**1. Introduction**

In a general context, the welding area currently develops on the conceptual and technological foundations of Industry 4.0, as well as the latter actively collaborates to advance the former. Welding joining processes have never presented so many changes in their techniques as in recent years. This is mainly due to the insertion of arc welding in additive manufacturing [1, 2], as robotic systems have sought to improve the manufacture of parts and components by deposition or coating of flat and tubular surfaces for any type of materials. Whether carbon steel [3] or special alloys [4, 5]. However, the difficulty of implementation and the cost of additive manufacturing favor more

traditional processes such as the electric arc to remain for longer acting as the front line of the metalworking industry in the welding segment.

Thus, processes such as GMAW (Gas Metal Arc Welding) and FCAW-G (Flux Cored Welding with Gas Protection), among others, still remain firm, being used in the manufacturing and heavy assembly industries such as shipbuilding, oil industry, in addition to construction of structures and pipelines for the power generation industry, for example. Thus, it is known that the mentioned processes have high deposition rates, weld metal quality, in addition to versatility in their applications, which consolidates them in the market. However, due to the excessive need to increase productivity, many variants have emerged in order to assist in this procedure.

Currently, there are some aspects of the GMAW process, with the Cold Wire–Gas Metal Arc Welding (CW-GMAW), the Double Cold Wire–Gas Metal Arc Welding (DCW-GMAW), and the Hot Wire–Gas Metal Arc Welding (HW-GMAW). CW-GMAW and DCW-GMAW welding contributed significantly to increased productivity [6, 7]. CW-GMAW welding allowed for narrow bevel welding with 4 mm gap [8]. Furthermore, it is responsible for reducing the level of residual welding stresses [9] and increasing fatigue strength [10]. On the other hand, the HW variant presents as its main characteristic the increase in productivity, with the possibility of variation in penetration [11]. These variants have the possibility to achieve deposition rates ranging from small percentages from 10% to more than 100% of extra molten metal, also influencing the formation of microstructures such as acicular ferrite, which contribute to increase the properties, mainly mechanical, of the weld metal. In this context, some variants of the GMAW process will be presented below, some of which have already been tested, and others are still being tested for possible field applications, thus showing the importance of these welds for the academy and the manufacturing industry, assembly, and maintenance.


**Fig. 3.1** Aquatic organisms collected from waters around Japan and other localities, showing high levels in the maximum value (>1.0 ppm = μg g−1 wet wt). Minimum, maximum, and mean values and the sample size (italics in the figure) are indicated. Solid lines show the range of values, while broken lines are used when minimum values are unknown. Among the numbers that precede, the scientific names in the figure, nos. 1–5, 8–10, 12, 13, 22, 23, 25–27, and 29 animals, were collected from waters around Japan, while nos. 6, 7, 11, 14–21, 24, and 28 animals were collected from other oceans. English names are as follows: 1, bottlenose dolphin; 2, Indo-Pacific blue marlin; 3, shortfinned pilot shale; 4, bluefin tuna; 5, sperm whale; 6, tilefish; 7, swordfish; 8, bigeye tuna; 9, Baird's beaked whale; 10, southern bluefin tuna; 11, marlin; 12, alfonsino; 13, swordfish; 14, king mackerel; 15, Spanish mackerel; 16, halibut; 17, tuna; 18, snapper; 19, perch (freshwater); 20, scorpionfish; 21, American lobster; 22, yellowfin tuna; 23, Malabar grouper; 24, grouper; 25, Dall's porpoise; 26, gnomefish; 27, bastard halibut; 28, butterfish; and 29, banded grouper (Data source: MHLW 2005a)

**Table 3.2** Three kinds of aquatic products categorized by FDA and EPA, USA, for avoiding lowdose methylmercury hazards (EPA 2007)

max = 1.1–1.5 ppm). Among these species, the splendid alfonsino is known as a deepwater fish (depth, > 200 m), while perch (no. 19, max = 1.4 ppm) is a freshwater fish. These data show that carnivorous aquatic animals can accumulate methylmercury irrespective of their habitat and the taxonomic group to which they belong.

