Does Fertigation With Fish Farming Effluent Alter the Morphophysiology and Biochemistry of Lippia Gracilis (Verbenaceae)?


 In order to evaluate the response mechanisms of L. gracilis fertigated with saline effluent from fish farming, L. gracilis plants were nourished with fish farming effluent with electrical conductivity of 0.45, 2.68, 4.60, 5.55 and 7.02 dS m− 1 for 60 days. The experiment was carried out in the open field using a completely randomized design with five treatments and four replicates. The salinity levels of the nutrient solution containing fish farming effluent did not affect the leaf and stem biomass production, relative water content and leaf area of the studied species. The activity of antioxidant enzymes varied when the nutrient solution salinity level was increased, which also stimulated the breakdown of starch reserves, but did not interfere with the biochemical parameters proline, photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoids), levels of membrane damage and malondialdehyde, indicating that the plant showed no stress symptoms when fertigated with high-salinity effluent. The anatomy of L. gracilis leaf cross-sections shows a unistratified epidermis with glandular and non-glandular trichomes in the adaxial and abaxial sides. Plants that received only fish farming effluent (7.02 dS m− 1) showed a 25% reduction in the number of xylem bundles in the midrib region, compared to the control. The yield, chemical composition and antimicrobial activity of the essential oil extracted from L. gracilis leaves did not differ between treatments. Thus, the saline effluent from fish farming can be used in the fertigation of L. gracilis without compromising plant yield, avoiding environmental contamination with disposal in soil and/or water bodies.


Introduction
Due to the low availability of surface water in arid and semi-arid areas, it is necessary to use technologies of coexistence based on sustainable water management, especially in agriculture. Among these technologies, the reuse of water is a very common practice, and the use of aquaculture e uent stands out because it can be used not only as a source of water, but also in the practice of fertigation, due its contents of nutrients.
Aquaculture activity has grown 87.1% in the last decade and sh has become the animal protein of greatest human consumption, about 16.7% higher compared to other meat (beef, pork, sheep and goat) (FAO 2014). In Brazil, according to the Brazilian Association of Fish Farming (Associação Brasileira de Psicicultura), sh production reached 722,500 tons in 2018, with tilapia (Oreochromis niloticus) being the sh most frequently produced in breeding tanks.
It is worth pointing out that this activity consumes a lot of water, demanding about 143.7 m³/ha every day due to the in ltration and evaporation of the breeding nurseries. For the production of one ton of sh, a water demand from 50 to 740,000 m³ is estimated (Oliveira and Santos 2011). In the sh harvesting process, the amount of e uent is equally high and its improper disposal in water bodies can cause eutrophication and reduce and change biodiversity (Henry-Silva and Camargo 2009).
In semi-arid environments, water is a natural resource of extreme importance, mainly due to its role in agricultural development, which highlights the need for developing strategies that enable the use of e uents rich in dissolved organic and inorganic compounds that can be used for the bene t of plant nutrition, ensuring e cient and low-cost irrigation. However, this type of water from sh farming is usually saline and, depending on the management, can hamper crop growth and production. The selection of salinity-tolerant species is the main practice used to ensure pro table crops when the goal is to take advantage of saline e uents as a water and nutritional source for agricultural crops and, when there is availability, the e uent can also be diluted to reduce electrical conductivity and attenuate salt stress (Dantas et al. 2019).
Saline e uents can be used to stimulate some plant species to produce bioactive compounds, according to some reports in the literature, which describe that plants irrigated or nourished with saline e uent are stimulated to produce secondary metabolites as a mechanism of defense and adaptation to the saline medium. Species belonging to the genus Lippia stand out in the production of bioactive molecules, since most of them produce a large amount of essential oils with proven antimicrobial properties (Albuquerque et al. 2006;Fernandes et al. 2015), hence being of pharmacological interest. Published studies with L. gracilis mainly cover the effects of the oil and its properties, but the records about the in uence of abiotic stresses on oil composition and yield are still scarce. In addition, the studies are limited to controlled environments, which do not re ect the actual behavior of the plant grown in the open eld.
As the species focused on in the study produces an essential oil with antimicrobial property and has shown tolerance to salinity in studies conducted in greenhouse (Ragagnin et al. 2013; Oliveira et al. 2019), it is important to enable its planting using saline e uent and/or in areas already salinized. The use of this native species of the Caatinga that is tolerant to salinity and maintains its chemical compounds potentially active opens perspectives to offer a product that can be marketed in the chemical industries, especially in pharmaceutical industries. In addition, it contributes to local economic development, especially in soils that are naturally saline or salinized of the Northeastern semi-arid region. Thus, the objective was to evaluate the response mechanisms of L. gracilis as a function of irrigation with the saline e uent from sh farming.

