Copper is an essential micronutrient that plays vital roles in various physiological processes such as neurotransmission, cellular metabolism of iron ions and Fe (II) and Fe (III), and as a cofactor of enzymes involved in cellular respiration, among other important metabolic functions. However, Cu (II) can be exerted toxic effects on humans when ingested in large quantities (Pedrozo and Lima, 2001). Aedes aegypti is an important species in toxicity studies, because of its short life cycle and resistance to various conventional insecticides. It can be used as a model to assess the impacts of toxic metals on aquatic environments. Aedes aegypti control strategies involve the use of both synthetic and biological insecticides such as Bacillus thuringiensis var. israelensis (OPAS, 1995) and pyrethroids, organophosphates, and carbamates, respectively, as well as sanitation and the elimination of insect breeding (Lefevre et al., 2003). However, synthetic insecticides used intensively both in agriculture and public health, can lead to the development of physiological resistance in target insects when applied at incorrect doses, with short intervals between applications, or using non-calibrated equipment. All of these errors can be detrimental to the success of important programs, such as those aiming to control mosquito populations (Brogdon and McAllister, 1998). Other factors, such as the biochemical mechanisms of resistance and the insects’ ability to adapt to insecticides, require further study via periodic surveys of insect populations. Various plant compounds have been evaluated as potential insecticides for use against mosquitoes. These compounds may induce alterations in and/or the death of the insect’s cells. Extracts of Camellia reticulata, Magonia pubescens, and Sapindus saponaria cause damage, either partial or complete, to the intestinal cells of mosquitoes via vacuolation, which causes an increase in the number intercellular spaces and triggers changes in the microvilli, nuclei, and nucleoli of the cells (Abed et al., 2007; Arruda et al., 2003). Larval intestinal cells exposed to Acacia coriacea extracts exhibit an increase in the production of cytoplasmic columnar cells. In this type of cell, cytoplasmic vacuolation results in osmotic instability. The Aedes aegypti larvae subjected to treatment with an extract of Derris urucu experienced changes in their peritrophic membrane structures, and the consequent disintegration of cells and separation of basement membranes (Gusmão et al., 2002). These observed effects suggest that different plant-derived insecticides act on different mosquito cell target sites. These physiological responses are commonly observe in the cells in the intestinal lumens of insects (Chapman, 1998; Lehane, 1996), occurring apocrine secretion of digestive enzymes (Caetano et al., 1994; Serrão and Cruz-Landin, 1995; Cristofoletti et al., 2001), renewal of the cytoplasmic contents (Cruz-Landin et al., 1996) or apoptosis (Rost-Roszkowska, 2008; Azevedo et al., 2009). Research conducted using Cu (II) has shown that the exposure of Aedes aegypti to the ions of transition metals leads to the disorganization of the peritrophic matrix (PM). The use of this metal has been proposed as a means of controlling insect vectors, as PM and epithelia constitute the first line of defense against larval and adult mosquitoes, often being used to prevent the spread of pathogens. In addition, transition metals are responsible for the selective transport of molecules. It is well established that copper is bactericidal, and thus it is possible that the metal could control microorganisms involved in the breeding of Aedes aegypti, impacting the larval food chain and thus making conditions unfavorable for insect reproduction (Rayms-Keller et al., 1998). Recent studies have shown that the reactivity of metal ions can be reduced and their toxicity increased via the formation of metal complexes between, for example, the combination of Cu (II) and EDTA, forming Na₂[Cu(EDTA)], which, produces [Cu(EDTA)]^2- complex ions when dissociated in an aqueous solution. This compound is toxic to Aedes aegypti (Arruda et al., 2011). Other studies have established the genotoxic effects of Cu (II) on fish and mice; these effects can be modulated by encapsulating the active compounds in natural polymers (Jacobowski et al., 2013). The determination of the complex’s target site in the digestive system of Aedes aegypti is essential for studies investigating the complex’s insecticidal effects for use in the development of a new insecticide (Arruda et al., 2011; Barreto et al., 2006; Costa et al., 2012; Valoto et al., 2014). Therefore, morphological studies are an important means of obtaining an understanding of the insecticidal activities and mechanisms of action of aminopolycarboxylic (APCAs) and complex ions, APCs. This study evaluated the mechanism of action of the metal complex [Cu(EDTA)]^2-, as well as the morphological changes that occur in the epithelial cells of the average larval Aedes aegypti gut after exposure.