Based on the results on mercury concentrations in aquatic products, the Food and Drug Administration (FDA) and EPA in the USA issued advice regarding amounts of seafood ingested (EPA 2007). This advice was geared toward helping women who are pregnant or may become pregnant as well as breastfeeding mothers and parents of young children. FDA/EPA categorized aquatic products into three groups (Table 3.2) and recommended that (1) people can eat 227 g to 340 g a week of fish in the "best choices" category, (2) people can eat 113 g a week of fish in the "good choices" category, and (3) people should not eat fish in the "choices to avoid" category or feed them to young children.

The Japanese government published a document about the mercury risk of consuming seafood (MHLW 2005b). The summary is as follows: (1) fish and shellfish are an important diet; (2) mercury contaminations in fish and shellfish are very small, but some species contain higher levels in the mercury concentration; (3) reported mercury effects on fetuses are minor ("no more than degree" in original), for example, auditory response is delayed by less than one-thousandth second, and its intake through normal meals will not affect the baby's future life; (4) this advice is intended not to ask pregnant women to avoid fish and shellfish that contain high levels of mercury; and (5) women who are or may be pregnant should follow the recommendations in order to reduce the health risk to the minimum. MHLW (2005b) also categorized aquatic products having risks into four groups (A–D, Table 3.3) mainly based on mean values shown in Fig. 3.1 as well as on 2.0 μg kg−1 week−1 of PTWI which was established by themselves (FSC 2005). The objects of the recommendations are women who are or may be pregnant, while lactating women, infants, children, and adults are removed from consideration, because MHLW (2005b) considered that they are not affected by normal consumption of any aquatic food. The

**Table 3.3** Recommendations for pregnant women on consumption of aquatic products concerning mercury contamination by MHLW (2005b)

See Fig. 3.1 for scientific names of species that are assigned numbers from 1 to 25 in parentheses. Scientific names of species that are assigned numbers 30–34 are as follows: 30, *Buccinum striatissimum*; 31, *Dentex tumifrons*; 32, *Helicolenus hilgendorfii*; 33, *Prionace glauca*; and 34, *Scombrops gilberti*

observed maximum concentrations of whales, marlins, and tunas, however, were at the same level as those found in Minamata around 1960, suggesting that if these food was consumed by humans freely, even adults would become the acute type of MPM. The document includes the sentence "you can eat other tune species (yellowfin tuna, Albacore and juvenile bluefin tuna) than listed ones and canned tuna without any particular restriction." Reasoning of this recommendation is supposed to be relatively low mean values of the mercury concentration in these species; however, we can find a high maximum value, 1.25 ppm, in yellowfin tuna. The observed mean and maximum values are usually affected by the sample size and locality collected; we should be careful when listing risky species. Thus, the recommendation of MHLW seems to emphasize the advantage of consuming aquatic food and to dispel concerns about consuming aquatic food by underrepresenting the risk of mercury. Should we think to follow international standards not the laxest standard (2.0 μg kg−1 week−1 ) that MHLW established by themselves, to list risky aquatic organisms not at a species level but at a trophic level, and to recommend avoiding the consumption of highly risky organisms during the gestational period?

Consumption of seafood provides risks and benefits for humans. Seafood is the primary source of protein for many populations. It has many nutrients including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which give benefit by reducing heart diseases. Therefore, balanced assessments of the risks and benefits of seafood are required. In general, as trophic level increases, methylmercury concentrations in marine animals increase, whereas there is no relationship between trophic level and nutrient concentrations, suggesting that consuming lower trophic level seafood can minimize the risk of mercury exposure without reducing the benefits of nutrient intake.