Cultivation conditions, experimental design and irrigation water quality
The experiment was conducted in an experimental area of 90 m² belonging to the Center for Environmental Sciences of the Federal Rural University of the Semi-Arid Region -UFERSA, Mossoró -RN, Brazil (5°12'02.4"S; 37°19'37.3"W). Four-month-old L. gracilis seedlings were transplanted to 40-cm-deep planting holes, with spacing of 1.0 m between plants and between rows. During the period of acclimation to the eld conditions, about 90 days, the seedlings were irrigated daily with public-supply water from the Water and Sewage Company of Rio Grande do Norte (CAERN).

Plant material, growth conditions, and treatments
At three months after planting, the seedlings were fertigated with a nutrient solution containing public-supply water and saline e uent from tilapia (Oreochromis niloticus Linnaeus) growing nurseries at different concentrations and with different physical-chemical compositions (Table 1). Table 1 Physical-chemical composition of sh farming e uent (FFE) and public-supply water (PSW) used in the experiment. Mossoró-RN, 2018.  Liquid nitrogen was used for cryopreservation of samples intended for biochemical analyses.
To obtain dry mass, the harvested material was washed, separated into stem and leaves, placed on Kraft paper and dried in a forced air circulation oven at 70°C, until reaching constant weight. Leaf area was measured at the end of the experiment through images analyzed by Image J software. For this variable, branches were collected from the middle region of the plant, placed in refrigerated container and taken to the laboratory for the reading procedure.
To evaluate the relative water content (RWC), one branch was collected from the central region of ve plants in each treatment, placed in ice and transported to the laboratory. Three discs of known diameter were removed from the leaves, with a cork borer. The discs were immediately weighed on an analytical scale to determine their fresh mass and then placed in a Petri dish on lter paper soaked with distilled water. The Petri dishes were placed in B.O.D chamber at 25°C and 80% relative humidity, in the dark for 24h. After this period, the discs were weighed again to determine the turgid mass and soon after they were placed in the oven for drying until reaching constant weight. Dry mass was then determined, and RWC was calculated using the following equation:

Sodium and potassium analyses
The extracted and exported contents of potassium (K) and sodium (Na) were determined in roots, stems and leaves after opening the owers of each material. All plants of the plot were collected, then oven dried at 60 ºC and ground in a knife mill. In the extracts obtained by digestion with sulfuric acid, the element K was determined. The element Na was extracted with nitric acid. Na and K contents were determined by ame emission photometry. Total N content was determined by the Nessler colorimetric method, after the samples were digested with concentrated H 2 SO 4 . The chemical analyses of the nutrient contents were carried out at the Soil and Plant Laboratory (LASAP), belonging to UFERSA. The ionic ratios were determined by the K/Na ratio of roots, stems and leaves.

