Being established in 1988 in the vicinity of Isfahan city, Mubarakeh Steel complex has imposed adverse environmental and health effects within the area. The study area is covered by lots of farms through which major crops like wheat and rice are provided.Considering the imposed pollution load of the complex, the current study has monitored the concentration of metals Fe, Al, Cd, Cr, Ni, Pb, Cu, Zn, Mn, Co, Mo, As in 14 soil samples within the study area. Furthermore, human health hazards of mentioned metals due to consumption of domestic rice and wheat have also been evaluated through different scenarios. In order to evaluate the mobility of metals in soil samples the sequential chemical analysis is performed.Regarding the accumulation of metals in loose phases the order of metals bioavailability risk level is estimated to be as follows:Co > Cd > Mo > Ba > As > Pb > Mn > Cu > V > Zn > Cr > NiAn index approach is also considered to evaluate the severity of metal contamination. Regarding geochemical accumulation index, only cadmium is detected to be in a moderately contaminated status while other metals declare an unpolluted condition. Index of pollution pays more attention to mobility potential of metals and accordingly detects metals Co, Mn, As, Pb, Cd, Ba and Mo to be in a moderately contaminated level. On the other hand, enrichment factor declares all toxic metals except for Co, Ba and V to be enriched.Considering human health hazard assessment, except for Fe, Ba, Cu and Zn, all metals intakes in different scenarios are considered as hazardous while their CDI values are much more than the respective oral reference doses. Metal contaminated sites contain a potentially hazardous risk for human beings and the environment. The bioavailability of such pollution is sophisticatedly used as an indicator of potential risk , 2. MetaKnowledge on metals bulk concentrations alone may not be sufficient enough to evaluate the adverse effects of contaminated soils while toxic metals are present in different chemical forms in soil , which determine their mobilization capacity and bioavailability –5.Knowledge of metal’s speciation may help to evaluate the metal tendency to remain in soil or sediments and how easily it may be released into the water phase.Sequential extraction procedures are among the most widely used methods through which several extractants are used consecutively to separate the portion of phases from the bulk metallic concentration in a soil sample.A vast variety of extraction processes providing information about the strength of bonds between metal species and soil fractions have been used by different researchers , 6–8. HoBeing established in 1988 in the vicinity of Mubarakeh county, Mubarakeh Steel complex has imposed adverse environmental and health effects within the area. A remarkable increase in the number of reported disease cases in Mubarakeh and Zarrinshahr in recent years may be attributed to the mentioned complex activities. The study area is covered by lots of farms through which major crops of Isfahan province are provided. The study area within Iran is demonstrated in Fig. Considering the imposed pollution load of the complex, the current study has monitored the concentration of metals iron, aluminum, cadmium, chromium, nickel, lead, copper, zinc, manganese, cobalt, molybdenum and arsenic in soil samples within the study area.The current study presents not only the bulk concentration of eight metals  in superficial soil samples of Mubarakeh steel complex vicinity, but also the chemical partitioning of mentioned elements. The latter findings create a clear view of metals potential mobility and consequently the tendency to be absorbed by plants or animals within the bioaccessibility phenomenon. For this study, a modified sequential extraction procedure has been considered , 11.Soil samples were collected from 14 stations in the vicinity of Mubarakeh steel complex during the summer of 2012 using a Peterson grab sampler . Being powdered using an agate mortar and pestle, about 0.5 g of the sample was placed in a Teflon beaker containing 10 mL aqua regia.3:H2SO4:HClO4 1:1:5 was added to 1 gram of powdered rice/wheat and heated to 80° centigrade. The mixtures were further cooled and filtered before injection process [The mixture was heated until most of the liquid had evaporated, and allowed to cool before 5 mL of hydrogen fluoride (HF) were added. The samples were further cooled to room temperature before being filtered. The filtrates were transferred to 50 mL volumetric flasks and brought to volume with 1 N HCl . Slightl process .The analysis of metals in solutions was carried out by an inductively coupled plasma atomic emission spectrometer. Certified reference material (CRM 320) was used to check the accuracy of the analytical procedures. Replicate analysis of this CRM showed an acceptable accuracy, with recovery rates for metals between 92 % and 105 %. Furthermore, two samples were analyzed in triplicate to determine the precision of the analytical processes. The average values of the variation coefficients were estimated to be less than 10 % which may be considered acceptable for such studies .To have an estimate of background status, according to soil samples collected from the vicinity of Mubarakeh steel, metals concentrations in shale have been considered. Such assumption is normally used as background values in sediment studies –16.In order to determine the value of metal concentrations in different sedimentary phases, the modified sequential chemical partitioning method was considered .To evaluate the severity of metallic pollution within the study area, geochemical accumulation index, enrichment factor and index of pollution were taken into consideration.An index approach was