不同价态铬在不同水分条件下的生物有效性及其对水稻的毒性

氧化还原过程在铬的形态转化中起了重要作用,而铬形态的转化能够影响其生物有效性及毒性。通过温室土培试验研究了六价铬(Cr(Ⅵ))与三价铬(Cr(Ⅲ))在淹水与不淹水条件下在土壤溶液中的动态变化及水稻对其吸收的变化。结果表明,土壤中添加Cr(Ⅲ)时,土壤溶液中检测不出Cr;而随着土壤中添加Cr(Ⅵ)浓度的增加,土壤溶液中Cr(Ⅵ)的浓度增加,但是溶液中检测不出Cr(Ⅲ);淹水处理总体上降低了土壤溶液中Cr(Ⅵ)的浓度。而土壤添加Cr(Ⅲ)、Cr(Ⅵ)和水分处理对土壤溶液p H没有显著影响,p H在7.08.0之间变动。土壤添加Cr(Ⅵ)处理的水稻中,只有90 mg·kg-1Cr(Ⅵ)淹水处理的水稻成活,而其余处理水稻没有成活。土壤中添加Cr(Ⅲ)处理,水稻幼苗生物量随Cr(Ⅲ)浓度的增加而显著降低;除了200mg·kg-1Cr(Ⅲ)处理外,其余淹水处理的水稻幼苗生物量明显高于不淹水处理的。土壤添加Cr(Ⅲ)处理的水稻,在不淹水条件下水稻空壳率比较高,淹水条件下,随着土壤中添加Cr(Ⅲ)浓度水平的增加,水稻各部位Cr含量有增加的趋势,但增加不显著,秸秆最高Cr含量达到33.80 mg·kg-1,籽粒中Cr含量最高0.30 mg·kg-1。土壤固定Cr(Ⅲ)的能力远强于Cr(Ⅵ),添加Cr(Ⅵ)处理的土壤溶液中Cr(Ⅵ)的浓度很高,对水稻表现出较强的生长抑制。     

Bioavailability and metabolism of hexavalent chromium compounds †

Intratracheal instillation of ⁵¹CrCl₃ in anaesthetized rabbits resulted in partial absorption. In blood, the absorbed material was entirely confined to the plasma compartment. Only trace amounts were deposited in liver and kidney. By contrast, after similar application of Na, ⁵¹CrO₄ the bulk of blood radioactivity was present in red blood cells (RBC). Substantial deposition occurred in liver and kidneys. It is concluded that Cr(VI) may enter the body unreduced via the lung and is partially deposited in cells over a prolonged period of time.

Since chromium was accumulated in liver after administration of Cr(VI) we investigated the intracellular disposition of Cr(VI) in the isolated perfused liver. No significant sex differences in chromium distribution were observed. At the end of the experiments (1 h), 60% of the applied dose (312μg Cr/liver) was located in the cytosol, whilst 14% was in the mitochondria, 9% in the microsomal pellet and 2% was associated with the nuclei. Gel chromatography of the cytosolic compartment showed that the overwhelming part of chromium was eluted in fractions with an apparent molecular weight of 6,000 dalton. These fractions exhibited absorption maxima at 410nm and 548nm. It is concluded, that cytosolic reduction might be the main intracellular redox pathway for chromates. This view was confirmed by monitoring the reaction of Cr(VI) with GSH in vitro. GSH reduced Cr(VI) without further cofactors under formation of GSH‐chromium complexes, which possibly represent major intermediates in the metabolism of Cr(VI).     

Phytoextraction of Cr(VI) from soil using Portulaca oleracea

Cr(VI) represents an environmental challenge in both soil and water as it is soluble and bioavailable over a wide range of pH. In previous investigations, Portulaca oleracea (a plant local to the United Arab Emirates (UAE)) demonstrated particular ability for the phytoextraction of Cr(VI) from calcareous soil of the UAE. In this publication, the results of the evaluation of P. oleracea phytoextraction of Cr(VI) from UAE soil at higher concentrations are reported. P. oleracea was exposed to nine different concentrations of Cr(VI) in soil from 0 to 400 mg kg^?1. The uptake of Cr(VI) increased as its concentration in soil increased between 50 and 400 mg kg^?1, with the most efficient removal in the range from 150 to 200 mg kg^?1. The total chromium concentrations exceeded 4600 mg kg^?1 in roots and 1400 mg kg^?1 in stems, confirming the role of P. oleracea as an effective Cr(VI) accumulator. More than 95% of the accumulated Cr(VI) was reduced to the less toxic Cr(III) within the plant.     