 

A strain of Aedes aegypti from Campo Grande, MS, Brazil, was established in the lab. The F1 and F2 generations were used for the realization of bioassay toxicity carried out according to WHO (1981) protocols. The treatment concentrations of [Cu(EDTA)]^2- used were 500 mg∙L^-1, 250 mg∙L^-1, 125 mg∙L^-1, 62.5 mg∙L^-1, and 31.25 mg∙L^-1, while the treatment concentrations of Cu (II) ions used were 46.95 mg∙L^-1, 24.48 mg∙L^-1, 11.74 mg∙L^-1, 5.87 mg∙L^-1, and 2.93 mg∙L^-1. Four replicates of 25 3^rd instar larvae each were subjected to each treatment. Bioassays were carried out at temperature, or 27 ± 2˚C. After 24 h of exposure, larval mortality was determined by counting the larvae that did not move or otherwise respond to the touch of a Pasteur pipette. Bioassays were performed in triplicate on different dates.

 

The larvae killed in the above bioassay were fixed in 4% formalin solution for 2 h at room temperature. They were then dehydrated in a graded alcohol series, cleared in xylene, embedded in paraffin, cut into 4-mm sections, and stained with hematoxylin and eosin (HE) (Pilat, 1935). Histological sections of the stomodeum and mesenteron of the digestive system were analyzed. The images were analyzed using a Samsung camera coupled to a Carl Zeiss microscope and ImageLab™ software.

 

The results of the mortality experiment showed that at the concentrations 31.25 mg∙L^-1 and 62.5 mg∙L^-1. There was no mortality after 24 h. However, mortality percentages of 30% to 60% were obtained with concentrations of 125 mg∙L^-1 and 250 mg∙L^-1, and from 90% to 100% at 500 mg∙L^-1. Behavioral changes were also observed, with the control group larvae making regular serpentine movements, while the larvae treated with [Cu(EDTA)]^2- exhibited little or no movement when touched with a Pasteur pipette. Morphological changes in the digestive system (the stomodeum and mesenteron) in longitudinally sectioned larvae were exposed at [Cu(EDTA)]^2-concentrations of 31.25 mg∙L^-1 to 500 mg∙L^-1. At 31.25 mg∙L^-1 and 62.5 mg∙L^-1, there was a detectable increase in weakness between the junctions of the cylindrical stomodeum cell coating, with an increase in intercellular space (Fig. 1a). Histopathological analysis revealed a reduction in the size of the digestive systems of larvae treated with [Cu(EDTA)]^2-. The columnar epithelium cytoplasmic understands the basis of stomodeum with small spherical nucleus visible vacuoles in some cells, brush-shaped edge in heart valve apical and composed of a circular crease stomodeum located at the end projecting into the lumen of the mesenteron (Fig. 1a). The mesenteron has three regions called previous, median and posterior to another structure that is located in the side portion of the call gastric cecum, which carries out part of the food absorption process. This structure is devoid of PM. Figure 1b shows the partially cut mesenteron and the presence of wrinkles. The gastric caeca, structures between the mesenteron and stomodeum, contained compound lining cells flatter than those of the control group. Figure 1c shows the increase in the amount of brush border epithelium. Figure 1d and 1e show the previous mentioned regions and the median of the mesenteron. Note that the coating of living cells degenerates, into agglomerates of flat epithelial cells, cytoplasm containing small vacuoles, large nuclei that occupy most of the cellular cytoplasm, and condensed chromatin. The PM is thicker than that of control larvae with food still collecting within the lumen. Figure 1f shows the rear region, where the cylindrical lining cells are more elongated, some being more pyramidal in shape than those of the control, and in which some cells are without the brush border. The cytoplasm contains small vacuoles, cores of various shapes, and disorganized chromatin. The PM creases in this region contain the remnants of food.