used to assess the severity of metal contamination in the vicinity of Mubarakeh steel complex. Accordingly, the geochemical accumulation index was calculated using:Where Igeo stands for geochemical accumulation index, Cn is the bulk metal concentration and Bn is the average metal concentration in shale , 18.The enrichment factor (EF) values was calculated for metals using:Where (Cn/CFe) sample indicates the ratio of the concentration of a specific element (Cn) to that of Fe (CFe) in soil sample and (Cn/CFe) crust is the same ratio in an unpolluted reference sample .Finally, index of pollution which is a modification of Igeo is referred by the following formula:The study area is a major agricultural site in the province and crops like wheat and rice are widely cultivated within the region. In order to evaluate the probable crops metal uptake, metals concentration in eight different rice and 15 different wheat samples were measured.To evaluate the hazard level threatening local habitants due to consumption of rice and wheat products, nutritional pattern and physiological characteristics of residents should be taken into consideration. Accordingly six different scenarios are defined Tables  and 3.TaChronic daily intakes of different metals within the framework of defined scenarios are compared with respective chronic oral reference doses to assess the existent hazards. Details for exposure assessment and hazard analysis are fully described within the literature .Total concentration of mentioned metals in soil samples collected from the vicinity of Mubarakeh steel complex are shown in Table In comparison with average shale values, all metals show lower concentrations except for Cd. Average cadmium concentration within the study area is more than five times higher than that of shale which indicate an intensified enrichment. On the other hand, although the concentration of other metals is lower than that of shale, such condition should not be considered as a low-risk and safe one. Potential mobility of existing metallic concentration in soil should be regarded before any judgment.The results of chemical partitioning for metals are demonstrated in Figs. The percent of metals concentration bound to different phases are grouped in a descending order as follows:Loose ions :Ba (31.53) > As (29.17) > Cd (26.73) > Pb (25.82) > Mo (20.37) > Co (19.21) > Mn (12.23) > Cu (10.98) > Zn (8.27) > V (4.68) > Ni (3.53) > Cr (2.48)Sulfide ions :Mn (36.97) > Co (30.51) > Ba (25.88) > Cd (20.26) > Pb (19.22) > As (17.48) > V (10.92) > Zn (8.96) > Mo (7.74) > Cu (3.67) > Ni (3.36) > Cr (1.25)Organic ions :Mo (35.39) > Co (26.30) > Cd (26.10) > Cu (16.12) > Cr (13.72) > V (12.33) > Pb (7.48) > As (6.38) > Ni (4.65) > Ba (3.75) > Mn (3.18) > Zn (1.15)Residual ions :Ni (88.41) > Cr (82.55) > Zn (81.61) > V (72.07) > Cu (69.23) > Mn (47.62) > Pb (47.48) > As (46.97) > Ba (38.84) > Mo (36.50) > Cd (26.91) Co (23.98)In order to have an overall estimate of metals mobility potential, the results of the sum of loose, sulfide and organic ions are grouped as the anthropogenic portion of metals are shown in Table As was explained in methodology, IPOLL, Bc, and Lp refers to pollution intensity, bulk concentration, and lithogenous portion, respectively. Average indices values for each metal in different stations are shown in Table The result of metal concentration in agricultural crops (wheat and rice), are shown in Tables According to defined scenarios in part of material and methods, respective chronic oral reference doses to assess the existent hazards are shown in Tables As it is seen except for Fe, Ba, Cu and Zn, all metals intakes in different scenarios are considered to be hazardous while their CDI values are much more than the respective oral reference doses.Evaluating the metallic pollution of soil and the potential mobility of metal species in the vicinity of Mubarakeh steel complex is considered in this study. Fourteen soil samples were collected and analyzed in summer 2012.Bioavailability and health risks linked to toxic metals are tightly related to the mobility of metal species. aqueous organisms and plants are more vulnerable towards free ions and labile complexes , 21–23. Sum of loose, sulfide and organic phases in bulk metallic concentration is considered as manipulated contamination. The higher the anthropogenic portion of a metal is, the higher the mobility risk would be occurred. Generally, the mean anthropogenic portions of the metals, as a percent of their bulk concentrations, obey the following pattern:Co > Cd > Mo > Ba > As > Pb > Mn > Cu > V > Zn > Cr > Ni.Considering index approach, Igeo deals with bulk concentration and is constructed upon the principle of comparison with shale values. As it is expected, only cadmium is detected to be in a moderately contaminated status regarding this index while other metals declare an unpolluted condition. Index of pollution pays more attention to mobility potential of metals and accordingly detects metals Co, Mn, As, Pb, Cd, Ba and Mo to be in a moderately contaminated level. On the other hand, enrichment factor which compares the bulk concentrations with earth crust values declares all toxic metals except for Co, Ba and V to be enriched.Considering human health hazard assessment, all toxic metals intakes in different scenarios are considered to be hazardous. The status is much more severe for scenarios dealt with children eating more portions. It must be noted that current hazard levels are calculated as single exposures and for more realistic conditions cumulative exposures should be taken into consideration. In other words, a variety of ingredients  exists in diurnal diet of residents within the study area and the overall hazard would exceed the above-mentioned levels.