Biochemical speciation in chromium genotoxicity

The biochemical speciation of chromium compounds in mammalian cells is discussed with respect to uptake, metabolism, DNA binding and damaging. Whereas soluble hexavalent chromium is taken up rapidly and accumulated intracellularly after its reduction, compounds of trivalent chromium penetrate biomembranes about three orders of magnitude slower. Cr(VI) after its uptake is metabolised by electron donating compounds via Cr(V) to Cr(III) compounds. Chromium from various Cr(III) compounds, but not chromate, binds to chromatin in isolated cell nuclei. The DNA‐protein crosslinks and DNA strand breaks observed in rat liver and kidney after chromate administration are also found in vitro, when Cr(III) compounds (but not chromate) interacts with isolated nuclei. In the Chinese Hamster cell HGPRT mutation assay, three out of four tested Cr(III) complexes were found to be mutagenic. In a direct DNA strand break assay with supercoiled bacteriophage PM 2 DNA, neither chromate nor the four Cr(III) compounds tested caused nicks. However, the combined action of chromate plus glutathione as well as the isolated complex of pentavalent chromium, Na₄Cr(glutathione)₄, did cause DNA breaks. Reactive oxygen species are inferred to be the ultimate DNA nicking agents in this assay. In conclusion there appear to be two mechanisms of chromate genotoxicity; one with direct DNA damage caused by Cr(V) species and one via DNA‐protein crosslinks formed with Cr(III), the final reduction state of chromate.     

Effects of Cr(III) organic compounds on Lactobacillus plantarum

Biotransformation of Cr(VI) to less toxic Cr(III) has been known to produce insoluble Cr(III) compounds and soluble Cr(III) organic complexes. However, recent research reports have indicated that Cr(III) organic complexes are relatively stable in the environment. Little has been reported on the fate and toxic effects of Cr(III) organic compounds on organisms. In this study, the toxic effects of the soluble Cr(III) organic complexes [Cr(III) citrate, Cr(III) histidine, Cr(III) lactate and Cr(III) glutamate] to a local strain of Lactobacillus plantarum isolated from sauerkraut was investigated. Growth inhibition, viable cell count and lactic acid inhibition were measured to determine the toxicity potential of the test compounds. The EC₅₀ values of Cr(III) citrate, Cr(III) histidine, Cr(III) lactate, and Cr(III) glutamate, calculated from the percent growth inhibition were found to be 56 mg L^?1, 70 mg L^?1, 81 mg L^?1, and 85 mg L^?1, respectively. Similar trend was observed in the viable cell counts and lactic acid production. Cr(VI) was observed to be more toxic than the Cr(III) organic compounds, while inorganic Cr(III) was the least toxic. The severity seemed to increase with increase in chromium compounds’ concentration. The results showed that Cr(III) citrate was the most toxic Cr(III) organic compound, while Cr(III) glutamate was the least.     

Statistical interpolation model for the description of ground pollution due to the TCDD produced in the 1976 chemical accident at seveso in the heavily contaminated zone “A”;

The influence of NTA on chromium genetic activity was studied in the D7 strain of Saccharomyces cerevisiae. At low dose (subgenotoxic) of sodium chromate (CrVI) (5mM), NTA increased the point mutation while at higher dose (25 mM) of chromate in the presence of NTA a decrease of point reverse mutation was observed. Probably NTA affected either the uptake of Cr(VI) favouring the intracellular reduction to Cr(III), or the recombinational repair of DNA breaks induced by chromate oxiding activity.

An increase of point (reverse) mutation was obtained in the experiments with NTA and chromium chloride, suggesting the hypothesis that NTA might interact with Cr(III) forming NTA‐Cr(III) complexes able to permeate cellular membranes and bind to DNA. In addition NTA genetic inactivity was confirmed.     