 

Figure 1 Micrograph of the digestive system of 3rd instar larvae of Aedes aegypti of the group treated with concentrations of 31.25 mg·L-1 and 250 mg·L-1 of [Cu(EDTA)]2- complex ions stained with HE, longitudinal section. 1a. Stomodeum, magnified 400X; 1b. General view of the mesenteron, magnified 100X; 1c. Gastric caecum; 1d. Anterior mesenteron; 1e. Median mesenteron; 1f. Posterior mesenteron. (400X magnification). Nucleus (N), Lumen (L), Peritrophic matrix (PM), Epithelial cells (EC), Cardiac valve (CV), Gastric caecum (GC), and Brush border (see arrow)

 

Figure 2 shows Aedes aegypti larvae treated with 125 or250 mg∙L^-1[Cu(EDTA)]^2-. The analysis of the digestive system revealed deleterious changes in the cells of the stomodeum and mesenteron, including different histopathological changes such as the total or partial destruction of the cells (Fig. 2a and 2d), intense cytoplasmic vacuolization (Fig. 2c), and increased ectoperitrophic space containing accumulated acidophilic material (Fig. 2e). The endoperitrophic space was filled with exfoliating cells. The lining in the median region of the mesenteron is made up of acidophilic pavement cells with small vacuoles and the remains of secretions in the tube (Fig. 2e). The posterior mesenteron contained rounded cells that were acidophilic, vacuolated, and rich in brush borders. The nuclei of the cells were in the process of degeneration (Fig. 2f).

Figure 3 analyzes the digestive systems Aedes aegypti larvae treated with 500 mg∙L^-1 [Cu(EDTA)]^2-. Note the reduction in the size of both the lumen and larvae (Figure 3a). The lining of the stomodeum undergoes intense degeneration (Figure 3b). As shown in the image, the gastric caecum is atrophied and the cells vacuolated. The linings of the anterior and median regions of the mesenteron are composed of flat, thin string-like cells (Figure 3d and 3e) with cellular remains located in the endoperitrophic region (Figures 3e and 3f). Some of the cells in the posterior mesenteron were globular, while others were “tear drop” shaped, with a tapered apical surface indicating either a process of the elimination or secretion of the contents. The lining of the digestive tract was thin, with flat, string-like cells, and the lumen was without signs of secretions, food, or cellular remains (Figure 3f).

