Archives Of Environmental Contamination And Toxicology Template

This Authorea document template can be used to prepare documents according to a desired citation style and authoring guidelines. Abstracts are not always required, but most academic papers have one and writers should know how to produce a useful abstract. An abstract should be a very short, clear and concise summation of the entire paper. An abstract should provide enough of a preview that a typical reader will know whether or not they wish to read the paper. It should reveal both the purpose and conclusions of the paper.

The effect of productive activities in the integrity of ecosystems, particularly intensive agricultural activity, is especially interesting due to its consequences on the environment, such as the deterioration of the soil quality and native flora. Besides, the application of pesticides can have negative effects in the ecosystem, even on the human health, so that more research is necessary in order to understand agrochemicals effects Casabé 2007.
In the last decades in Argentina, the use of pesticides has increased due to the expansion of genetically modified soybean, which achieved between 2010 and 2012 an average local production of 33.8 millions tons (Ciani 2012). These kinds of transgenic crops are associated with different pesticide formulations, the most used worldwide are glyphosate-based formulations (Pazos 2008), followed by the insecticides Endosulfan and Cypermethrin (CASAFE, 2013).
Glyphosate [N-phosponomethyl glycine (GLY)] is a nonselective herbicide that inhibits plant growth, and is the active ingredient of Roundup® (RU) and Panzer Gold (PANZ), the most widely used formulated herbicides Aparicio 2013 Vera-Candioti 2013. This compound is associated to drift phenomenon, due to its high water solubility and its adherence to soil particles, it can be found both in superficial water and in soil samples affecting wild species (Aizen, Garibaldi, and Dondo 2009; Aparicio 2013 Peruzzo 2008. As regards Endosulfan (END), it is an organochlorine insecticide and acaricide used for pest control, highly toxic to non-target organisms (Pazos 2008). Since 2010, its production in Argentina is banned, but its commercialization and the use of the remainder is allowed, so that it continues being applied in many places, without any control (Mendoza, 2010). Finally, Cypermethrin (CYP), pyrethorid, is a synthetic insecticide, which act by contact and ingestion (Pazos, 2008).
Pesticides tend to be very reactive substances known to cause oxidative damage to biomolecules, such as proteins, lipids and nucleic acids, due to the production of Reactive Oxygen Species (ROS) Monteiro 2006 Halliwell 2007.
Organisms produce ROS and other free radicals (FR) constantly. ROS are derived from subproducts in the mitochondrial respiratory chain; likewise, organisms generate ROS from exogenous sources such as smoke, radiation, UV light and contamination (Södergren, 2000). These reactive species can react with DNA, proteins and lipids, causing oxidative damage, which would lead to the alteration and destruction of membranes, enzymes and other proteins Slater 1984; Halliwell & Chirico, 1993; Halliwell 2012.
The negative imbalance between ROS generation and the capacity of the biological systems to eliminate the reactive intermediaries or repair the damage, is called Oxidative Stress (OS).
In order to keep ROS in healthy levels to the cells, organisms have antioxidants defenses (AOD) which work together: antioxidant molecules as Glutation (GSH), C and E vitamins, carotenoids, among others; and antioxidant enzymes: catalase (CAT), superoxide dismutase (SOD), GSH reductase (GR), GSH peroxidase (GPX) and glutathione-S-tranferase (GST) Limón-Pacheco 2009 Valko 2007. Antioxidants form stable complexes in order to avoid the action of reactive species in the cellular membrane and other cellular compounds Halliwell 1990.
Pesticides can act as pro-oxidant in a variety of tissues; they produce ROS accumulation, DNA damage, alteration of the antioxidants defenses and lipid peroxidation, causing a great perturbation at intra and intercellular homeostasis Franco 2009 Mena 2009. These xenobiotic interact with the plasmatic membrane and generate lipid peroxidation, resulting in phospholipid’s degradation, membrane damage and alteration of its functionality.
Meanwhile, cellular protein damage is evidenced as an alteration of antioxidant enzymes, such as CAT and SOD, which activity can increase or decrease depending on the intensity and duration of the exposure and susceptibility of the exposed species. A decreased on CAT and SOD’s activities leads to OS and stimulates lipid peroxidation Oru 2007. Both enzymes are part of the antioxidant system that act in the presence of high concentrations of hydrogen peroxide (Gutiérrez, 2002).
Different studies evaluated OS induced by pesticides in vertebrates Costa 2007 Cattaneo 2011 Rossi 2011; Davico et al, 2012; Crupkin 2013, but there is no information in reptiles.
In a recent study,  Poletta 2016 characterized a new set of OS biomarkers in Caiman latirostris blood so that OD induced by exogenous agents can be evaluated without any damage to the animals. Previous works demonstrated genotoxic and immunotoxic effects of pesticides and pesticide mixtures on C. latirostris Poletta 2009 Poletta 2011 Latorre 2012, under review; González 2013, under review). Taking into account this, together with the lack of information about pesticide oxidative stress in reptiles, the aim of the present study was to evaluate damage to lipids through thiobarbituric acid reactive substances (TBARS) and antioxidant defenses through CAT and SOD activities on C. latirostris neonates exposed to Glyphosate, Endosulfan and Cypermethrin formulations during embryonic stage.