Picea abies and Pinus sylvestris branches as well as the effect of dynamic bending on the vulnerability. We hypothesized this mechanical stress to substantially impair xylem hydraulics. Intense static bending caused an only small decrease in hydraulic conductance (−19.5 ± 2.4% in P. abies) but no shift in vulnerability thresholds. Dynamic bending caused a 0.4 and 0.8 MPa decrease of the water potential at 50 and 88% loss of conductivity in P. sylvestris, but did not affect vulnerability thresholds in P. abies. With respect to applied extreme bending radii, effects on plant hydraulics were surprisingly small and are thus probably of minor eco-physiological importance. More importantly, results indicate that available xylem hydraulic analyses (of conifers) sufficiently reflect plant hydraulics under field conditions.The xylem hydraulic efficiency and safety is usually measured on mechanically unstressed samples, although trees may be exposed to combined hydraulic and mechanical stress in the field. We analysed changes in hydraulic conductivity and vulnerability to drought-induced embolism during static bending of  Tree life is based on the formation of wood, which fulfils manifold functions. Metabolic processes and storage pools are provided by parenchyma cells, while dead cells with lignified cell walls facilitate the formation of complex axis systems. The latter enable trees to withstand gravitational and dynamic forces and to transport water into the crown. In angiosperms, the hydraulic function is provided by vessels, while fibre cells withstand self-loading and compression as well as tension forces. In contrast, tracheids fulfil both hydraulic and mechanical function in conifer xylem. Angiosperm and conifer conduits are connected by countless pits and are thus part of a complex transport network within the wood  induced by transpiration and transmitted by continuous water columns to roots and soil Boehm . Under tet al. et al. et al. et al. In situ, axes xylem is often exposed to combinatorial stresses and has to withstand various mechanical forces during drought or freeze-thaw stress. For instance, wind may cause enormous dynamic loads, and snow  within tropical trees. Spicer & Gartner  decrease in sapwood conductivity after deflection were found  and vulnerability to embolism when thawed samples were measured after the bending/freezing treatment. Please note that in all studies mentioned, hydraulic parameters were analysed after exposure to mechanical stress while an analysis of changes in hydraulic efficiency and safety at the time of mechanical stress, to our knowledge, is lacking.Mechanical stress may have consequences for xylem hydraulics at two levels. Firstly, plants react (in the long term) on mechanical stress by changes in xylem structures Telewski , which, et al.  and Chri  Bending exposes the xylem to tensile and compressive forces leading to deformation of conduits. Changes in conduit shape may affect the hydraulic conductivity as it changes with the fourth power of the conduit radius . We chose conifers because of the close link between hydraulic and mechanical function in their tracheid system (see above). We hypothesized that mechanical stress would affect xylem structures and/or the water column and thus result in reduced hydraulic efficiency and safety.In the present study, we therefore tested the direct effects of static bending on the hydraulic conductivity and direct effects of static and dynamic bending on the vulnerability to drought-induced embolism of two conifer species (P. abies L. Karst) and Scots pine (P. sylvestris L.) were harvested, wrapped in plastic bags and transported to the laboratory. Branches were re-cut under water and rehydrated for at least 24 h. Up to 25-cm-long segments of the main shoot with a mean diameter of 7.3 ± 0.6 mm (P. abies) and 7.3 ± 0.5 mm (P. sylvestris) were used for conductivity measurements during static bending. For vulnerability analyses (static and dynamic bending), intact branches with a basal diameter of up to 1.5 cm and a length of up to 120 cm were taken.In a mixed conifer forest in Natters, Austria , south-exposed branches of Norway spruce (Static bending for conductivity measurements (hydraulic efficiency) was performed with plastic tubes with radii of 80, 62.5 and 55 mm Fig. a. Fully Static bending for vulnerability analysis (hydraulic safety) was performed with a tube of 80 mm radius, mounted to a metal frame. Central sections of branches were bent around the tube and branch base and tip fixed on the frame with clamps Fig. b. BranchFor dynamic bending, a metal holder allowing bending of five branches in parallel was constructed. The base of branches was fixed in metal tubes and branches connected to a lever arm  with a chain. The chain was fixed on branches with a cable strap whereby branches were protected by a foam sleeve. The lever was moved forward and backward by a wiper  powered by a car battery. Branches were bent about 39 times per minute. The maximum deflection radius was about 67 cm whereby no bending stress acted on branches when the wiper arm reached its initial position. Branches were dehydrated to various extents while bending (for 1 week at maximum). As a control for time effects (branches dehydrated longer were moved over a longer period), some fully hydrated or slightly dehydrated branches were wrapped in plastic bags and moved for up to 7 d without dehydration.Bending of branches was always achieved according to their position in the field. Therefore, the compression wood side  was oriented to the centre of the bending circle. It was not possible to analyse the hydraulic efficiency during dynamic bending as required branch length would not allow measureable flow rates and movement of branches would cause biased flow patterns.