Biochemical analyses
The percentage of membrane integrity was estimated from electrolyte leakage (adapted from Azevedo et al., 2008). Five leaf discs with known diameter remained immersed in test tubes with 20 mL of water for 24 hours at 25°C. After this period, free electrical conductivity (R1) was measured with a portable conductivity meter. Then, the test tubes were placed in a water bath for one hour at 100°C to perform the second reading of electrical Lipid peroxidation was determined according to Heath and Packer (1968), with modi cations. The reaction was determined by the production of MDA, a metabolite reactive to 2-thiobarbituric acid (TBA), from readings performed in spectrophotometer at 535 and 600 nm. Plant tissue was macerated in 0.1% trichloroacetic acid (TCA) along with 20% polyvinylpolypyrrolidone (PVPP). The samples were homogenized and centrifuged at 10,000 g for ve minutes at a temperature of 4 ºC. A 0.25-mL aliquot was collected from the supernatant and transferred to a 1.0-mL Eppendorf tube with solution containing 0.5 %of TBA and 20% TCA. The solution was put in a water bath (95 ºC) for 30 minutes and then cooled for 10 min. Prior to the reading, the samples were centrifuged again for more 10 minutes at 10,000 g. The enzymatic activity of superoxide dismutase (SOD) was determined according to Giannopolitis and Ries (1977). This method consists in the inhibition of NBT (nitro blue tetrazolium) by the enzymatic extract, preventing the formation of the chromophore. The sample together with the solution composed of a mixture of 3 mL of phosphate buffer at 85 mM (pH 7.8), 75 µM of NBT, 5 µM of ribo avin, 13 mM of methionine, 0.1 mM of EDTA and 50 µL of enzymatic extract were placed in glass tubes and exposed to white light ( uorescent lamp) in a closed box covered with aluminum foil, to better re ect light throughout the environment, for 5 minutes. After this period, reading was performed in a spectrophotometer at 560 nm.
The enzymatic activity of catalase (CAT) was determined by the method of Havir and Mchale (1987). The solution was prepared with 1 mL of 100 mM potassium phosphate buffer at pH 7.5 and 25 µL of 1 mM hydrogen peroxide. The reaction was initiated by the addition of 25 µL of the sample, monitoring the H 2 O 2 consumption reaction for 1 min. The reaction was monitored in a spectrophotometer at 240 nm at temperature of 25 ºC.
The enzymatic activity of Ascorbate Peroxidase (APX) was determined according to the method proposed by Nakano and Asada (1981). The reaction medium consisted of 650 µL of potassium phosphate buffer at 80 mM, pH 7.5, 100 µL of ascorbate at 5 mM, 100 µL of EDTA at 1 M, 100 µL of H 2 O 2 at 1 mM and 50 µL of protein extract. APX activity was determined by the ascorbate oxidation rate for 60 seconds, in spectrophotometer at 290 nm at 30 ºC.
Starch content was determined by the colorimetric method standardized by Appenroth et al. (2010). Fresh plant material was macerated in 18% HCl (w/v), incubated at 5 ºC for 1 hour. Then, the suspension was centrifuged at 6682 rpm for 20 minutes. An aliquot of the supernatant was added to another aliquot with equal volume of Lugol's solution [0.5% KI (w/v) and 0.25% I2 (w/v) in distilled water] to determine absorbance at 530 and 605 nm.
The absorbance values were applied in the equation described to determine the starch content.
Reducing sugars were determined using the 3,5-Dinitrosalicilic (DNS) colorimetric method, described by Miller (1959), using in the analysis 250 mg of the samples, diluted in water and mixed with the DNS reagent. The mixture was kept under heating in a water bath at 100°C (boiling) for 5 minutes, then cooled with ice bath, to determine the absorbance of the compound formed, at 540 nm. The standard curve of the spectrophotometric determination of reducing sugars was prepared with standards of 1 g.L − 1 of glucose.
Proline content was determined according to the method proposed by Bates et al. (1973). Proline was extracted from 0.3 g of leaf sample crushed in porcelain crucible with 10 mL of 3% sulfosalicylic acid. The reaction mixture was extracted with 5.0 mL of toluene and stirred for 15 s in vortex mixer.
The tubes were kept for 20 min in the dark at room temperature to allow separation of the aqueous phase from the toluene. The toluene phase was collected, and absorbance was measured at 520 nm in spectrophotometer.

Anatomical analysis
For the morphoanatomical evaluation of the median region of L. gracilis leaf blade, slides were prepared from plant material xed in FAA 70 solution and stored in 70% alcohol. Leaf segments were dehydrated in alcoholic series and included in para n (Johansen, 1940 Initially, 100 µL of each essential oil, solubilized in Tryptone Soya Broth (TSB; Lio lchem, Italy), were added to the wells of the microtitration plates at concentrations ranging from 5 to 0.04%, and then 100 µL of bacterial suspension (1 x 10 6 CFU/mL) were added to the wells. The plate was incubated for 24 h at 37°C and later the optical density of each well was measured at 620 nm wavelength with a microplate reader (SpectraMax i3).
The lowest concentration of essential oil capable of visually inhibiting bacterial growth was considered as MIC. For the determination of MBC, 10 µL of the solution contained in the wells that did not have microbial growth were collected, followed by inoculation in Petri dishes with Tryptone Soya Agar medium (TSA; Lio lchem, Italy) and subsequent incubation for 24 h at 37°C. After the 24-hour period, the presence of colony-forming units (CFU) was evaluated and the lowest concentration of essential oil that inhibited the growth of CFUs on the plates was considered as the MBC.