As noted in Sect. 3.2.2, there is competent evidence for the fetal neurotoxicity of methylmercury at low-dose exposure (Gribble et al. 2016). That is, the positive association of mercury with neurodevelopment was found in four of nine populations, while five populations did not show any evidence (Kjellström et al. 1989; Davidson et al. 1998; Myers et al. 2003; Valent et al. 2013; Tatsuta et al. 2014; Strain et al. 2015). Further studies are necessary to clarify the relationship between low-dose mercury exposure and health hazards and to make appropriate suggestions regarding the acceptable dose level.

#### **3.3 Outstanding Issues**

Dr. Masazumi Harada, who contributed to the study on MPM as well as to MPM patient care, published a book entitled *Minamata Disease Not Settled Yet* in 1985. In those days, many patients were not acknowledged officially and sought redress from the court. As of 2017, legal battles over MPM still persist. This is mainly due to a strict criterion for diagnosing MPM which was established by the national government in 1977. In this criterion, multiple symptoms among (i) sensory disturbance, (ii) ataxia, (iii) equilibrium disturbance, (iv) visual field constriction, (v) central visual disturbance, (vi) central auditory disturbance, and (ii) others are needed for the diagnosis of MPM, resulting in discarding many patients. Such a conflict seems to be caused from a lack of understanding the whole context of victims through detailed surveys by the government. Urgent and thorough surveys are necessary, although long overdue. Besides, pathology of MPM should be elucidated further. If methylmercury damages the vascular system and/or acts as an endocrinedisrupting agent as suggested by Shiraki (1979), lesions will extend over the whole body resulting in a variety of symptoms, and even tiny amounts of methylmercury may affect the whole human body. Such studies may provide a useful clue as to resolving low-dose mercury issues in the global mercury cycle.

Gworek et al. (2016) described that the average residence time of mercury in oceanic waters, 20–30 years, is much longer than that in the atmosphere (0.8–2 years), resulting in that the mercury discharged into the ocean is removed from there much more slowly than the mercury emitted into the atmosphere and that an increase in the mercury concentration level in oceanic waters will be very slow and may take hundreds of years. This fact means that the mercury concentration will continue to increase gradually, even if anthropogenic emissions of mercury to the atmosphere stop right now. Methylmercury concentrations in some aquatic animals have reached the threshold level to cause neurodevelopmental deficits in fetuses and infants already; therefore, we have to minimize the mercury emissions as soon as possible. In this context, it is gratifying that the "Minamata Convention on Mercury" entered in force on August 16, 2017. Its major highlights include a ban on new mercury mines, the phasing out of existing ones, the phasing out and phasing down of mercury use in a number of products and processes, control measures on emissions to air and on releases to land and water, and the regulation of the informal sector of artisanal and small-scale gold mining.

An American photographer, William Eugene Smith, took excellent pictures of MPM patients. Among these, a picture titled "Tomoko Uemura in Her Bath," which depicted a mother cradling her severely deformed, naked daughter in a traditional Japanese bathroom, is impressive. The mother called Tomoko as "Takara-ko" in Japanese which means "treasure daughter" for the reason that the mother was able to keep her health because Tomoko had drawn all mercury and accumulated it into Tomoko's own body and that her six children grew up healthy and helped each other as a result of learning the importance of mercy from assisting their elder sister (Harada 1985). Tomoko passed away on December 12, 1977, at the age of 20. We can provide a description of the assailant who took the life of Tomoko. On the other hand, presently, we may be living in the world full of toxicants including mercury. In this world, we have a high probability of becoming a victim, and at the same time, we can be an assailant. Let's consider what we can do under such circumstances.

#### **References**

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**5. Lower and upper limit of achievable feature size**

So far, only structures with feature size of about 100 nm were described. Laser-assisted processes for much smaller and larger feature sizes will be discussed in this section, with a focus on factors that limit the achievable feature sizes of the techniques.