Rayms-Keller et al. (1998) studied the toxic effects of Cu(II) ions in aqueous solution on the 3^rd instar larvae of Aedes aegypti. Cu(II) ions form aqueous [Cu(H₂O)₆]²⁺ complex ions that prevent the eggs from hatching and induce cell damage and mortality in the larvae of Aedes aegypti. They determined that Cu (II) ions are more reactive when in the form of aqueous complexes. However, the experimental results indicate that although Cu(II) was not in an aqueous complex form (reactive), but in a negative ion complex with EDTA, [Cu(EDTA)]^2-, its larvicidal activity was intense. Low concentrations of 31.25 mg∙L^-1 and 62.5 mg∙L^-1 of [Cu(EDTA)]^2- (corresponding to 2.93 and 5.87 mg∙L^-1 of Cu (II), respectively) did not induce mortality in the larvae of Aedes aegypti over a period of 24 h. However, the histopathological analyses revealed the presence of cellular lesions, demonstrating that when the metal ion was complexed with EDTA, it can induce cell damage and lead to the death of larvae, even at low concentrations. This is more likely to occur when the time of exposure to the compound is increased, as this causes the continued to infliction of cellular damage via a mechanism based on the active metabolism of the larvae (observations not shown here). At concentrations of 250 mg∙L^-1 and500 mg∙L^-1, mortality of the 3^rd instar larvae reaches 100%. The experimental results of the toxicity and the histopathological analyses presented here confirm the expected larvicidal activity of Cu (II) (in the form of EDTA complexes), and indicate that the digestive system is the main target of metal ions and their complexes. The experimental results suggest that [Cu(EDTA)]^2- complex ions exhibited greater cellular activity and permeability than other forms of Cu, possibly because of its ability to internalize metal ions in the cells, then release and reduce them. This increases the toxicity of the complexed metal ion by allowing for the alteration of homeostasis, increasing competition with other essential metal ions, and facilitating the complex’s permeation and internalization by the biological membrane. In addition, this process leads to the reduction of Cu (II) to Cu (I), and the production of free radicals and reactive oxygen species (ROS) via oxidative stress (Raes et al., 2000; Benite et al., 2007; Kim et al., 2008; Bertini and Cavallaro, 2008). During the toxicity bioassays, the larvae displayed intense changes in behavior, particularly as regards to their capacity for movement in through the aqueous medium and their delayed larval development. These results confirm the larvicidal activity of the active complex, and are consistent with the results of previous toxicity studies (Abed et al., 2007; Arruda et al., 2003; Rayms-Keller et al., 1998; Barreto et al., 2006). The cells of the mesenteron participate in the secretion of various substances and absorption of nutrients, as demonstrated by Levy et al., (2004). A morphological study of the midgut of Anticarsia gemmatalis (Lepidoptera) larvae revealed that the absorption of Cu (II) ions caused deleterious changes in the cells of the mesenteron that were irreversible when the concentration of ions was increased. This finding is consistent with the results reported by Raes et al. (2000), who analyzed Aedes aegypti cells treated in vitro with Cd (II), Hg (II), and Cu (II) metal ions and observed that the occurrence of cell apoptosis was dependent on the concentration of Cu (II) ions applied. At all the concentrations, the greatest cell damage occurred in the posterior mesenteron and gastric caecum. The pH in the region lies within the 7.0 to 8.0 range. The results suggest that at this pH, Cu (II) ions are either made available by the complex or internalized in the form of a complex and released inside the cell (Costa et al., 2014). The damage found in the anterior mesenteron, where the pH is 11, may be explained by the stability of the complex when it is formed among EDTA and Cu (II)ions. In this case, it is suggested that the [Cu(EDTA)]^2- complex is internalized, and that the metal undergoes a reduction from Cu (II) to Cu (I), producing free radicals and ROS (oxidative stress reaction) (Raes et al., 2000). The histopathological analyses of the digestive system of the treated larval group were consistent with the results of other previous studies (Abed et al., 2007; Barreto et al., 2006; Arruda et al., 2008). However, no previous studies have reported on the effects of such treatments on the globular cells in the digestive tube (visible along the lumen of the treated larvae), which this study found to exhibit evidence of ruptured cellular membranes when treated with 11.74 mg∙L^-1, 24.48 mg∙L^-1, or 46.95 mg∙L^-1 Cu (II)ions. In a study of Aedes aegypti larvae treated with an ethanol extract of Sapindus saponaria (Sapindaceae), Barreto et al. (2006) found that the median mesenteron was the most important region for toxicological studies of Aedes aegypti larvae. However, our study revealed that intense cell and tissue changes also occur in both the stomodeum and the anterior and posterior regions of the mesenteron. Barreto et al. (2006) also reported the formation of a constriction in the digestive tubes of larvae treated with active insecticidal compounds derived from vegetable extracts. Similar observations were made during studies of the toxicity of [Cu(EDTA)]^2- complex ions. It was hypothesized that the constriction is related to the larvae’s defense mechanism against toxic compounds, which involves the creation of compartments inside the digestive system in order to prevent the toxin from being absorbed. The destruction of the cells in the basal region observed in the digestive system of Aedes aegypti larvae subjected to [Cu(EDTA)]^2- ion concentrations exceeding 125 mg∙L^-1 is characterized by an irreversible, intense and rapid. The posterior region of the mesenteron contained cylindrical cells with plasma membranes containing evaginations and exhibiting intense secretory activity, forming droplets in the apical portion of the cell that are the released into the lumen. These cells possessed brush borders. All these observations can likely be attributed to the absorption carried out in this region of the digestive system (Abed et al., 2007; Barreto et al., 2006; Arruda et al., 2008). Abed et al. (2007) described the effects of Copaifera reticulata resin oil on Aedes aegypti larvae, highlighting the formation of stratified epithelium to replace dead cells in response to the toxic effects of an active vegetable extract insecticide, and also as a defense strategy. The modification and stratification of the epithelium seems to occur when larvae are treated with insecticides of vegetable origin; however, these effects were not observed in larvae treated with [Cu(EDTA)]^2- complex ions. Costa et al. 2012 and Costa et al., 2014 describe morphological changes that occur in the midgut of 3^rd instar Aedes aegypti larvae following treatment with either a methanolic extract of Annona coriacea (Magnoliales: Annonaceae) or acetogenins with larvicidal and cytotoxic effects, such as squamocin, from Annona squamosa. In these studies, the midguts were shown to be subdivided into anterior and posterior regions, and analyzed via light and scanning electron microscopy. Insects exposed to the extract or acetogenins experienced intense, destructive cytoplasmic vacuolization of the columnar and regenerative midgut cells. The apical surfaces of the columnar cells exhibited cytoplasmic protrusions oriented toward the lumen, suggesting that these cells could be involved in apocrine secretory processes, cytotoxicity and/or apoptosis. The amides piplartine and piperlonguminine, isolated from piper species, were toxic to 