Kinetics of hexavalent chromium reduction by iron metal

The kinetics of Cr(VI) reduction to Cr(III) by metallic iron (Fe⁰) was studied in batch reactors for a range of reactant concentrations, pH and temperatures. Nearly 86.8% removal efficiency for Cr(VI) was achieved when Fe⁰ concentration was 6 g/L (using commercial iron powder (< 200 mesh) in 120 min). The reduction of hexavalent chromium took place on the surface of the iron particles following pseudo-first order kinetics. The rate of Cr(VI) reduction increased with increasing Fe⁰ addition and temperature but inversely with initial pH. The pseudo-first-order rate coefficients (k _obs) were determined as 0.0024, 0.010, 0.0268 and 0.062 8 min^?1 when iron powder dosages were 2, 6, 10 and 14 g/L at 25°C and pH 5.5, respectively. According to the Arrehenius equation, the apparent activation energy of 26.5 kJ/mol and pre-exponential factor of 3 330 min^?1 were obtained at the temperature range of 288–308 K. Different Fe⁰ types were compared in this study. The reactivity was in the order starch-stabilized Fe⁰ nanoparticles > Fe⁰ nanoparticles > Fe⁰ powder > Fe⁰ filings. Electrochemical analysis of the reaction process showed that Cr(III) and Fe(III) hydroxides should be the dominant final products.     

Removal of chromium (VI) from water streams: a thermodynamic study

The chemical speciation of water solutions containing chromium oxyanions in various ratios is calculated (with the aid of available computer code Mineql Plus, Geochemist Workbench and Visual Minteq) and discussed. The effect of solution pH, total concentration of each species studied, and the presence of other species in solution are calculated and presented in the form of thermodynamic speciation diagrams. Electrochemical diagram (correlating pH to Eh) has been calculated as well. The conditions of Cr(VI) reduction to Cr(III) and the subsequent solid phase precipitation are given as a function of redox potential and pH. Surface reactions that might occur between the chromate ions in the solution and redox couples as Fe²⁺/Fe³⁺ on the surface of sorbents are also discussed. Moreover, experimental sorption data of hexavalent chromium onto synthetic magnetite are presented and discussed with the aid of the calculated diagrams. A mechanism is elucidated for the efficient removal of Cr(VI) from water streams at low pH values, and zeta potential data of synthetic magnetite with and without Cr(VI) are presented, which agree well with the mechanism proposed.     

Exposure-response of Cr(III)-organic complexes to <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Hexavalent chromium [Cr(VI)] bioreduction produces soluble Cr(III)-organic complexes. The Cr(III)-organic complexes are relatively stable once they are formed, and no data about their toxicity were reported. Therefore, this study aims to investigate the bioavailability and toxicity of the soluble Cr(III)-organic complexes. Saccharomyces cerevisiae L-1 wild type yeast strain was chosen as the model organism and Cr(III)-citrate was selected as the representative compound of the Cr(III)-organic complexes. The short-term chronic aquatic toxicity tests of the Cr(III)-citrate was explored by measuring growth inhibition, direct viable cell count, dry biomass, biosorption, and the amount of CO₂ production. Cr(III)-citrate exerted a toxicity of 51 mg/L with an EC ₅₀, which was calculated from the percent growth inhibition. These toxicity data would be helpful to define the toxic potential of the organo-chromium-III compounds in the environment.     

Role of the vitamin E model compound Trolox in the prevention of Cr(VI)-induced cellular damage

Given the wide industrial use of chromium (Cr) and its environmental contamination, chromium represents a risk to humans exposed to the metal. Considering that Cr(VI) is a potent oxidizing agent that increases intracellular oxidation and DNA damage, it would be worth considering the pretreatment of cells with antioxidants as a means of preventing Cr(VI)-induced toxicity. The objective of this study was to pretreat yeast cells with the water-soluble vitamin E analogue Trolox in an effort to increase cell tolerance against reactive chromium and reactive oxygen species formed during Cr(VI) reduction. Results revealed a decrease in Cr(VI)-induced cytotoxicity and mitotic gene conversions in Trolox-pretreated cells. The protective effect of Trolox in Cr(VI) induced genotoxicity was confirmed also with the prokaryotic Salmonella typhimurium SOS/umu test. Pretreatment of cells with Trolox (1) increased total Cr bioaccumulation, (2) decreased Cr(VI)-induced intracellular oxidation, (3) decreased Cr(V) persistence and (4) increased OH^? formation in yeast extracts. These findings might be useful in directing future investigations concerning the use of Trolox as a human antioxidant supplement, and in clinical applications related to Cr-induced genotoxicity in occupational and environmental situations where chromium is a problem.     