Experimental design and statistical analysis
The experimental design was completely randomized (CRD) with ve treatments (levels of electrical conductivity) and 10 replicates per treatment. After the experimental period, the plants were collected and subjected to analysis, 5 replicates per treatment for growth and nutrition analyses and 5 replicates per treatment for biochemical, anatomical and oil yield analyses. The samples for biochemical analyses were frozen in liquid N and kept in a vertical freezer at -30 ºC. The data obtained were subjected to analysis of variance and the means were compared by Tukey test at 5% probability level, expressed as mean and standard deviation of the mean (Mean ± SD, n = 3). The statistical analysis was performed using the program ASSISTAT, beta version 7.6 (Silva, 2015).

Biomass, osmoregulators and pigments
The addition of sh farming e uent in irrigation water did not signi cantly in uence (p < 0.05) leaf dry biomass, stem dry biomass and RWC (Table 3). Leaf RWC ranged from 64 to 72% on average. In general, the plants of all treatments showed vigorous appearance, with no signs of damage or senescence. Means followed by the same letters do not differ statistically by Tukey test at 5% probability level.
Data of chloroplast pigments indicate that the photochemical apparatus was not damaged by the variation in electrical conductivity (Table 4), which is considered important for the development of plants subjected to abiotic stress. Means followed by the same letters do not differ statistically by Tukey test at 5% probability level.

Osmoregulation
The levels of reducing sugars differed as a function of the salinity of the sh farming e uent. In treatments 2 and 3, higher sugar production was observed; while at the highest salinity, sugar levels were lower (Fig. 1A). On the other hand, when the salinity levels of the e uent increased, the starch content decreased progressively, with reductions ranging from 37 to 19% in the control and T5 treatments, respectively (Fig. 1B). On the other hand, the content of proline, an important osmoregulator in stress situations, was statistically lower only at the highest salt level (Fig. 1C).

Ion concentrations in stem, leaves and roots
The Na + and K + levels differed between the different salt concentrations of the sh farming e uent. It is observed that K levels were higher in leaves than in the other organs. Despite the increase in salt concentration, T5 did not differ statistically from the control. The roots showed lower concentrations of this macronutrient, even in the control treatment ( Fig. 2A, B and C). On the other hand, sodium was more signi cantly concentrated in the stem, but in the treatment with highest salinity, this element remained concentrated in the leaves (Fig. 2B). The Na + /K + ratio con rms that plants tend to accumulate sodium in the stem, contributing to stress modulation; however, at higher levels of salinity, sodium was translocated to the leaves (Fig. 2C), probably as a defense strategy, through leaf senescence.

Oxidative Stress
No increments were detected in the percentage of membrane damage in the leaf tissues of L. gracilis, also accompanied by the maintenance of the content of malondialdehyde (MDA), a product of the peroxidation of polyunsaturated fatty acids of biomembranes ( Fig. 3A and B). However, there was an increase in H 2 O 2 levels with signi cant increments (p < 0.05) at all salinity levels of the e uent compared to the control. The antioxidant enzymatic system, represented by the activity of the enzymes SOD, APX and CAT, showed signi cant differences with the salinity of the sh farming e uent. It was possible to observe maintenance of SOD activity, generating H 2 O 2 , in parallel with an increase in APX and/or CAT. These antioxidant responses were su cient to maintain MDA levels in all treatments (Fig. 4). The observed increase in H 2 O 2 content suggests the generation of this ROS from other metabolic processes.

Anatomy
The cross-sections of L. gracilis leaves show a unistrati ed epidermis with glandular and non-glandular trichomes in the adaxial and abaxial sides. The mesophyll is dorsiventral, with the palisade parenchyma involving the vascular system, which has a half-moon shape in the leaf midrib (Fig. 5). In addition, the presence of raphides in parenchyma cells was veri ed, suggesting the presence of a calcium-rich compound, probably oxalate, in all treatments evaluated ( Fig. 5F and P).
The number of auxiliary vessels (NAV) in the leaves of plants subjected to different concentrations of the e uent did not differ between the treatments tested (Table 5). In this respect, plants that received only sh farming e uent (T5) showed a 25% reduction in the number of xylem bundles in the midrib region when compared to plants of the control treatment (Table 5). To compensate for this reduction, there was an increase of about 171% in the number of xylem vessels in the leaves of plants irrigated with different combinations of sh farming e uent. In addition, the use of e uent was related to the apparent increase in the ligni cation of xylem cells, which were identi ed by wall thickening and intensi cation of safranin staining (Figs. 6B, E, H, K). Means followed by the same letters do not differ statistically by Tukey test at 5% probability level.