μm) and degassed water containing 0.005% (v/v) ‘Micropur Forte MF 1000F’  to prevent microbial growth  and re-cut under water several times prior to the experiment Fig. a. The baThe water potential (Ψ) of branches from static or dynamic bending experiments was determined on up to 10-cm-long end twigs with a Scholander apparatus . Three twigs per branch were measured and Ψs averaged.et al. et al. (μm) and degassed water containing 0.005% (v/v) ‘Micropur’ (see above). Flushing was repeated until measurements showed no further increase in conductivity. PLC was calculated from the ratio of initial to maximal conductance  were fitted with an exponential sigmoidal equation (Pammenter & Vander Willigen a is a constant related to the curve slope. Ψ50 corresponds to Ψ at 50% PLC. Data (average PLC versus average Ψ of each branch) of treatments were pooled for construction of vulnerability curves. Curves were calculated using Fig.P 2.98 software . Ψ at 12 and 88% PLC  were calculated from Ψ50 and parameter a, calculated standard errors refer to the regression fitting.where PLC is the loss of conductivity (%), Ψ is the corresponding water potential, and ks):From maximum flow rates of unbent samples, we also calculated the specific hydraulic conductivity (Q is the volume flow rate (m3 s−1), l is the segment length (m), Ac is the xylem cross-sectional area , and ΔP is the pressure difference between the segment ends (Pa). As flushing of samples with high PLC sometimes cannot completely restore the maximum conductivity, only samples with PLC < 60% were used for ks determination. Measurements were corrected to 20 °C to account for changes in fluid viscosity with temperature.where t-test after testing for Gaussian distribution (Kolmogorov–Smirnov test) and variance homogeneity (Levene test) of data. Vulnerability parameters were tested with the Welch's test. All tests were performed pairwise at a probability level of 5%.Conductivity measurements were performed on six branches per species. Vulnerability analysis was based on up to 27 branches per species and treatment, respectively. For control measurements, six to seven (static bending) and three (dynamic bending) branches were used per species. Results of conductivity measurements and vulnerability parameters are given as mean ± SE. Differences in conductivity were tested with paired Student's −1.Applied bending stress intensities were high and in some cases exceeded the mechanical resilience of branches, which broke during experiments. These branches were excluded from further measurements and analyses. The maximum force (depending on the size and elasticity of branches) for static and elastic bending of branches was 10–20 N and 8.5–18.5 N per branch, respectively. For dynamic bending, the branch was moved from its initial to maximally bent position in about 0.77 s. The deflection was 18 cm and thus approximately 34.6% , which gives a deflection rate of approximately 45% sP. abies had slightly lower ks (9.3 ± 1.0.10−4 cm2 s−1 MPa−1) than P. sylvestris (10.1 ± 1.1.10−4 cm2 s−1 MPa−1). In both species, bending caused a decrease in hydraulic conductance, with P. abies showing a more pronounced effect . P. sylvestris showed a similar trend but due to higher variation between samples, differences in conductance were not significant. When samples were unbent, the conductance increased but was overall 10% lower than before bending treatments , we did not observe major implications upon bending of branches: the hydraulic efficiency was hardly affected by static bending. We observed a significant but small decrease (−20%) in conductance only in es Table . No signn et al. . OverallP. abies and in P. sylvestris, which indicates that relevant structures, like the pits, were not substantially deformed and could maintain their function for hydraulic safety. Observed vulnerability thresholds thereby were similar to values published in other studies  was in the range of natural swaying of conifers. Rudnicki & Burns (−1 in Pinus strobus branches and Moore & Maguire (P. abies and P. sylvestris trees (0.39 and 0.35). Spatz et al. (−1 in P. menziesii branches (0.6–1.6 m in length). However, within bending cycles of our dynamic experiments, deflection rates were high due to the strong bending. We reached a deflection rate of 45% s−1 (see Results) while, for example, Christensen-Dalsgaard & Tyree (−1 (1 cm deflection at 15 cm sample length). In conclusion, our experiments simulated high mechanical stress intensities. From an eco-physiological point of view, observed hydraulic effects might thus be of relevance only during extreme situations such as storm events or extreme winter precipitation. Moderate wind speeds or moderate snow loads will not affect the hydraulic efficiency and safety.Please note that branches in our experiments were exposed to high mechanical stress intensities with bending radii down to 55 mm  and 67 cm (dynamic bending 1.5 cm diameter). For example, Bründl et al.  reported & Burns  reported Maguire  showed az et al.  reported & Tyree  used a det al. et al. et al. et al. et al. et al. Probably more important is a methodical point of view: there are countless vulnerability analyses available (e.g. Cochard