#### **5.1 Resolution limit**

In LADI, planarization, and via-hole filling processes, squeezing liquid metals or semiconductors into small trenches or via holes will be ultimately limited by the pulling pressure due to surface tension. Fig. 15 shows schematic drawing illustrating how much pressure is needed to fill trenches or via holes. For all cases, the pulling pressure is proportional to the surface tension constant and inversely proportional to feature size. Therefore, there is no fundamental resolution limit provided the applied pressure is high enough. In practice, the liquid metal and silicon generally have surface tension on the order of 1 N/m (for water, it is 0.076 N/m), which leads to a pressure of 200 bar to fill in 100 nm-wide trenches, or 2000 bar for 10 nm-wide trenches. This pressure is considerably higher

solidification across the gap should be uniform (since the melting time is order 100 ns that is much longer than pulse duration). Nano-tips are formed at the broken bridge necks before or during solidification due to surface tension and volume shrinkage, or after the two wafers

Fig. 14. Schematic of the physical mechanism behind the nano-tip formation. (a) Build-up of

condensation. (c) Nano-bridge formation due to attractive electrostatic force. (d) Bridging of approaching peaks and charge neutralization. (e) Break of bridges upon solidification or

So far, only structures with feature size of about 100 nm were described. Laser-assisted processes for much smaller and larger feature sizes will be discussed in this section, with a

In LADI, planarization, and via-hole filling processes, squeezing liquid metals or semiconductors into small trenches or via holes will be ultimately limited by the pulling pressure due to surface tension. Fig. 15 shows schematic drawing illustrating how much pressure is needed to fill trenches or via holes. For all cases, the pulling pressure is proportional to the surface tension constant and inversely proportional to feature size. Therefore, there is no fundamental resolution limit provided the applied pressure is high enough. In practice, the liquid metal and silicon generally have surface tension on the order of 1 N/m (for water, it is 0.076 N/m), which leads to a pressure of 200 bar to fill in 100 nm-wide trenches, or 2000 bar for 10 nm-wide trenches. This pressure is considerably higher

free charges on the two wafers. (b) Metal transfer to lower wafer by evaporation-

**5. Lower and upper limit of achievable feature size**

focus on factors that limit the achievable feature sizes of the techniques.

were separated.

after separation.

**5.1 Resolution limit**

than that used for regular thermal nanoimprint lithography (order of 10 bar). However, considerably lower applied pressure would be sufficient for patterning metals and silicon if: (1) the molten metal partly wets the SiO2 substrate; and/or (2) the mold consists of sparse protruded patterns to create sparse recessed metal features (such as a periodic hole array having diameter ~100 nm and pitch 500–600 nm in Au for extraordinary optical transmission devices (Lesuffleur 2008)), because the effective local pressure at the protruded mold features is significantly higher than the applied pressure (applied force divided by wafer surface area).

Fig. 15. Schematic drawing showing the calculation of minimum pressure to squeeze a liquid into (a) a trench, (b) a trench with liquid partly squeezed against the bottom, and (c) a via hole. The pressures are calculated assuming 90o contact angle and no wetting/adhesion.

#### **5.2 Upper limit of achievable feature size**

The maximum feature size that can be patterned depends on how far the molten metal can flow before re-solidification. For a rough estimation, the dynamic flowing process is modeled as in Fig. 16. It is assumed that the grating structure has a period 2L and a trench depth h0; and the entire layer is melted at t=0 and re-solidification takes place simultaneously throughout the film thickness at time t=. The mold will travel a distance of h0/2 and the residual molten layer thickness will decrease to h0/2 when the trench is fully filled. Since L is large, the effect of surface tension can be ignored and thus the internal pressure at x=L/2 is zero. There are three forces acting on the liquid: the pressing force from the mold, the viscous force, and the inertial force.

