Alteration of antioxidant enzymes and impairment of DNA in the SiO2 nanoparticles exposed zebra fish (Danio rerio)

The incorporation of nanoparticles in industrial and biomedical applications has increased significantly in recent years, yet their hazardous and toxic effects have not been studied extensively. While standard toxicological test methods are generally capable of detecting the toxic effects, the choice of relevant methods for nanomaterials is still discussed. Among the various oxide nanomaterials, silica nanoparticles are widely used in biological applications that include nano-medicine. But studies on adverse effects of silica nanoparticle exposure to fish remain unclear. Therefore, the present study was designed to investigate the oxidative toxic effects of silicon dioxide nanoparticles using fish model. The size of the SiO₂ nanoparticles was between 68 and 100 nm which was confirmed by X-ray diffractometer, dynamic light scattering, scanning electron microscope and transmission electron microscope. The zebra fish were exposed to sub-lethal concentrations (5 and 2.5 mg/L) of characterized SiO₂ nanoparticles for a period of 7 days. After 7 days, SiO₂ nanoparticle-treated fishes were sacrificed, and tissues such as liver, muscle and gill were dissected out for the analysis of antioxidant enzymes and DNA fragmentation. The DNA profiles were analysed in the tissues of zebra fish that treated with SiO₂ nanoparticles. Tissues of fish from clean water were used as control, and DNA profiles were analysed. It is found that DNA from control tissues was intact, whereas the tissues treated with SiO₂ were all fragmented. SiO₂ nanoparticle-mediated antioxidant enzymes activities, such as catalase, superoxide dismutase, glutathione (GSH)-S-transferase, glutathione reductase and GSH, in the tissues of zebra fish were measured. The results revealed that alteration of antioxidant enzymes due to SiO₂ nanoparticle can be considered as a biomarker to SiO₂-mediated oxidative stress in biological samples.     

Oxidative stress in Nile tilapia (Oreochromis niloticus) and armored catfish (Pterygoplichthys anisitsi) exposed to diesel oil

Considering that diesel oil is one of the most common aquatic contaminants, we compare the oxidative stress between two species of fish with different habitats (Pterygoplichthys anisitsi, benthic and Oreochromis niloticus, nektonic) exposed to diesel oil. Malondialdehyde concentrations (MDA) and the activities of ethoxyresorufin-O-deethylase (EROD), glutathione S-transferase (GST), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase were all analyzed in the fishes' livers and gills after 2 and 7 days of exposure to different concentrations of diesel (0.1 and 0.5 mL/L). In the tilapia, MDA levels and the activities of EROD and GST activity in the liver, as well as MDA levels and the activities of GST and SOD in the gill had statistically significant differences between the treatments and between the times of exposure. For the catfish, the same occurred in the case of MDA, EROD, and SOD in the liver and in CAT and SOD in the gills. There were significant differences in the enzyme activity and lipid peroxidation between the species. Although the activity of most enzymes seemed to be more expressive and responsive to diesel in O. niloticus, diesel oil also caused significant effects on oxidative stress parameters in P. anisitsi, even though this species is benthic and thus has less access to insoluble fractions of diesel oil. Therefore, both species can be used as sentinel organisms in environmental biomonitoring of diesel contamination.     

Oxidative stress,glutathione level and antioxidant response to heavy metals in multi-resistant pathogen,Candida tropicalis

In this study, we explored the multiple heavy metal-resistant yeast isolated from heavy metal-polluted environment. The isolated yeast showed maximum growth at 30 °C, pH 7.0, and the strain was identified as Candida tropicalis through 18S ribosomal RNA (rRNA) gene sequence analysis. Yeast cells grew well in medium containing different concentrations of heavy metal ions [CdCl₂, Pb(NO₃)₂, NaAsO₂, CuSO₄ and K₂Cr₂O₇]. Minimum inhibitory concentration (MIC) against different metal ions was ranged from 5 to 19 mM, and the metal resistance value against each metal observed by yeast cells was 5 mM (Cr), 10 mM (Cd), 15 mM (As), 14 mM (Cu) and 19 mM (Pb) and increased in the following order: Pb > Cu > As ≥ Cd > Cr. The total cellular glutathione, GSH/GSSG redox couple and metallothioneins like protein (MT) were assayed by growing cultures for 24 h and exposed to 100 mg/L of each heavy metal ion. Remarkable increase in γ-glutamylcysteinylglycine (GSH) level was determined in arsenic and cadmium treatment followed by chromium, lead and copper. Stressed cells had much more oxidized GSH than unstressed cells. GSH/GSSG ratio was significantly increased in cadmium and copper treatment in contrast to chromium, arsenic and lead. Statistical analysis revealed significantly higher cysteine level in all metal-treated samples as compared to control. Antioxidant glutathione transferase activity was not detected in metal-treated and untreated yeast samples. One-dimensional electrophoresis of proteins revealed marked differences in banding pattern of heavy metal-exposed yeast samples. A prominent 20 kDa band was observed in all treated samples suggesting that some differential proteins could be over-expressed during heavy metal treatment and might be involved in cell resistance mechanisms.     