Catla catla sampled from ten sites of Ravi River between its stretches from Shahdara to Head Balloki were studied to know the level of contamination of the selected pesticides by GC-ECD method. All fish samples were found contaminated with different concentrations of DDT, DDE, endosulfan, and carbofuran; however, DDT and DDE concentrations were more than the maximum residue limits (MRLs) about food standards, while endosulfan sulfate and cartap were not detected. Pesticide concentrations in the fish flesh were ranged from 3.240 to 3.389 for DDT, 2.290 to 2.460 for DDE, 0.112 to 0.136 for endosulfan, and 0.260 to 0.370 μg g−1 for carbofuran. The findings revealed that the pesticide concentrations in the fish flesh decreased in the order: DDT > DDE > carbofuran > endosulfan. After Degh fall and After Hudiara nulla fall river sampling sites were found severely contaminated. It is proposed that a constant monitoring programs are needed to be initiated to overcome the present alarming situation.The levels of dichlorodiphenyltrichloroethane (DDT), dichlorodiphenyldichloroethylene (DDE), endosulfan, endosulfan sulfate, carbofuran, and cartap which were estimated in the flesh of  Persistent organic pollutants (POPs) are a major group of hazardous chemicals having anthropogenic origin and three main characteristics: persistence, bioaccumulation, and long range transport . In the Ravi River is the smallest among the five main eastern tributaries of the Indus, which takes its origin from India and in Pakistan it enters near village Tadyal, Kot Naina, Tehsil Shakargar of Sialkot. After flowing about 560 km, it joins River Chenab near village Sayyal Faqir, Sidhnai, Tehsil Kabirwala. In addition to its surface run-off upstream water, it also receives water from the Qadir Abad link canal and upper Chenab canal between its stretches from Shahdara to Balloki Headworks. Raw domestic sewage and untreated industrial effluents originating from the cities of Lahore, Sheikhupura, and Kalashahkaku are dumped into the Ravi River by main tributaries of Hudiara drain and Degh fall. Hudiara drain originates from Batala in Gurdaspur district, India, and enters into Pakistan at village Laloo which is approximately 44 km away from Lahore city. Catla catla in Ravi river which is one of the indigenous fish in the Indo-Pak regions. The results obtained may be made use of as a baseline data in developing effective remedial measures to improve the water quality status of the river.Keeping in view the environmental significance of organochlorine and carbamates in Pakistan context and highly polluted status of the Ravi River, the study was undertaken to assess the residual presence of selected pesticides the flesh of Catla catla from predetermined locations nearby Head Balloki in triplicates.The Ravi River along the India-Pakistan border meanders substantially in the alluvial plains of the Amritsar and Gurdaspur districts of Punjab before entering Lahore, Pakistan. The pollution levels in the river discharge are reportedly very high, which is attributed to careless disposal of large amounts of industrial and agricultural wastewater and faulty drainage system in both countries. A 72 km stretch of the Ravi River from Lahore Siphon to Bulloki Headworks indicates heavy contamination of the water and sediment with different pesticides. The river sediments are highly contaminated and have become a secondary source of pollution of the river water, even though some controls over unauthorized discharges into the river have been checked. The effect of pollution was evaluated on fish samples of Catla catla, fish samples were taken for one year on a fortnightly basis from ten sampling sites along right and left banks of Ravi River between its stretches from Shahdara to Head Balloki, namely, River site (R1): Shahdara bridge downstream, right bank (R B), R2: Shahdara bridge downstream, left bank (L B), R3: After Farrukhabad nulla fall (R B), R4: After Bakar mandi nulla (L B), R5: Before Degh fall (R B), R6: Before Hudiara drain (L B), R7: After Degh fall (R B), R8: After Hudiara drain (L B), R9: Balloki Headworks upstream (R B), and R10: Balloki Headworks upstream (L B), respectively. Map of the study area with sampling sites is presented in To find out the organochlorine  and nitrogen containing pesticide residues (carbofuran and cartap) inThe extraction and cleanup of dichlorodiphenyltrichloroethane (DDT), dichlorodiphenyldichloroethylene (DDE), endosulfan, endosulfan sulfate, carbofuran, and cartap in fish samples were done with some modifications in the method .Calibration curves of all standard pesticides were prepared with the help of computer software Turbochrome  and limit of detection was calculated by computer software Super cal-5. LOD for the OPs—was 0.1 ng/g lipid, and the limit of quantification (LOQ) was 0.3 ng/g. Quality-assurance measures applied in the laboratory included rigorous contamination-control procedures (strict washing and cleaning procedures), monitoring of blank levels of solvents, equipment and other materials, analysis of procedural blanks, recovery of spiked standards, monitoring of detector response and linearity, and analysis of a reference material.1 L (extract 1). 