Essential oil composition analysis
In the study, the yield of the essential oils of L. gracilis leaves varied from 1.54 (4.60 dS m − ¹) to 2.13% (2.68 dS m − ¹). The essential oils extracted from L. gracilis leaves were analyzed by GC/MS and GC/FID, and the constituents were identi ed and quanti ed (Table 6). A total of 25 compounds organized in order of elution in a DB-5 column were identi ed in the ve essential oil samples. The chemical composition of the essential oil from L. gracilis is mostly constituted by monoterpenes, phenylpropanoids and sesquiterpenes. The major components are: carvacrol (45.35 to 50.17%) (1), pcymene (14.16 to 16.81%) (2) and γ-terpinene (12.44 to 15.03%) (3) (Fig. 6). Moreover, the results showed that the variations in water salinity did not cause signi cant difference in oil yield per plant, between the ve plant samples (Table 6).

Antimicrobial activity
The antimicrobial activity of the essential oil from leaves of L. gracilis irrigated or not with sh farming e uent was tested against clinically relevant bacterial strains. The results showed MIC and MBC of the essential oil against Gram-positive bacteria, with values ranging from 0.625 to 2.5%. Interestingly, E. coli ATCC 11303 showed greater sensitivity to the action of the essential oil. On the other hand, L. gracilis essential oil showed only MIC at 2.5 % against P. aeruginosa ATCC 10145 (Table 7).