We first consider only the effect of viscous force by assuming that a steady flow develops momentarily at t=0 (i.e. ignore inertial force). Then the maximum grating half pitch L1 that can be patterned (trench fully filled) before the liquid metal solidifies is given by (Heyderman 2000, Cui 2006):

$$L\_1 = h \sqrt{\frac{P\tau}{\mu}} \tag{11}$$

where *h* is the metal film thickness (200 nm), P is the applied pressure (order 100 bar), is the viscosity, and is the melting duration (~ 200 ns) that depends on laser fluence. The

Ultrafast Fabrication of Metal Nanostructures Using Pulsed Laser Melting 135

Thus even within a very short melting time of order 100 ns, the calculated L2 values are

The critical length can also be estimated by experiment. For example, Fig. 17a shows a backside-irradiated 200 nm-thick Ni film by a single laser pulse with fluence 0.60 J/cm2 that partly blew the film off the substrate. The surface tension was the force driving the flow, and the driving pressure can be estimated by 2/h (h is the film thickness, and the factor 2 accounts for the top and bottom surface/interface), which was 180 bar. The SEM image indicates that Ni flowed about 4 m before the film re-solidified. For LADI, we achieved 17 m isolated Cu mesas with 100 nm height (Fig. 17b), but it was found that a pattern of several tens of micrometer size required multiple pulses. Therefore, the maximum patternable feature size using one laser pulse lies on the lower tens of m, close to the

Fig. 17. (a) A 200 nm-thick Ni film irradiated from backside by a single laser pulse with a fluence of 0.60 J/cm2 that partly blew the film off the substrate. The liquid traveled about 4 m before it froze. (b) AFM image of isolated Cu mesas patterned by LADI at laser fluence of 0.45 J /cm2. The mesas have a length on each side of 17 μm and a height of 100 nm.

(a) (b)

In this chapter, five nanofabrication processes using pulsed excimer laser were described. These methods share the common advantage of being orders faster than most other fabrication techniques. They are also very suitable to patterning metals, which are more difficult to process than semiconductors by conventional lithographies and

The first was laser-assisted direct imprinting, which produced 200 nm-period Cu, Ni and Al gratings over about 1 mm2 area within ~100 ns. LADI of W failed due to its high thermal stress. The second was wafer planarization using a flat and smooth "mold". With this technique, Cu surface was planarized by laser melting under pressure, which also squeezed the molten film to fill completely voids under the film. Cu conducting lines embedded in a dielectric matrix were created by an additional etching step. Third, a similar process could also fill 100 nm-wide and 500 nm-deep via-holes with Si and Cu. Fourth, sub-100 nm diameter Cr dots were melt-transferred to the receiving substrate using a laser-induced

order 20 µm for applied pressure of 100 bar.

theoretical prediction.

4m

**6. Conclusion**

etching.

viscosity of molten Au and Cu is around 4 cp (only 5 that of wafer (Yaws 1998)), leading to L1~ 5 m. To pattern isolated metal mesas, the liquid flows from four sides to fill a square-shaped hole in the mold, so the maximum patternable mesa size should be roughly doubled to ~10 m, which qualitatively agrees with the experiment (17 m). Moreover, it is interesting to see how far molten SiO2 can flow under similar conditions. For example, its viscosity at 2200 K is 8107 cp (a very tacky "liquid"), 7 orders higher than that of Au and Cu, leading to a calculated L1 of only ~1 nm if assuming a top 200 nm SiO2 layer is molten. This explains why the quartz mold can be used to pattern a metal at temperature slightly higher than its melting point, though the mold could not be used repeatedly for >>10 times.

Fig. 16. Dynamics of molten metal flowing into trenches during LADI. Assume that grating line-width and trench-width are both equal to L, and trench depth and molten layer thickness are both equal to h0.

Next, to estimate the inertial force which decides how fast the steady flow develops, it is assumed that the liquid metal is inviscid (μ = 0) for simplicity. The applied pressure is to accelerate the flow along the x-direction with acceleration given by (note that the effective pressure is doubled for a grating with equal line and trench width, and z is the direction perpendicular to the paper):

$$a = \frac{F}{m} = \frac{2P \times hz}{\rho \times hz \text{(L/2)}} = \frac{4P}{\rho L} \tag{12}$$

The velocity is then u=at at time t, and the flow-rate Q=uh=ath (ignoring the z-dimension). On the other hand, the volume of the liquid changes at a rate

$$Q = -\frac{L}{2}\frac{dh}{dt}\tag{13}$$

Therefore, solving the equation

$$\frac{4P}{\rho L}t\text{h} = -\frac{L}{2}\frac{dh}{dt}\tag{14}$$

gives (h=h0, h0/2 at t=0, )

$$L\_2 = 2\sqrt{\frac{P}{\rho \ln 2}} \tau \sim \sqrt{\frac{P}{\rho}} \tau \tag{15}$$

Thus even within a very short melting time of order 100 ns, the calculated L2 values are order 20 µm for applied pressure of 100 bar.