Histopathological changes in zebrafish embryos exposed to DLPCBs extract from Zhanjiang coastal sediment

Dioxin-like polychlorinated biphenyls (DLPCBs) are ubiquitous persistent pollutants that cause adverse effects in many environmental organisms. DLPCBs in marine sediments can be absorbed by benthic organisms, bioaccumulate, and biomagnify through the food chain and threaten animal and human health. There are no reports of DLPCBs concentrations in the Zhanjiang Gulf seabed. This study was designed to investigate the concentration of DLPCBs in the Zhanjiang coastal sediment and histopathological changes in zebrafish (Diano rerio) embryos exposed to environmentally relevant concentrations of DLPCBs. Of the five sites selected, two sites TS and JSW contained DLPCBs at concentrations of 0.08 and 22.54 ng/g dry sediment, respectively. Two groups of zebrafish embryos were used. One group was exposed to 3.75, 7.5, 15, 30, and 60 mg/ml of DLPCBs extracted from the sediments sampled from the TS site and the second group to 4.375, 8.75, 17.5, 35, and 70 mg/ml of DLPCBs from JSW site from 0.75 h post-fertilization (hpf) to 96 hpf. The zebrafish exposed to 60 and 70 mg/ml of DLPCBs at 96 hpf displayed gross histopathological changes with cardiac lesions including pericardial edema being the most deleterious. Other changes observed were hydropic degeneration of gill filaments and hepatocytes, loss of intestinal folds, and uninflated swim bladder. It appears that only a few sites of the Zhanjiang gulf are contaminated with DLPCBs. This is the first report of histopathological changes in the gills, hepatocytes, intestines, heart, and the swim bladder in zebrafish embryos exposed to DLPCBs from a coastal sediment. Further studies with sampling at different stages of development are required to identify which organ/tissue is most sensitive to DLPCBs.     

Genotoxic effects and gene expression in <Emphasis Type="Italic">Danio rerio</Emphasis> (Hamilton 1822) (Cypriniformes: Cyprinidae) exposed to mining-impacted tributaries in Manizales,Colombia

The zebrafish (Danio rerio) is one of the most studied aquatic organisms for water biomonitoring, due to its sensitivity to environmental degradation and resistance to toxic substances. This study determined the presence of micronuclei and nuclear abnormalities in peripheral blood erythrocytes, and assessed the gene expression of caspase-3 (CASP-3) and metallothionein 1 (MT-1) in the gills and liver of D. rerio. The study fish (n = 45) were exposed to water collected from two stations with mining impact (E2 and E3) and a reference station without evident mining contamination (E1), all located in La Elvira stream (Manizales-Colombia). In addition, a positive control (PC) with HgCl₂ (50 μg/L) and negative control (NC) with tap water were included. The fish from the PC and E2 and E3 treatments displayed genotoxic effects and changes in gene expression, with significant differences in micronuclei formation and the presence of blebbed nuclei. The cytochrome oxidase subunit I (COI) gene was used as reference and proved to be stable compared to the β-actin and 28S ribosomal RNA (28S) genes. In gills, CASP-3 expression was higher in the PC, and MT-1 expression was higher in the PC and E3 treatment. In liver, CASP-3 was expressed in the E2 treatment, and MT-1 expression was low. These results show that the genotoxic effects and differential gene expression observed in fish exposed to water from La Elvira stream could also be affecting the organisms present in this habitat.     