3^rd and 4^th instar Aedes aegypti larvae at concentrations ranging from 1 to 300 µg∙mL^-1 (ppm). Piplartine reduced the mosquito development period, and caused larval mortality only at concentrations >100 µg∙mL^-1; meanwhile piperlonguminine slowed the larval development of mosquitoes (10 µg∙mL^-1) and caused 100% larval mortality (30 µg∙mL^-1) within 24 h. The toxicity and cytotoxic effects of piperlonguminine on the epithelial cells of the Aedes aegypti digestive system were observed using transmission electron microscopy, which revealed the vacuolization of the cytoplasm, mitochondrial swelling, and leaking of nuclear material. Piperlonguminine was the more effective amide, exhibiting toxic activity with LD₅₀ of 12 µg∙mL^-1 against the larvae of Aedes aegypti (Maleck et al., 2014). The mortality observed in this study is caused by the rupture of the peritrophic matrix (PM) in the region of the mesenteron, and by cell damage caused by [Cu(EDTA)]^2- complex ions through the production of free radicals and ROS. Similar results were reported by Gusmão et al (2002), who treated larvae of Aedes aegypti with Cu (II) ions and observed an increase in membranous sacs containing food, suggesting that the toxicity of Cu (II) was based on the formation of a large quantity of PM produced to protect the lining of the mesenteron. Abedi and Brown (1961) reported that strains of DDT (dichlorodiphenyltrichloroethane)-resistant Aedes aegypti produced up to nine times more PM components than susceptible individuals. Bradshaw (1992) suggests that the reinforcement of PM induced by the absorption of DDT is a mechanism of primary resistance. Kato et al. (2008) showed that in Aedes aegypti, PM is as a physical barrier for the defense of the midgut epithelium during invasion by mosquito-borne pathogens. Therefore, the elimination of the toxic material via the PM seems be related to a physiological response to insecticidal compounds (Gusmão et al., 2002). It is believed that the disorganization of the PM facilitates the internalization of the [Cu(EDTA)]^2- complex ion by the cell membrane, thereby increasing the efficacy of its insecticidal activity and enabling continued damage to the cells. Another important observation made by this study is the presence of small vacuoles in the cellular cytoplasm of the control larvae, which had previously only been observed in the cells of Aedes aegypti larvae treated with active compounds derived from vegetable extracts. However, as a result of the treatment with [Cu(EDTA)]^2- complex ions, these vacuoles increased in quantity and size in a manner proportional to the increasing concentration of [Cu(EDTA)]^2- ions. These results are similar to those reported by Arruda et al. (2003), Barreto et al. (2006), and Abed et al. (2007) in their studies of the use of active insecticidal compounds for the control of Aedes aegypti larvae. Therefore, the formation of cytoplasmic vacuoles suggests that the cells of the mesenteron undergo cellular lysis under these conditions (Arruda et al., 2008). Copper toxicity (excess) disrupts normal function, and so does copper deficiency. Having the proper balance of essential nutrient minerals is what is most important for healthy function. Copper is an essential trace element that is required in enzyme systems. And enzymes are responsible for countless metabolic processes required to sustain life. For example, enzymes are indispensable in cellular activity for signal transduction, and cell regulation. Copper is an essential trace element that is vital to the health of all living things (humans, plants, animals, and microorganisms). In humans, copper is essential to the proper functioning of organs and metabolic processes. The human body has complex homeostatic mechanisms which attempt to ensure a constant supply of available copper, while eliminating excess copper whenever this occurs. However, like all essential elements and nutrients, too much or too little nutritional ingestion of copper can result in a corresponding condition of copper excess or deficiency in the body, each of which has its own unique set of adverse health effects (Scheinberg and Sternlieb, 1976; Aspin and Sass-Kortsak, 1981; Watts, 1989; Casper and Malter, 2015). Studies for population control Aedes albopictus have demonstrated the effectiveness of copper against these mosquitoes. Romi et al. (2000) has demonstrated that copper concentrations > 1,000 ppb inhibited larval development completely killing all the larvae and affected adult weight in laboratory. In field trials, 20 g/liter reduced the number of larvae in treated pots by 90%, and 40 g/liter completely prevented oviposition. Moreover, the persistence of the toxic action of metallic copper in the field lasted for several months. Studies suggest the use of metallic copper as a practical alternative method for preventing development of Aedes albopictus in small containers such as flower saucers found in urban areas (Bellini et al., 1998). The cost of illness associated with dengue fever in Indonesia is about 1.4 billion dollars, whereas the cost for preventing eclosion of Aedes aegypti by using copper fiber was low at about 45 million dollars. Studies demonstrated that measures to eradicate Aedes aegypti by using copper ions to be a useful and economical approach (Hisaya et al., 2015). The use of copper as biactive for population control aquatic insects comes up on the issue of biosafety. Study Schat and Bookum (1992) demonstrated that the copper tolerance for chicken and pig, in confined conditions, is 20 and 50 g/t. These results demonstrate that the concentrations of the compounds used in this study are far below those that could cause toxicity to vertebrates. Another factor favoring the use of copper is the deficiency of the metal in many farmland soils. Study of DAF (2015a) showed that most soils in Western Australia were copper deficient in their natural state. Copper is essential for pollen formation and has a role in formation of chlorophyll and lignification (cell wall strength). Deficiency causes sterile pollen, which, in turn causes poor grain formation and high yield losses. Besides, copper is essential for pollen formation, and has a role in formation of chlorophyll and cell walls. Deficiency can cause grain abortion and high yield losses. With the exception of loam or clay salmon gum or York-gum soils, most soils required copper and zinc when cleared for agriculture. Canola has a lower copper requirement than wheat or barley. Copper is readily available in the soil for many years, but is relatively immobile and can become unavailable to crops in dry soil particularly in no-till systems copper deficiency can be induced by heavy liming; increased plant nitrogen status; use of zinc fertilizer; and where root growth is restricted (DAF, 2015b). Copper toxicity is known for the production of natural cupricide by algae. Cupricide is a chelated copper product that kills algae with fewer toxic effects than copper sulphate and does not precipitate out when there is a high concentration of carbonate. Water treated with cupricide can be used on plants. Despite its reduced toxic effects, it should not be used in hard water containing trout, native fish or crustaceans, or in water used for livestock where the livestock are grazing plants that may cause liver damage (for example caltrop, lupins, heliotrope or ragwort). Copper sulphate is no longer recommended for treatment of water in dams as it can kill crustaceans, fish and aquatic life. Blocks may be used to treat water in troughs but these can give the water a copper taste that livestock will refuse to drink. It may also cause toxicity in livestock especially if they have pre-existing liver damage (DAF, 2015c).