The critical length can also be estimated by experiment. For example, Fig. 17a shows a backside-irradiated 200 nm-thick Ni film by a single laser pulse with fluence 0.60 J/cm2 that partly blew the film off the substrate. The surface tension was the force driving the flow, and the driving pressure can be estimated by 2/h (h is the film thickness, and the factor 2 accounts for the top and bottom surface/interface), which was 180 bar. The SEM image indicates that Ni flowed about 4 m before the film re-solidified. For LADI, we achieved 17 m isolated Cu mesas with 100 nm height (Fig. 17b), but it was found that a pattern of several tens of micrometer size required multiple pulses. Therefore, the maximum patternable feature size using one laser pulse lies on the lower tens of m, close to the theoretical prediction.

Fig. 17. (a) A 200 nm-thick Ni film irradiated from backside by a single laser pulse with a fluence of 0.60 J/cm2 that partly blew the film off the substrate. The liquid traveled about 4 m before it froze. (b) AFM image of isolated Cu mesas patterned by LADI at laser fluence of 0.45 J /cm2. The mesas have a length on each side of 17 μm and a height of 100 nm.


**2. Materials and Methods**

### *2.1. Food Materials and Sample Preparation*

Colombian fresh lulo fruits (*Solanum quitoense* Lam.) were purchased at the Central Market of Valencia (Spain) and stored at 4 ◦C until processing.

The whole fruits were processed as follows for their analysis: after removing the peduncle, they were washed and cut into pieces, which were crushed in a mortar. This whole crushed fruit is hereafter called "fruit".

The procedure for obtaining the lulo juice was as follows: after removing the peduncle, the whole fresh fruit was washed and blended (Phillips Advance Collection Standmixer, 800 W, 2 L) for 10 min. Then, the blend was filtered and sieved with a 500 μm stainless-steel mesh sift (200/50, CISA). The filtered liquid thus obtained is hereafter called "juice".

The sample preparation for VI experiments was as follows: the fruit was washed and cut transversally into 5 mm thickness and 64 mm diameter slices (FAGOR CF-150 slicer). The fruit skin was not removed, so as to prevent any absorption of materials through the lateral surface of the slices, which allowed assuming unidirectional matter flow. The

impregnation experiments were carried out on three lulo fruit batches at a very similar ripeness stage, and three samples per batch were characterized.

Figure 1 summarizes in a flow chart the sample preparation and treatments.

**Figure 1.** Flow chart summarizing sample preparation and treatments.


**2. Current obstacles in biota monitoring**

Starting from the early of XX century, scientific communities at worldwide scale started to put questions regarding the possibility of humans' exposure to unwanted chemical contaminants as well on the facts that could resulted after exposure events to such chemical contaminants. Thus in 1973, was initiated through World Health Organization the Environmental Health Criteria Programme which presented at that time these emerging objectives:

Nowadays, in medicine and ecotoxicology fields, it is widely accepted that contaminants effects testing must involve animals because in most cases researches performed on animals

Non-Invasive Matrices Use in Pollution Evaluation

increasingly difficult to be fulfilled in researcher studies.

environmental contaminants.

were chosen on the following principles:

the studied organisms

environment.