Materials and methods
Pesticides formulations tested were obtained by courtesy of Establecimiento La Matuza S.A., Santa Fe, Argentina and included: (1) Roundup® Full II (RU, 66.2% GLY), a liquid water soluble (12000 mg/l) herbicide, containing GLY potassium salt [N-(phosphonomethyl) glycine monopotassium salt, C3H7KNO5P] as its active ingredient (a.i.) (CAS No. 70901-12-1); (2) PanzerGold® (PANZ; 60.2% GLY), isopropylamine salt of glyphosate-based [N-(phosphonomethyl) glycine; CAS 1071-83-6] commercial formulation; (3) CYP Atanor® (25% CYP), a liquid water-insoluble (0.01 mg/l) mixture of different CYP isomers (C22H19Cl2NO3, CAS No. 52315-07-8); and (4) END Galgofan® (35% END) a liquid practically water- insoluble (0.32 mg/l) formulation, containing END as a.i. (C8H6Cl6O3S, CAS No. 115-29-7) (EXTOXNET, updated 2015). Ethanol was used as a vehicle control for END and CYP formulations.
Hydrogen peroxide (H2O2), trichloroacetic acid (TCA), 2-thiobarbituric acid (TBA), butylatedhydroxytoluene (BHT), and SOD Kit (19160-1KT) were from Sigma-Aldrich (St. Louis, MO, USA). Potassium dihydrogenphosphate (KH2PO4) and potassium hydrogen diphosphate (K2HPO4) were form Cicarelli (Argentina).
Caiman latirostris eggs collection
This study was evaluated and approved by the ‘Institutional Committee of Animal Use and Care of Universidad Nacional del Litoral (Santa Fe, Argentina)’ (N° 05-11). C. latirostris eggs from different nests harvested in the Natural Managed Reserve “El Fisco” (30° 11’ 26’’ S; 61° 0’27’’W; Dpto. San Cristobal, Santa Fe Province, Argentina, as part of “Proyecto Yacare” (PY) ranching program activities (Larriera et al., 2008), were used. This is a Protected Natural Area, free of farming and urban activities that belong to the natural distribution of the species, and was chosen to ensure that eggs had not been environmentally exposed to any xenobiotic, as no contaminating activity is carried out there. A total of six nests, collected during 2013-2014 nesting season (December 2013) with a minimum of 32 eggs each, were used to carry out the experiment. All nests were collected within 5 days after oviposition, under the same conditions from harvest to treatment assignment, and egg viability was determined by analyzing the opaque eggshell banding (Donayo et al., 2002). The average weight of eggs used in experiments was 67.8 ± 4.75 g.
Experimental design and treatments
One hundred and ninety-two eggs from six nests (32 eggs per nests), were randomly distributed into 16 experimental groups of 12 eggs each (with two replicates of six eggs each): 1) a negative control (NC) group, treated with distilled water; 2-4) three groups exposed to 500, 750, 1000 µg/egg of RU; 5-7) three groups exposed to 500, 750, 1000 µg/egg of PANZ Poletta 2009; 8-11) 4 groups exposed to 1, 10, 100 y 1000 µg/ egg END formulation Sinha 1997;