Coelomocyte biomarkers in the earthworm Eisenia fetida exposed to 2,4,6-trinitrotoluene (TNT)

Contamination by 2,4,6-trinitrotoluene (TNT) is a global environmental problem at sites of former explosive production, handling, or storage, and could have deleterious consequences for human and ecological health. We investigated its sublethal effects to Eisenia fetida, using two nonspecific biomarkers. In coelomocytes of earthworms exposed 24, 48, or 72 h, we evaluated DNA damage (comet assay) and neutral red retention time (NRRT), using the filter paper contact test. Both percentage of damage (D%) and calculated damage index showed significant DNA damage at almost all concentrations, at all time points assayed. Along exposure time, two different patterns were observed. At the lower TNT concentrations (0.25?C0.5 ??g/cm²) an increased DNA migration at 48 h, with a decrease close to initial levels after 72 h exposure, was observed. This decrease could be attributed to activation of the DNA repair system. At higher concentrations (1.0?C2.0 ??g/cm²), the high DNA damage observed remained constant during the 72 h exposure, suggesting that the rate of DNA repair was not enough to compensate such damage. Analysis of NRRT results showed a significant interaction between time and treatment. After 48 h, a significant decrease was observed at 4.0 ??g/cm². After 72 h, NRRT presented a concentration-dependent decrease, significantly different with respect to control at 0.5, 1.0, 2.0, and 4.0 ??g/cm². The two assayed methods, performed on the same sample, showed clear responses to sublethal TNT exposure in E. fetida, providing sensitive unspecific biomarkers of cell injury and DNA damage.     

Oxidative stress in Cyprinus carpio to analyze microcystin impact in eutrophic shallow lakes: a preliminary study

Microcystins (MCYSTs) are toxins produced by cyanobacteria in aquatic environment and are of high potential risk to aquatic organisms. The physiological responses and pathobiological developments that they elicit in fish have been extensively studied, mainly through acute toxicity experiments. This study was designed to examine the seasonal fluctuation of biochemical markers of oxidative stress in different tissues of a natural population of Cyprinus carpio inhabiting a shallow Mediterranean lake, along with the respective MCYSTs concentrations in blood and tissues at environmentally relevant MCYSTs values. MCYSTs content was assessed in liver, kidney, intestine, brain and muscle along with the MCYSTs in lake water and scum applying ELISA technique. Catalase activity, GSH/GSSH relative concentrations and lipid peroxidation were used as biochemical markers. Our results suggest that common carp of Lake Pamvotis exposed to naturally fluctuating concentrations of MCYST in water and scum contained stably high MCYST concentrations in all tissues that might pose a threat to public health. Liver and kidney were the primary target organs. Tissue concentrations did not correlate with the response of any of the elements of the antioxidant defence system. Hepatic catalase, GSH content and TBARS in all tissues tested followed the fluctuations of major limnological parameters, i.e. water temperature and oxygen concentration, chlorophyll-a, MCYST in water and scum, suggesting that they should be cautiously used to monitor exposure to MCYSTS in natural freshwater ecosystems.     

Heat shock protein 47 stress responses in Chinese hamster ovary cells exposed to raw and reclaimed wastewater

As wastewater reclamation and reuse becomes more widespread, risks of exposure to treated wastewater increase. Moreover, an unlimited number of pollutants can be identified in wastewater. Therefore, comprehensive toxicity assessment of treated wastewater is imperative. The objective of this study was to perform a comprehensive toxicity assessment of wastewater treatment systems using stress response bioassays. This powerful tool can comprehensively assess the toxicity of contaminants. In this study, samples from conventional activated sludge treatment, membrane bioreactors (MBRs) with different pore sizes and sludge retention times (SRTs), rapid sand filtration, coagulation, nano-filtration (NF) and reverse osmosis (RO) were investigated. The results of stress response bioassays confirmed that the secondary effluent showed higher stress response than influent indicating that biological treatment generates toxic compounds. The results obtained from molecular weight fractionation of water samples demonstrated that organic matter with a higher molecular weight fraction (>0.1 μm) causes toxicity in secondary effluent. Furthermore, supernatant from MBR reactors showed toxicity regardless of SRT. On the other hand, stress response was not detected in MBR permeates except for an MBR equipped with a larger pore size membrane (0.4 μm) and with a short SRT (12 days). While rapid sand filtration could not remove the toxic compounds found in secondary effluent, coagulation tests, operated at an appropriate pH, were effective for reducing stress response in the secondary effluent. Experimental findings also showed that stress response was not detected in cases of NF and RO permeate subsequent to MBR treatment.     