50 mL of acetonitrile was again added to the mixture and shaken for 2 minutes, composite was taken, and 10 g of anhydrous sodium sulpate (Merck) was added to disintegrate it. A mixture of fish flesh and anhydrous sodium sulphate was blended and mixed with a spatula until both were well mixed. A 20 mL of hexane (Merck) was added to the mixture, followed by the addition of 100 mL of acetonitrile  saturated with hexane. The mixture was shaken for 2 minutes. Acetonitrile was decanted into a separatory.For further two times after adding 50 mL of acetonitrile followed by shaking for 2 minutes in both cases, acetonitrile was then drained into separatory funnels (extracts 3 and 4). All extracts were combined and 500 mL of distilled water was added to wash them. A 40 mL of saturated sodium chloride (Merck) solution and 50 mL of hexane were added to the extract. Separatory funnel was kept in a stand until two distinct layers were formed. The aqueous layer was drained into another 1L separatory funnel. A 50 mL of hexane was added to this aqueous layer (hexane extract 1). The mixture was again extracted with 50 mL of hexane (hexane extract 2). Combined hexane extracts (hexane extracts 1 and 2) were passed through a plug of 10 g of anhydrous sodium sulphate. The solvent (hexane) was evaporated using a rotary evaporator (Buchi-R-200) to get the residue. A 10 mL of methanol (Merck) was added to the residue and diluted by adding 25 mL of distilled water.Solution of sample extract was transferred on to preconditioned C-18 solid phase extraction bond column. Pesticide residues were recovered by eluting the column with 5 mL portions of hexane for three times. Elute was concentrated to 2 mL using rotary evaporator and the concentrated elute was now ready for injection into a gas chromatograph .2) at the flow rate of 30–32 mL/minute and with variable temperature arrangements (injector temperature (220°C); oven temperature 150°C maintained for 4 minutes, then raised to 290°C at a rate of 8°C/minute, and then hold for 10 minutes; detector temperature: 300°C). Gas chromatograph was turned on for the detection and quantification of pesticides. First of all 1 μL standard solution of pesticides under this study was injected and their retention times were determined. Calibration curves of all standard pesticides were prepared with the help of computer software Turbochrome and limit of detection was calculated by computer software Super cal-5 . After running standard solutions of pesticides, 1 μL aliquot of concentrated elute of fish flesh was injected. Residue peak(s) of elute(s) injected was identified on the basis of retention time. The retention time of all test solutions was within ±2% of standard pesticide solutions. Height of residue peak(s) was measured, and residue amount of test solution was determined, by comparison with the height/area obtained from a known amount of appropriate reference/standard solution in the chromatograms.The determination of 6 pesticides in fish flesh was detected by following multiresidue method  using ga Catla catla was analyzed by using two-way classification . Analysis of Variance and Duncan's Multiple Range tests was performed to analyze differences between the parameters under study [Data on pesticide residue concentrations iner study . Catla catla fluctuated between a minimum of 3.240 ± 0.0274 μg g−1 at Before Hudiara nulla fall (RB) and maximum of 3.389 ± 0.0166 μg g−1 at After Degh fall (RB) river sampling sites, respectively. The difference between these two sites, for the concentration of DDT in fish muscle, was statistically significant (P < 0.05), while the differences between other river sampling sites, Bakar Mandi nulla fall (LB), Before Hudiara Nulla fall (LB), After Hudiara nulla fall (LB), Balloki Headworks (LB), Shahdara bridge (RB), After Farrukhabad nulla (RB), before Degh fall (RB), and Balloki Headworks (RB), were statistically (P > 0.05) nonsignificant (P < 0.05). The lowest mean concentration of 3.041 ± 0.010 μg g−1 of DDT in Catla catla was detected during the month of September 2009, while the highest mean concentration of 3.351 ± 0.004 μg g−1 of DDT in Catla catla was detected during the month of December 2009, respectively (P < 0.01).Mean annual DDT concentrations innificant . The difectively . The dif Catla catla collected from Balloki Headworks (RB) river sampling site showed minimum mean annual DDE concentration of 2.290 ± 0.0196 μg g−1, while at After Degh fall (RB) river site maximum mean annual DDE concentration of 2.460 ± 0.0340 μg g−1 was detected. The difference between these river sampling sites, for the toxicity of DDE, was statistically highly significant (P < 0.01). The lowest mean concentration of 2.180 ± 0.015 μg g−1 of DDE in Catla catla was detected during the month of September 2009, while the highest mean concentration of 2.509 ± 0.014 μg g−1 of DDE in Catla catla was detected during the month of December 2009, respectively (P < 0.01).ectively . The dif Catla catla was detected as 0.112 ± 0.003 μg g−1 at the Shahdara bridge (LB), whereas maximum mean annual concentrations of endosulfan (0.136 ± 0.0031 μg g−1) was detected at After Degh fall (RB) river sampling site, respectively (P < 0.01). The lowest mean concentration of 0.099 ± 0.002 μg g−1 of endosulfan was detected in Catla catla during the month of August 2010, while the highest mean concentration of 0.137 ± 0.003 μg g−1 of endosulfan in Catla catla was detected during the month of January 2010, respectively (P < 0.01).Mean annual minimum endosulfan concentration inectively . The difectively . The dif Catla catla from all the ten selected sampling locations of the Ravi river.Endosulfan sulfate is a persistent environmental metabolite of endosulfan, an organochlorine insecticide-acaricide. Toxicity is partly dependent on the manner with which the pesticide is administered. Undiluted endosulfan is slowly and incompletely absorbed into the body whereas absorption is more rapid in the presence of alcohols, oils, and emulsifiers.This organochlorine pesticide was not detected in fish muscle of Catla catla fluctuated between a minimum of 0.260 ± 0.0158 μg g−1 at the Shahdara bridge (LB) and maximum of 0.370 ± 0.0191 μg g−1 at After Degh fall (RB) river sampling sites, respectively. The difference between these two rivers sampling sites, for the toxicity of carbofuran, was statistically (P < 0.01) highly significant .The mean annual carbofuran concentrations innificant . The lowectively . The dif7H15N3O2S3HCl). Cartap is essentially a contact and stomach poison. It is used for the control of chewing and sucking pests and results in insect paralysis. It has been categorized as a high-effectiveness, low-toxicity, and low-residue pesticide used in rice and sugarcane fields. Cartap was not detected in fish muscle of Catla catla from all the ten selected sampling locations of the Ravi river.Cartap hydrochloride is a nereistoxin analog and is a commonly used low-toxicity insecticide. It is commonly used as a hydrochloride . Out of ten sampling sites on Ravi river, After Degh fall river sampling site (RB), was found with the highest pesticide contamination level in fish samples followed by After Hudiara drain fall river sampling site (LB). Catla catla collected from After Degh fall river sampling site (RB) showed the highest contamination level of 3.389 ± 0.0166 μg g−1 of DDT, 2.460 ± 0.0340 μg g−1 of DDE, 0.136 ± 0.0031 μg g−1 of endosulfan, and 0.370 ± 0.0191 μg g−1 of carbofuran, respectively. The maximum level of pesticide contamination at after Degh fall river sampling site (RB) is due to the entry of Degh fall nulla into Ravi River. After Degh fall river sampling site (RB) was found severely contaminated by pesticides probably this tributary is bringing a lot of untreated waste from various sources. This is supported by a study on the chemical contaminants in water and sediments of Degh nulla by [In the present study fish samples ofnulla by , who repnulla by , but DDTnulla by . The finnulla by  are in lnulla by . The levnulla by . No pres Catla catla. The above findings are in agreement with the findings of [In the present study, maximum contamination levels of pesticides in fish samples were detected during the months of low water supply (dry season) of the year, especially December 2009 and January–May 2010, when less water was flowing in Ravi River. It is due to the fact that, during these low water supply months, pesticides get concentrated in water and sediments of river bodies and chances for the contamination of fish and other flora and fauna increase and maximum level of pesticide was detected in muscle ofdings of . Referendings of  also repdings of , accumulOrganochlorine water contamination pathways in the Ravi river are likely to be nonpoint sources, including runoff, atmospheric deposition, and leaching due to agricultural applications and vector control practices. Sediments can act as a sink for persistent contaminants, whose resuspension at the sediment-water interface, especially in storm events and during river mixing, increased pesticide bioavailability and accumulation in the food chain in particular DDT and DDE. Pesticide pollution is, therefore, likely to pose a threat to both aquatic organisms and humans. Generally, it is believed that contaminants taken in by aquatic organisms are from water, rather than from their food, and may vary with seasonal variation in contaminant availability within the water column . There wIt was concluded that pesticide toxicity level of Ravi River is due to the pesticide toxicity levels of its tributaries. These tributaries carry a huge burden of concentrated industrial, domestic, and agricultural waste substances and pour them as such into the main river water. Unfortunately, before the fall of these tributaries into the Ravi river, no treatment for the removal or breakdown of pollutants is done. The stretch of Ravi River from Shahdara to Head Balloki has been found polluted and rendering the water unfit for aquatic life. Awareness should be developed among the general public through the print and electronic media so that they may realize the harmful effects of pesticides and stop/reduce their use. Currently, a constant monitoring program needs to be initiated to reform the present situation.


Little is known about how this conversion alters soil carbon (C) and nitrogen (N). We selected three sites, each with three plots arrayed along the bamboo encroachment pathway: moso bamboo forest (BF); transition zone, mixed forest plots (MF); and broad-leaved forest (BLF), and examined how bamboo encroachment affects soil organic C (SOC), soil total N, microbial biomass C (MBC), microbial biomass N (MBN), water-soluble organic C (WSOC), and water-soluble organic N (WSON) in three forests. Over nine years, moso bamboo encroachment leads to a decrease in SOC and total soil N, an increase in MBC and WSOC, and a decrease in MBN and WSON. Changes in soil C and N occurred mainly in the topsoil. We conclude that moso bamboo encroachment on broadleaved forest not only substantially altered soil C and N pools, but also changed the distribution pattern of C and N in the studied forest soils. Continued bamboo encroachment into evergreen broadleaved forests seems likely to lead to net CO The soil organic carbon (SOC) and nitrogen (N) pools, an important component of terrestrial carbon and nitrogen pools, play a crucial role in global carbon and nitrogen cycles1457Phyllostachys edulis) is an economically important plant that is often cultivated for its delicious shoots and versatile culms131415Moso bamboo  and nitrogen (N), although such vegetation changes have important implications for local ecosystem dynamics, as well as regional C and N cycles21The study area is the Tianmu Mountain National Nature Reserve  situated within the northwestern region of Zhejiang Province, in southeastern China. Established in 1956 with the felling ban approval of the Central Government, Tianmu Mountain was promoted as a national reserve in 1986 and included by UNESCO in the