Chromium phytoextraction and physiological responses of the hyperaccumulator Leersia hexandra Swartz to plant growth-promoting rhizobacterium inoculation

● Improved Cr phytoextration efficiency was achieved by B. cereus inoculation. ● B. cereus could produce plant-beneficial PGPR factors at diverse Cr stresses. ● Enhanced resistance of inoculated L. hexandra towards elevated Cr stress. ● The majority of Cr existed in the stable forms in the tissues of L. hexandra. Phytoextraction is a promising option for purifying hexavalent chromium (Cr(VI))-laden wastewater, but the long remediation period incurred by poor growth rate of Cr hyperaccumulators remains a primary hindrance to its large-scale application. In this study, we performed a hydroponic experiment to evaluate the feasibility of promoting the growth and phytoextraction efficiency of Cr hyperaccumulator Leersia hexandra Swartz (L. hexandra) by inoculating plant growth-promoting rhizobacteria (PGPR) Bacillus cereus (B. cereus). In batch tests, the Cr(VI) removal rates of L. hexandra and B. cereus co-culture were greater than the sum of their respective monocultures. This was likely due to the microbial reduction of Cr(VI) to Cr(III), which is amiable to plant uptake. Besides, the PGPR factors of B. cereus, including indoleacetic acid (IAA) production, 1-aminocyclopropane-1-carboxylic acid deamination (ACCd) activity, phosphate solubilization capacity, and siderophore production, were quantified. These PGPR factors helped explain the biomass augmentation, root elongation and enhanced Cr enrichment of the inoculated L. hexandra in pot experiments. Despite the increased Cr uptake, no aggravated oxidative damage to the cell membrane was observed in the inoculated L. hexandra. This was attributed to its capacity to confront the increased intracellular Cr stress by upregulating both the activities of antioxidative enzymes and expression of metal-binding proteins/peptides. Moreover, L. hexandra could always conserve the majority of Cr in the residual and oxalic integrated forms with low mobility and phytotoxicity, irrespective of the B. cereus inoculation. These results highlight the constructed Cr hyperaccumulator-rhizobacteria consortia as an effective candidate for decontaminating Cr(VI)-laden wastewater.     

Response of indigenous Cd-tolerant electrochemically active bacteria in MECs toward exotic Cr(VI) based on the sensing of fluorescence probes

Electrochemically active bacteria (EAB) on the cathodes of microbial electrolysis cells (MECs) can remove metals from the catholyte, but the response of these indigenous EAB toward exotic metals has not been examined, particularly from the perspective of the co-presence of Cd(II) and Cr(VI) in a wastewater. Four known indigenous Cd-tolerant EAB of Ochrobactrum sp X1, Pseudomonas sp X3, Pseudomonas delhiensis X5, and Ochrobactrum anthropi X7 removed more Cd(II) and less Cr(VI) in the simultaneous presence of Cd(II) and Cr(VI), compared to the controls with individual Cd(II) or single Cr(VI). Response of these EAB toward exotic Cr(VI) was related to the associated subcellular metal distribution based on the sensing of fluorescence probes. EAB cell membrane harbored more cadmium than chromium and cytoplasm located more chromium than cadmium, among which the imaging of intracelluler Cr(III) ions increased over time, contrary to the decreased trend for Cd(II) ions. Compared to the controls with single Cd(II), exotic Cr(VI) decreased the imaging of Cd(II) ions in the EAB at an initial 2 h and negligibly affected thereafter. However, Cd(II) diminished the imaging of Cr (III) ions in the EAB over time, compared to the controls with individual Cr(VI). Current accelerated the harboring of cadmium at an initial 2 h and directed the accumulation of chromium in EAB over time. This study provides a viable approach for simultaneously quantitatively imaging Cd(II) and Cr (III) ions in EAB and thus gives valuable insights into the response of indigenous Cd-tolerant EAB toward exotic Cr(VI) in MECs.