A flounder (Paralichthys olivaceus) gill cell line as in vitro acute assay system of nonylphenol cytotoxicity

A cell line (FG cells) derived from a gill of the flounder, Paralichthys olivaceus were used to determine the cytotoxic effects of nonylphenol (NP). Cytotoxicity was measured by three endpoint systems: neutral red (NR) uptake assay, methyl thiazolyl tetrazolium (MTT) assay and cell protein assay. The result showed that NP was cytotoxic to FG cells at all tested concentrations, and toxicity increased as the concentration of NP was progressively increased. The 24 h-IC₅₀ values of NP were 39.81, 37.76 and 38.22 ??mol/L for NR uptake, MTT assay and cell protein assay, respectively. Moreover, the morphological changes of FG cells were also studied at the concentration of 30 ??mol/L for 24 h. Cells morphology were markedly altered by NP observed under a scanning electron microscopy, as evidenced by swelling cells, two and more nucleolus and an increased number of lipid particles. This would suggest that the FG cell line is a suitable bioindicator for the screening of the acute toxicity of NP.     

The response of nematode assemblages to intensive mussel farming in coastal sediments (Southern Brazil)

The impact of mussel farms on the benthic environment was investigated using free-living nematode assemblages as indicators of environmental condition of the Enseada do Brito, a shallow embayment in South Brazil. The studied area is located at the State of Santa Catarina, responsible for 90% of the production of mussel farmed in the country. Samplings were carried out quarterly, from July 2004 to May 2005, below mussel ropes and at control sites located 300 m apart from the longlines. The results suggested that the mussel culture changed ecological functioning of the benthic environment at studied coastal embayment. The intense organic loads due to the biodeposition of the mussel farms, which had been in production for more than 10 years, have turned fine and well-sorted sand bottoms into a muddy deposit with significantly higher total organic content than the control. The number of nematode genera, diversity, dominance and trophic diversity were significantly lower under the mussel farms than at the control sites. The mussel farms biodeposition had also affected the temporal variability of nematode assemblages, which was detected only for samples at the control sites. The low depth and environmental energy, together with the age of the farms, were probably responsible for the observed changes in the nematode assemblages.     

Bacterial response to dynamic metal concentrations in the surface sediments of a solar saltern (Goa, India)

The Ribandar solar saltern, situated adjacent to the Mandovi estuary is influenced by the barge transport of ferromanganese ore to the Mormugao harbour (Arabian Sea). The current study focuses on the distribution of metals and related heterotrophic bacterial populations in the surface sediments (0–10 cm) of the Ribandar salterns (Goa, India) during the salt-making (January to May) and non salt-making seasons (August and November). The concentrations of heavy metals in the sediments ranged from 17.2?±?2.8 to 26.3?±?6.7 % Fe; 0.6?±?0.2 to 0.9?±?0.2 % Mn; 27.6?±?7.3 to 51?±?8.3 ppm Ni; 28.4?±?8.9 to 35.2?±?10.6 ppm Co; 44?±?21.6 to 62.8?±?23.6 ppm Zn; 0.1?±?0.01 ppm Cd and 1.7?±?0.1 to 2.6?±?0.7 ppm Pb and were much higher than those reported at the same site in a previous study by Kerkar (2004). Hg concentrations were below detection limits. In general, computation of “geoaccumulation index” revealed the sediments as ‘uncontaminated to moderately contaminated’ with Fe, Mn, Ni, Co, Pb and Zn during the salt-making season. The abundance of metal-tolerant bacteria was comparatively restricted to the salt-making season and was higher than the non salt-making season. Fe-, Mn-, Ni-, Co- and Pb- (200 ppm) tolerant bacteria were retrieved and restricted to the surface sediments (0–5 cm), Cd and Fe being the two most regulatory elements governing bacterial populations in the non salt-making season. However, during the salt-making season, the concentration of Zn was found to be pivotal in regulating the counts of Fe-, Mn- and Ni-tolerant bacteria. In general, the strength of correlation of metals and microbes was higher in the non salt-making season as compared to the salt-making season. This would probably indicate metal-induced limitations in microbial populations in the non salt-making season and the absence of this effect during the salt-making season. In this study, we test the hypothesis that solar salterns behave as ecological sinks with a potential to transform native bacterial populations to metal-resistant strains, in relation to the dynamic changes in the surrounding metal concentrations.     