study

**2.2 Current trends in living things exposure monitoring**

**3. Choice of sample matrix type collected non-invasively**

at Nanoscale Levels: A Way Forward in Ecotoxicological Studies 171

Moreover, in terms of ecotoxicological studies performed on animal subjects in most situations the performed studies were completed by their sacrifice. Considering these, in the recent years at world wide it has brought up repeatedly in discussions the ethical aspects throughout these studies are conducted (Watson, 2009). But considering the importance of such kind of study (mainly as regards environmental contaminants biomonitoring or their toxicological evaluation on living things) by which obtained information make possible to take measures (pollutants presence regulation through law, contaminated environment remediation) or get treatment measures (in case of diseases) made them until now

Today a large number of countries introduce regulation regarding the way through that are performed these studies, imposing clear conditions as limiting the number of animal subjects subjected for study as well regarding the mode through that they are treated during the experiment. Unfortunately, considering these requirements in most cases the necessity of a large number of data (which obviously involve uses of a large number of subjects) is

Taking into account the requirements imposed by legislation but also by the need of a large number of subjects submitted for study so that to could draw realistic and convincing conclusions induced the need to seek new ways for ecotoxicological evaluation of

In an attempt to meet these ideas has emerged and formed a new path in ecotoxicological assessment of chemical pollutants, namely qualitative and quantitative evaluation of chemical contaminants from biological matrices from living beings using noninvasive sampling ways. This new direction in ecotoxicological biomonitoring of environmental contaminants not only allows the monitoring of animal subjects exposed but make also possible to assess the impact of those chemical contaminants on humans that leave in contaminated areas. Also this way of sampling of biological matrix from living beings allows an extension of studied subjects in terms of both as species, age, gender and living

As in any case of chemical pollutants monitoring a sample must to render an accurate picture about chemical compounds composition present in the environmental media from which the sample was taken at the sampling moment. In order of eliminating the problems of invasive sampling method of biological matrix from living beings, now sample matrixes

primarily to not cause any kind of discomfort, pain or sacrifice to subjects submitted for

 to be stable in time without altering the sample and the chemical structure, physicochemical properties and quantities of the monitored chemical contaminants to be a representative "mirror of the footprint" leaved by the chemical contaminants on

acceptable and necessary even in conditions of sacrifice of studied animal subjects.

helped scientists to discover and developed lifesaving treatments and medications, and to observe which chemical compounds might be dangerous to humans if they get contact with it through ingestion, inhalation and/or dermal contact. But even if we consider all benefits that were obtained in humans health science through animal testing, not everyone consider animal testing as the best method for toxicology. There are two major reasons for that they are not totally accepted, once due to the fact that toxicological study performed on animal subjects induce terrible pain on them, most of the time resulting in the animal subjects death, and secondly due to the fact that not always the approximation between the selected testing animal species physiology and their anatomical profile is similar enough with that of humans (Watson, 2009).

Leaving aside whether if the chosen animal subjects in the study correspond entirely in term of physiological and anatomical structure to humans, when we want to have certainty that a chemical compounds is able to leave some imprint on a living things, study on any kind of animal subject becomes priority. Based on the presence of a chemical compounds on a animal subject, scientist can made analogy about the possibility of this chemical compound presence in human body.

Taking into account all that were presented before as well the controversies that have arisen in the recent years regarding testing on animal subjects, today more and more researchers are trying to develop novel testing methods that are not invasive to the subjects in question.

Development of such of kind methods is beneficial even for human biomonitoring, permitting a larger number of studies on a wider range of subjects as age, gender and geographical living position.

#### **2.1 "Old sampling methods" in monitoring of living things exposure to organic contaminants**

Human biomonitoring used for determination of chemical substances in body fluids was for the first time introduced in the early of 1930's by occupational medicine for health protection of exposed workers (Angerer et al, 2007). The first biological sample matrixes that were used was blood and urine, (blood being the first biological sample collected through invasive mode while urine was the first biological sample matrix that was sampled through non-invasive ways). The benefits of using biological samples in order to get information regarding human exposure made that this mode of humans health evaluation in terms of exposure to chemical contaminants to expand, so today it is used in areas like public health politics, environmental medicine, toxicology and other science that relate to the impact of chemical compounds on human health.