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Cr (VI) and Fe (III) removal using Cajanus cajan husk

Husk of tur dal (Cajanus cajan) was investigated as a new biosorbent for the removal of Fe (III) and Cr (VI) ions from aqueous solutions. Parameters like agitation time, adsorbent dosage and pH were studied at different initial Fe (III) and Cr (VI) concentrations. The biosorptive capacity of the Tur dal husk was dependent on the pH of the chromium and iron solution, with pH 2 and 2.5 respectively being optimal. The adsorption data fit well with Langmuir and Freundlich isotherm models. The practical limiting adsorption capacity (qmax) calculated from the Langmuir isotherm was 96.05 mg of Cr(VI)/ g of the biosorbent at an initial pH of 2.0 and 66.65 mg/g at pH 2.5. The infrared spectra of the biomass revealed that hydroxyl, carboxyl and amide bonds are involved in the uptake of Cr (VI) and Fe (III) ions. Characterisation of tur dal husk has revealed that it is an excellent material for treating wastewaters containing low concentration of metal ions.     

Chromium sorption on grape stalks encapsulated in calcium alginate beads

A novel sorbent based on 2% grape stalk wastes encapsulated in calcium alginate beads was investigated for the removal of Cr(VI) and Cr(III) from aqueous solutions. The sorbent proved to be efficient for chromium removal in both hexavalent and trivalent forms from aqueous solution at pH 3.0. In binary mixtures, the presence in solution of one of the forms enhances the sorption of the other which results in an increase of the amount of total chromium removed. This study suggests that the proposed sorbent can be used for the removal of chromium from wastewaters through a cost-effective and environmentally friendly process.     

Evaluation of metabisulfite and a commercial steel wool for removing chromium(VI) from wastewater

Efficiency of metabisulfite and a commercial steel wool as reducing agents in the removal of Cr(VI) from wastewaters was evaluated. Chromium(VI) was converted to Cr(III), precipitated with NaOH, and removed by filtration. A reduction of more than 1.0 × 10⁵ and 4.0 × 10⁵ fold in total Cr and the Cr(VI) concentrations, respectively, was observed by employing steel wool masses as low as 0.4420 g to 30-mL solutions of wastewater. Chromium(III) hydroxide obtained after the treatment was recycled and used as marker in cattle nutrition studies. The liquid residue obtained after the treatment was reused as precipitation agent replacing NaOH.     

Biokinetic modeling of heavy metals in earthworms

Studies concerning bioaccumulation kinetics and bioconcentration factor (BCF) of heavy metals like zinc (Zn), lead (Pb), chromium (Cr), cadmium (Cd), and copper (Cu) in earthworm Eudrilus eugeniae tissues including integument, gizzard, clitellum, and head region were undertaken. Calculated BCF, predicted K _ow, and predicted K _oc showed a significant correlation between heavy metals in different earthworm tissues, in substrate spiked with heavy metals. The regression coefficient (r ²) between heavy metal uptake concentration and exposure time varied between 0.73 and 0.99, indicating significant correlation. The K _oc was a maximum of 13.9016 in case of Cu and integument at an exposure time of 100 days and a minimum of 0.1114 in case of Cr with respect to head at the same exposure time. Earthworms accumulated heavy metals following chronic exposure to municipal solid waste containing heavy metals. BCF and uptake rate kinetics of heavy metals were calculated and showed increased values in head tissue followed by integument.     

Preparation and characterization of <Emphasis Type="Italic">β</Emphasis>-FeOOH-coated sand and its adsorption of Cr(VI) from aqueous solutions

Batch adsorption experiments were conducted to explore the adsorption of Cr(VI) in aqueous solutions by β-FeOOH-coated sand. We investigated the key factors which affected the adsorption process such as adsorbent dosage, initial pH, initial Cr(VI) ion concentration, contact time and temperature. The uptake of Cr(VI) was very rapid and 44.3%, 51.6%, 58.9% of the adsorption happened during the first 180 minutes at 293K, 303K and 313K, respectively. The pseudo-second-order rate equation successfully described the adsorption kinetics. To study the adsorption isotherm, two equilibrium models, the Langmuir and Freundlich isotherms, were adopted. At 293K, 303K and 313K, the adsorption capacities obtained from the Langmuir isotherm were 0.060, 0.070 and 0.076 mg Cr(VI) per gram of the adsorbent, respectively. Thermodynamic parameters such as the change of energy, enthalpy and entropy were calculated using the equilibrium constants. The negative value of ΔG ⁰ and the positive value of ΔH ⁰ showed that the adsorption of Cr(VI) in aqueous solutions by β-FeOOH-coated sand was spontaneous, endothermic and occurred by physisorption.     