Modulations in the levels of respiration and ions in carp Cyprinus carpio (L.) exposed to cypermethrin

This paper reports an investigation of the sublethal toxicity of cypermethrin (20 µg/l) on the levels of respiration and selected ions in tissues of carp (Cyprinus carpio L.). A significant decrease in O ₂ consumption and ions was observed in gill, brain, liver and muscle tissues of cypermethrinexposed fish. The decreased rate of O ₂ consumption may be due to inhibition of oxidative enzymes during cypermethrin stress.     

Properties and erosional response of soils in a degraded ecosystem in Crete (Greece)

To study desertification processes relating to soil erosion, a climatological and altitudinal gradient from south to north was selected in Crete (Greece) and four locations were selected along the gradient. At the locations precipitation ranged from 1400 mm/year at the highest location to 400 mm/year at the lowest. All locations are affected by the actual land use: intensive grazing, small controlled fires, and abandoned agricultural terraces. Representative soil profiles were described in the field and analyzed in the laboratory, and rainfall simulation experiments in the field measured soil erosion over different soil surfaces and land uses. Data on physical and chemical properties were obtained from the soil profiles and soil hydrology, and erosion data were obtained from the rainfall simulation experiments. Soil aggregation was studied with samples taken from the soil in the rainfall simulation plots and special attention being paid to the aggregate size distribution and the water-stable microaggregation. The interaction between climatological conditions and land use seems to be the main factor controlling soil erosion. This paper describes how the expected erosion along the gradient (from the most humid to the driest site) can be affected and disturbed by specific processes derived from land use.     

Methane oxidation and abundance of methane oxidizers in tropical agricultural soil (vertisol) in response to CuO and ZnO nanoparticles contamination

There is worldwide concern over the increase use of nanoparticles (NPs) and their ecotoxicological effect. It is not known if the annual production of tons of industrial nanoparticles (NPs) has the potential to impact terrestrial microbial communities, which are so necessary for ecosystem functioning. Here, we have examined the consequences of adding the NPs particularly the metal oxide (CuO, ZnO) on CH₄ oxidation activity in vertisol and the abundance of heterotrophs, methane oxidizers, and ammonium oxidizers. Soil samples collected from the agricultural field located at Madhya Pradesh, India, were incubated with either CuO and ZnO NPs or ionic heavy metals (CuCl₂, ZnCl₂) separately at 0, 10, and 20 μg g^?1 soil. CH₄ oxidation activity in the soil samples was estimated at 60 and 100 % moisture holding capacity (MHC) in order to link soil moisture regime with impact of NPs. NPs amended to soil were highly toxic for the microbial-mediated CH₄ oxidation, compared with the ionic form. The trend of inhibition was Zn 20?>?Zn 10?>?Cu 20?>?Cu 10. NPs delayed the lag phase of CH₄ oxidation to a maximum of 4-fold and also decreased the apparent rate constant k up to 50 % over control. ANOVA and Pearson correlation analysis (α?=?0.01) revealed significant impact of NPs on the CH₄ oxidation activity and microbial abundance (p?<?0.0001, and high F statistics). Principal component analysis (PCA) revealed that PC1 (metal concentration) rendered 76.06 % of the total variance, while 18.17 % of variance accounted by second component (MHC). Biplot indicated negative impact of NPs on CH₄ oxidation and microbial abundance. Our result also confirmed that higher soil moisture regime alleviates toxicity of NPs and opens new avenues of research to manage ecotoxicity and environmental hazard of NPs.     