Usually, testing the qualitative as well quantitative presence of any kind of chemical compounds in living organisms, involve the use of sample matrixes as blood, tissues or bones. Although in terms of pollutants biomonitoring from living things it is considered that blood samples offer the most accurate information regarding the quantitative presence of a pollutants or its metabolites (due to the fact that it's get contact with the whole organism), this test specimen is done by venipuncture (piercing artery or vein) which cause pain and discomfort to subjects (Beebee, 2008). As regards bone or tissue sampling methods, these are more painful causing in most cases severe injuries.

helped scientists to discover and developed lifesaving treatments and medications, and to observe which chemical compounds might be dangerous to humans if they get contact with it through ingestion, inhalation and/or dermal contact. But even if we consider all benefits that were obtained in humans health science through animal testing, not everyone consider animal testing as the best method for toxicology. There are two major reasons for that they are not totally accepted, once due to the fact that toxicological study performed on animal subjects induce terrible pain on them, most of the time resulting in the animal subjects death, and secondly due to the fact that not always the approximation between the selected testing animal species physiology and their anatomical profile is similar enough with that of

Leaving aside whether if the chosen animal subjects in the study correspond entirely in term of physiological and anatomical structure to humans, when we want to have certainty that a chemical compounds is able to leave some imprint on a living things, study on any kind of animal subject becomes priority. Based on the presence of a chemical compounds on a animal subject, scientist can made analogy about the possibility of this chemical compound

Taking into account all that were presented before as well the controversies that have arisen in the recent years regarding testing on animal subjects, today more and more researchers are trying to develop novel testing methods that are not invasive to the subjects in question. Development of such of kind methods is beneficial even for human biomonitoring, permitting a larger number of studies on a wider range of subjects as age, gender and

**2.1 "Old sampling methods" in monitoring of living things exposure to organic**

Human biomonitoring used for determination of chemical substances in body fluids was for the first time introduced in the early of 1930's by occupational medicine for health protection of exposed workers (Angerer et al, 2007). The first biological sample matrixes that were used was blood and urine, (blood being the first biological sample collected through invasive mode while urine was the first biological sample matrix that was sampled through non-invasive ways). The benefits of using biological samples in order to get information regarding human exposure made that this mode of humans health evaluation in terms of exposure to chemical contaminants to expand, so today it is used in areas like public health politics, environmental medicine, toxicology and other science that relate to the impact of

Usually, testing the qualitative as well quantitative presence of any kind of chemical compounds in living organisms, involve the use of sample matrixes as blood, tissues or bones. Although in terms of pollutants biomonitoring from living things it is considered that blood samples offer the most accurate information regarding the quantitative presence of a pollutants or its metabolites (due to the fact that it's get contact with the whole organism), this test specimen is done by venipuncture (piercing artery or vein) which cause pain and discomfort to subjects (Beebee, 2008). As regards bone or tissue sampling methods, these are

humans (Watson, 2009).

presence in human body.

geographical living position.

chemical compounds on human health.

more painful causing in most cases severe injuries.

**contaminants**

Moreover, in terms of ecotoxicological studies performed on animal subjects in most situations the performed studies were completed by their sacrifice. Considering these, in the recent years at world wide it has brought up repeatedly in discussions the ethical aspects throughout these studies are conducted (Watson, 2009). But considering the importance of such kind of study (mainly as regards environmental contaminants biomonitoring or their toxicological evaluation on living things) by which obtained information make possible to take measures (pollutants presence regulation through law, contaminated environment remediation) or get treatment measures (in case of diseases) made them until now acceptable and necessary even in conditions of sacrifice of studied animal subjects.

Today a large number of countries introduce regulation regarding the way through that are performed these studies, imposing clear conditions as limiting the number of animal subjects subjected for study as well regarding the mode through that they are treated during the experiment. Unfortunately, considering these requirements in most cases the necessity of a large number of data (which obviously involve uses of a large number of subjects) is increasingly difficult to be fulfilled in researcher studies.