Role of NADH-dependent chromium reductases,exopolysaccharides and antioxidants by Paenibacillus thiaminolyticus PS 5 against damage induced by reactive oxygen species

ABSTRACT

Cr (VI) being used in various activities of industries and its improper treatment lead to contamination of environment. Among different methods, biological is the most efficient method to control pollution from soils affected with metals. Present study was designed to assess the role of Paenibacillus thiaminolyticus PS5 for adsorption, Cr (VI) reduction and mechanism of reduction. Sorption of chromium (VI) by strain PS5 was obtained by batch equilibrium method. Cr (VI) reduction in both free and immobilised cells were evaluated by 1,5-Diphenyl Carbazide method. The formation of biofilm by Paenibacillus thiaminolyticus PS5 was observed for colour change as well as quantified spectrophotometrically. Analysis kits were used to measure the amount of eDNA, superoxide and malondialdehyde (MDA). Metal resistant strain PS5 was characterised as P. thiaminolyticus using 16S rRNA gene sequence. Maximum biosorption of Cr (VI) by strain PS5 was found at pH 6–8 and 100–400 µgCr/mL within 24 hours of incubation. Complete reduction of Cr (VI) by strain PS5 was achieved at pH 6–8 and100–300 µg/mL Cr (VI). Paenibacillus thiaminolyticus PS5 immmobilisation on sodium alginate and polyvinyl alcohol facilitated complete reduction of Cr (VI) within 18 hours due to the formation of more biofilm under metal stress conditions. Strain PS5 reduced almost all Cr (VI) into Cr (III) in supernatant, most of which was immobilised by cell debris. Experiments confirmed the reduction of Cr (VI) by cytosolic cell-free extracts which is a mechanism of detoxification. The release of exopolysaccharides and antioxidants by strain PS5 played a protective role against cell damage by Cr (VI) as Cr (VI) could not release the significant amount of eDNA, superoxide and MDA. The present study proved strain PS5 to be a super bioinoculant which has great capacity for adsorption, biotransformation and can activate cytosolic reductases, exopolysaccharides and antioxidants against oxidative damage.     

Role of GSH in the amelioration of chromium-induced membrane damage

Membrane damage is one of the important consequences of chromium (Cr), an environmental toxicant, induced cytotoxicity. Reduced glutathione (GSH), a membrane protectant may be used to reduce the Cr-induced membrane damage. In the present study, the impact of Cr in presence and absence of GSH was studied on plasma membrane of the liver and kidneys in male Wistar rats. Significant increases in membrane cholesterol levels as well as significant decreases in membrane phospholipid levels in Cr exposed (0.8 mg per 100 g body weight, i.p., for 28 days) animals suggest structural alterations in both the liver and kidney plasma membranes. Alkaline phosphatase (ALP), total ATPase, and Na⁺–K⁺–ATPase activities of plasma membrane were significantly decreased in both the liver and kidneys after Cr treatment. This treatment also produced significant weight loss and increased Cr content in the liver and kidneys. However, GSH (8 mg per 100g body weight, i.p., daily at an interval of 6 h after injection of Cr for a period of 28 days) supplementation restored alterations induced by Cr in plasma membrane of both the liver and kidneys but was not able to eliminate the deposited Cr from the liver and kidney tissues.     

Impact of organic manure on the phytoremediation potential of Vetiveria zizanioides in chromium-contaminated soil

Chromium is a pollutant present in electroplating waste water and its removal is necessary for the protection of the environment. Vetiveria zizanioides (VZ) was grown in chromium effluent concentrations of 50, 100 and 200 mg kg ^?1 soil amended with organic manure and the potential for phytoremediation was determined. The amounts of Cr in plant tissues (root and shoot), soil and percentage electrolyte leakage of VZ roots were analysed. From the results, VZ amended with organic manure showed the greatest potential for Cr removal because of its faster growth and larger biomass achieved over the whole length of the experiment. In this study, 92.25% Cr removal efficiency was obtained with a Cr concentration of 50 mg kg ^?1 soil and removal efficiencies of 90.5% and 85% were obtained with 100 and 200 mg kg^?1, respectively after a period of two months of VZ growth.