Discussion
The The maintenance of growth levels, and consequently of biomass, may have been in uenced by the maintenance of adequate K + levels in the leaves up to T4. Plants often experience reduction of K + when there is exposure to higher levels of Na + due to the competition of this ion for the absorption channels of other ions, especially with K + (Kibria et al. 2017). However, this result was only observed in the treatment with highest salinity, in which there was a reduction in K + to the detriment of Na + . The Na + /K + ratio con rms that there was a higher allocation of sodium to the stem in all treatments, except for T5. The mobilization of Na + ions to the stem and the maintenance of adequate levels of K + in the leaves allowed greater protection and functionality of the photosynthetic organs, as well as the growth rate of the plant (Miranda et al. 2017). The change of sodium allocation to the leaves in the treatment of highest salinity can be explained as a strategy of defense of the plant, since the leaves expel sodium when they senesce.
Growth is in uenced by salinity and by osmotic imbalance and disturbances in the photosynthetic apparatus and osmoregulation (Negrão et al. 2017). Thus, the water retention capacity in leaf tissues, evaluated in this study by the relative water content (RWC) ( Table 3), favored the tolerance to salinity, as it kept the cells turgid, providing full functioning of physiological processes, which positively in uences the growth process, as shown in the parameters of leaf dry biomass and stem dry biomass ( Table 3). The maintenance of RWC levels in L. gracilis subjected to salinity had already been identi ed by Ragagnin et al. (2014) under greenhouse conditions with different salinity levels.
The maintenance of leaf tissue hydration is a behavior of plants that can adjust osmotically. Thus, some species invest in sugar synthesis or starch breakage in an attempt to mitigate damage caused by stress (Almodares et al. 2008;Santelia and Lawson 2016). In this study, starch contents decreased (Fig. 1B) in treatments with higher salinity levels, indicating that the species was using its polysaccharide reserves to produce sugars for osmoregulation in order to keep leaf tissue hydrated according to its osmotic potential, thus maintaining its metabolism in operation.
In situations of abiotic stress, plants accumulate in the cytosol or vacuoles low-molecular-weight solutes (proline, betaine glycine, sucrose) to maintain water balance and preserve the integrity of membranes, proteins and enzymes (Ashraf et al. 2013;Marijuan and Bosch 2013). However, depending on the intensity and duration of stress, the production of these osmoregulators may or may not be intensi ed. In this study, it was observed that proline levels decreased at the highest concentrations of the e uent (Fig. 1C). This nding indicates, once again, that sugar production served as the main regulatory mechanism for the maintenance of water potential and leaf tissue turgor. It is important to highlight that in saline treatments with up to 5.5 dS m − 1 the proline levels did not differ from those of the control. It is suggested that this result is probably attributed to the adaptation of L. gracilis to environments with water scarcity and high temperatures. Under these conditions, proline is generally produced to keep the tissues hydrated, avoiding stress by desiccation. Results similar to these were found by Hu et al. (2012)  Lipid peroxidation patterns can be modulated according to the amount of ROS generated and the defense capacity of the cells. One of the factors that can increase lipid peroxidation is excessive production and non-removal of H 2 O 2 . This compound is naturally synthesized by plants and its production occurs mainly through photorespiration or as a result of the dismutation of the superoxide radical, by superoxide dismutase (SOD  (Fig. 4). Increase in CAT activity was observed by Gondim et al. (2012) in a study with corn plants subjected to stress induced by NaCl, where the harmful effects of salinity did not compromise the growth variables. This result is indicated by the authors due to the performance of the antioxidant system, especially CAT.
The sh farming e uent did not interfere in the water balance of the cells in such a way to hamper the transport of water, so the mesophyll thickness was similar in all treatments evaluated (Table 5; Fig. 5), indicating that there was water maintenance in the cells, since water availability is one of the factors that rst affect the maintenance of cell turgor (Santos and Carlesso 1998), which may therefore affect the mesophyll thickness.
Essential oils are components of the secondary metabolism of plants that are extracted from several parts (Oussalah et al. 2007). These compounds guarantee some advantages, acting for example as antioxidants and in the ght against microbial agents (Gutierrez et al. 2008). However, the chemical composition and content of the essential oil of a same species are associated with a variety of factors. According to Morais (2009) The essential oil from L. gracilis, with different saline treatments, showed bacteriostatic and bactericidal activity against Gram-positive and Gramnegative bacteria. The antimicrobial activity obtained in this study may be explained mainly by the phenylpropanoids carvacrol and thymol, compounds that are usually found in Lippia species and have shown action against bacteria and fungi. The essential oil from L. origanoides showed activity against bacteria such as methicillin-resistant Staphylococcus aureus, and the essential oil from L. menosides showed activity against Escherichia coli, Enterococcus faecalis, Salmonella enteritidis, Serratia marcescens, Candida albicans and Mycobacterium smegmatis. The antimicrobial activity was associated with the presence of the phenolic monoterpenes, carvacrol (41.77%) and thymol (10.13%) (Girón et al. 1991;Lacoste et al. 1986;Oliveira et al. 1990).
Essential oils are compounds that, for being hydrophobic, are easily diffused through the cell wall of microorganisms and cause damage to the membrane, especially with regard to uidity and permeability (Millezi et al. 2012). Gram-negative bacteria, in general, have greater resistance to the action of essential oils, due to the greater complexity of their plasma membrane, which acts as a barrier to the diffusion of hydrophobic components of essential oils (Naik et al. 2010).
It is known that sh farming e uent has high levels of excrement and nutrients that can modify the characteristics of the fertigation solution in such a way to make its use unfeasible in other activities, such as in agriculture when using salinity-sensitive species (Mercante et al. 2004; Mainardes Pinto and Mercante 2003). The results of this study are relevant to the planning and management for the use of sh farming e uent, which is currently disposed of inadequately and without any utilization. In this context, the set of results obtained support the responses observed in the growth, anatomical and biochemical parameters, consolidating the indication of sh farming e uent used in this experiment for reuse in the irrigation of L. gracilis, an endemic species of the Caatinga and with pharmacological potential.

Conclusions
Acceptable biomass yield was maintained at all levels of salinity of the nutrient solution, composed of sh farming e uent; The high salinity of the e uent did not alter the leaf area and the relative water content associated with the production of sugars, pigments and activity of the enzymes of the antioxidant system, indicating that the fertigation of L. gracilis with saline e uent from sh farming did not affect the development of this species. The saline e uent from sh farming can be used in fertigation of L. gracilis without compromising the yield of plants, avoiding environmental contamination with disposal in soil and/or water bodies.

Declarations
Ethics approval and consent to participate Not applicable Consent for publication Not applicable  Means followed by the same letters do not differ statistically by Tukey test at 5% probability level.