C with a flow rate of 300 μL min−1. The mobile phase consisted of formic acid 0.1% in Milli-Q water for Channel "A" and acetonitrile for Channel "B". A 2 μL aliquot of the sample was injected into the column and kept at 80% B from 0 to 3 min. From 3 to 4 min, the gradient was linearly ramped to 20% B, where it was kept for 1.5 min. Then, the gradient was linearly ramped to 80%, and it was held for 2.5 min at 80% for re-equilibration. The NBPT was eluted at approximately 3.3 min.

Data quantitation was performed using Bruker Daltonic QuantAnalysis (ver. 4.4) software (Billerica, MA, USA). The ion chromatograms for NBPT were defined as [M + H]+ (168.0719 *m*/*z*). The concentrations of NBPT recovered in soil were determined from a calibration curve of known standard solutions of NBPT and their corresponding peak

areas. The quantity of NBPT recovered was expressed as a percentage of NBPT applied to the soils.

### *2.4. Kinetics and Statistical Analysis*

Model fitting and statistical analysis were performed with SAS software (SAS Institute 2014, ver. 9.4 [24]). PROC NLIN was used to fit an exponential decay function (Equation (1)) to determine the degradation rate constant (k) of NBPT in the soils as follows:

$$\mathbf{Y} = \mathbf{b}\_o[\exp(-\mathbf{k}\mathbf{t})] \tag{1}$$

where Y is the % of NBPT recovered in soils at time t, t is the time in days, k is the NBPT degradation rate constant, and bo is an empirical constant.

For ease of interpretation, the generated k was used to calculate the half-life (t1/2) of NBPT in the inhibitor treatments using Equation (2):

$$\mathbf{t}\_{1/2} = \ln(2)/\mathbf{k} \tag{2}$$

We used PROC GLIMMIX (beta distribution) for repeated measure analysis to determine the significant effect of time, inhibitor treatments, and their interaction on the % of NBPT recovered in each soil. Furthermore, analysis of variance with PROC GLIMMIX (gamma distribution) was performed on the degradation rate constant and half-life of the NBPT across soils and inhibitor treatments. The fixed effects in the model were soil and inhibitor treatment. Mean comparisons were deemed significant at a probability level of 0.05 Fishers' protected least-significant difference. The goodness of fit for the exponential decay model was tested using the Nash–Sutcliffe model efficiency (ME) and root means square error [25]. Stepwise regression with PROC REG was used to analyze the influence of soil properties on the half-life of NBPT in soils.

$$ME = 1 - \frac{\sum\_{i=1}^{n} \left(\mathbf{Y}\_i^{\rm m} - \mathbf{Y}\_i^{\rm p}\right)^2}{\sum\_{i=1}^{n} \left(\mathbf{Y}\_i^{\rm m} - \overline{\mathbf{Y}}\right)^2} \tag{3}$$

where *Ym i* is the measured NBPT recovered in soil, *Yp i* is the predicted NBPT recovered in soil, and *Y* is the mean of measured NBPT recovered in soil. When *ME* = 1, there is a perfect relationship measured and predicted NBPT recovery in soil; and when *ME* = 0, the model has the same precision as the mean of measured NBPT recovered.