 

Cellular changes caused by [Cu(EDTA)]^2- ions were observed in the digestive systems of Aedes aegypti larvae. The histopathological analyses showed that the mesenteron was the region most strongly affected by the metal ion, notwithstanding the impairment of the digestive system. The application of the Cu(II)-EDTA complex caused the death of the larvae by destroying the midgut cells through their cytoplasmic vacuolization, cellular and nuclear hypertrophy, degeneration the lip brush, apical vesicle formation with the release of the cells’ cytoplasmic contents, epithelial stratification, and the formation of folds in the peritrophic matrix. The treatment also affected the brush border, augmented cytoplasmic vacuoles in the cell of the gastric caeca and mesenteron, and ruptured cellular junctions originating from the stomodeum. The observed toxicity was concentration-dependent. At [Cu(EDTA)]^2- concentrations higher than 125 mg∙L^-1, extensive damage to the digestive system and a large mortality rate were observed. The lumen contained cellular remains and the destruction of cell tissue. These toxic effects appear to occur through contact with and internalization of the [Cu(EDTA)]^2- complex ion into the cells, leading to the alteration of homeostasis, induction of the oxidative stress reaction caused by a pH-dependent mechanism, and/or the lability of the metal complex within the inter- and intracellular environment. These results are as important resource for the development of new metallo-insecticide compounds based on for the prolonged and/or comprehensive control of Aedes aegypti in its immature forms (eggs, larvae, and pupae), and as a means of restrict the larval food chain in the insect’s breeding grounds while simultaneously ensuring human safety and having a low environmental impact.

 

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