Use of the Multispecies Freshwater Biomonitor to assess behavioral changes of Poecilia reticulata (Cyprinodontiformes: Poeciliidae) and Macrobrachium lanchesteri (Decapoda: Palaemonidae) in response to acid mine drainage: laboratory exposure

The behavioral responses of guppy Poecilia reticulata (Poeciliidae) and prawn Macrobrachium lanchesteri (Palaemonidae) individuals exposed to acid mine drainage (AMD) were monitored online in the laboratory with a Multispecies Freshwater Biomonitor? (MFB). These responses were compared to those to reference water acidified to the respective pH values (ACID). Test animals in the juvenile stage were used for both species and were exposed to AMD and ACID for 24 hours. The stress behaviors of both test animals consisted mainly of decreased activity in AMD and increased activity in ACID, indicating that the metals in the AMD played a role as a stress factor in addition to pH. The locomotor activity levels of guppies and prawns for the ACID treatment were higher than the locomotor activity levels for the AMD treatment with increasing pH value. For guppies, significant differences were observed when specimens were exposed to AMD and ACID at pH 5.0 and 6.0; the percentage activities were only 16% and 12%, respectively, for AMD treatment, whereas for ACID treatment, the percentage activities were 35% and 40%, respectively, similar to the value of 36% for the controls. Similar trends were also observed for prawns, for which the percentage activities were only 6% and 4%, respectively, for AMD treatment, whereas for ACID treatment, the percentage activities were 31% and 38%, respectively, compared to 44% in the controls. This study showed that both species are suitable for use as indicators for ecotoxicity testing with the MFB.     

Analysis of trace elements (Cu, Cd, and Zn) in the muscle, gill, and liver tissues of some fish species using anodic stripping voltammetry

Heavy metal (Cu, Cd, Zn) concentration of the muscle, gill, and liver of Otolithes rubber, Pampus argenteus, Parastromateus niger, Scomberomorus commerson, and Onchorynchus mykiss are determined by anodic stripping voltammetry method. The results show that the concentration of Cu, Cd, and Zn varied between 0.44 and not detected, 0.585 and 0.001, and 0.450 and 0.005?μg/g, respectively. It is interesting that in O. mykiss, the mean value of copper concentration was more than other marine organisms of this study, while in P. niger, the mean value of copper was minimum. The results of this study indicate that in the different tissues of fish species (O. rubber, P. argenteus, P. niger, S. commerson, and O. mykiss), the concentrations of Cu, Cd, and Zn were significantly lower than the permissible FAO/WHO levels.     

Evaluation of mentum deformities of Chironomus spp. (Chironomidae: Diptera) larvae using modified toxic score index (MTSI) to assess the environmental stress in Juru River Basin, Penang, Malaysia

Morphological mentum deformities which represent sublethal effect of exposure to different types of pollutants were evaluated in Chironomus spp. larvae inhabiting three polluted rivers of Juru River Basin in northwestern peninsular Malaysia. Using mentum deformity incidences, the modified toxic score index (MTSI) was developed based on Lenat??s toxic score index (TSI). The suggested MTSI was compared with TSI in terms of its effectiveness to identify different pollutants including heavy metals. The MTSI showed stronger relationship to total deformity incidence expressed as percentage. Additionally, the multivariate RDA model showed higher capability of MTSI to explain the variations in heavy metal contents of the river sediments. The MTSI was recommended in bioassessment of water and sediment quality using the mentum deformities of Chironomus spp. larvae from aquatic ecosystems receiving anthropogenic, agricultural, or industrial discharges.     

Immune response of earthworms (Lumbricus terrestris, Eisenia andrei and Aporrectodea tuberculata) following in situ soil exposure to atmospheric deposition from a cement factory

In order to reduce their energy costs, many cement plants use fuel product substitutes (old tyres and used oil). The combustion of these products generates a metal increase (e.g. Cu, Cd, Pb and Zn) in the atmospheric emissions. After their release, these elements are deposited into the environment and could eventually accumulate up to concentrations of concern. At the Saint-Laurent cement factory (Joliette, QC, Canada), maximum deposition of these elements occurs in the direction of prevailing winds (North-East). We evaluated the potential impact of these depositions upon the immune system of three earthworm species (Lumbricus terrestris, Eisenia andrei and Aporrectodea tuberculata) exposed in a natural environment. The exposure sites were 0.5, 1.0, and 2.0 km downwind from the cement factory, along with an upwind reference site. The immune parameters studied were the cell viability and phagocytic potential of the immune cells (coelomocytes). For both L. terrestris and E. andrei, after 7 d exposure, none of the measured parameters showed significant differences among the sites. On the other hand, for the indigenous worm A. tuberculata, in the most exposed zone (at 0.5 km), we observed an increase in cell viability and phagocytic potential. This increase could possibly be attributed to physicochemical effects such as the alkaline pH of the soil, or alternatively, it could result from beneficial effects induced by an increased calcium supply.