International Man and Biosphere Reserve Network in 1996. Tianmu Mountain National Nature Reserve lies in the northern limit of mid-subtropical zone covering a total area of 4,284 ha. The climate there is damp monsoon climate with an annual rainfall of 1390–1870 mm and an annual temperature of 8.8–14.8 °C. It is warmer in winter and cooler in summer than that of other areas in this location. The reserve is one of the sites with the richest subtropical higher plant species in China. There are 2,160 species of higher plants, 1,781 species of seed plants, 151 species of ferns, 222 species of mosses and 69 species of liverworts. Among them, more than 37 species are named after Tianmu Mountain and 1,200 species of medicinal plants. This area boasts a plethora of different kinds of vegetation, such as evergreen and deciduous broadleaved forest, bamboo forest, coniferous forests, marshes and aquatic vegetation. The soils were classified as Ultisols (according to USDA Classification System).Tianmu Mountain is a famous religious mountain with a long history of Confucianism, Taoism and Buddhism. It has been forbidden to cut any trees and disturb by human activities since established as a nature reserve in 1956. However, the moso bamboo planted originally below the mountain expanded naturally and has expanded from 55.1 ha in 1956 to 105.4 ha in 2014.Cyclobalanopsis glauca, Cyclobalanopsis gracilis, Castanopsis sclerophylla, Litsea coreana Var. Sinensis. The BLF is the climax community in this region and is mainly dominated by Cyclobalanopsis glauca, Cyclobalanopsis gracilis, Castanopsis sclerophylla, Litsea coreana Var. Sinensis. Aspect, slope, elevation, and parent material were consistent within each of the three sites.In order to quantify the effects of moso bamboo encroachment on soil C and N pools, we employed a complementary chronosequence /transition front approach, i.e., we selected three sites, each with three plots arrayed along the bamboo encroachment pathway: pure moso bamboo forest (BF); transition zone plots that were unencroached plots in 2005, that had been almost fully encroached by 2014 MF; see ; and heaThree replicate permanent plots (10 m × 20 m) were established in BF, MF and BLF forest types at each study site in 2005. Five soil cores were collected near the center of each plot using a soil core (diameter 2.5 cm) to 0–60 cm depth and divided into three layers: 0–20, 20–40, and 40–60 cm in each sample site see . Soil co2Cr2O7 and titration with ammonium ferrous sulfateSoil total organic C (SOC) was measured using the volumetric oxidation method with Ket al.Water-soluble organic C (WSOC) concentration was determined using the methods described by Jiang 4+-N and NO3−-N) concentrations colorimetrically. Water-soluble organic N was determined the amount WSN less extractable inorganic N. The soil C and N pool sizes were calculated respectively by multiplying soil C and N concentrations by the soil bulk density and then multiplying the soil depth.Water-soluble organic N (WSON) was extracted from 20 g moist soil with addition of 40 mL distilled water. The mixture was shaken for 0.5 h with 250 rpm, and centrifuged for 10 min at 8,000 rpm. The supernatant liquid was filtered through 0.45 μm filterable membrane . Then divided two subsamples. The one subsample was used to determine the total N concentration in the water-soluble organic N by an automated TOC-Vcph Analyzer . The other one subsample was used to determine inorganic nitrogen  (α = 0.05) and least significant difference (LSD) were used to test the differences among forest types for each layer  as well as the 0–60 cm separately, and to test the differences among layers  in the same forest type at each sampling date. Repeated measures analysis of variance (RM ANOVA) was applied to test the differences of measured variables between 2005 and 2014 for each layer as well as the 0–60 cm separately in the same plots with α = 0.05. All statistical analyses were conducted by using SPSS13.0 (Chicago USA).P < 0.05), but no significant difference between MF and BLF in 2005 (P > 0.05). Soil organic C was about 30% greater in MF and BLF plots than in BF. Nine years later, SOC decreased significantly in MF (~17%) and did not change significantly for BF and BLF plots. Trends in changes in soil total N was similar to that of SOC, and was about 21% greater in MF and BLF than BF in 2005, but there were no significant differences in 2014 between in MF and BF (P > 0.05).Encroachment of moso bamboo led to a large reduction of the total amount of soil organic C and N in newly established MF in 2014 relative to the MF that bamboo began encroaching in 2005 . There wP < 0.05). SOC at 0–20 cm depth accounted for 44.0%, 42.2%, and 42.2% of the total amount of soil organic C in 2005 in BF, MF, and BLF respectively, and accounted for 43.5%, 41.0%, and 41.8% in 2014. Total soil nitrogen at 0–20 cm depth accounted for 45.7%, 48.4%, and 49.3% of the total amount of total soil N in BF, MF, and BLF respectively, and accounted for 45.3%, 45.8%, and 48.5% in 2014. There was no significant difference in SOC and total soil N at the 40–60 cm depth among different forests (P > 0.05).Encroachment of moso bamboo also affected the distribution of soil organic carbon and soil total nitrogen through the soil profiles . Both SOP < 0.05). MBC was highest in BF, and lowest in BLF, and about 19% greater in BF than BLF. However compared to 2005, MBN decreased significantly in MF (8%) and BF (7%) plots, and had almost no changes in BLF plot in 2014.Details of the soil microbial biomass C concentrations in each forest are listed in P < 0.05). MBC at 0–20 cm depth contributed 46.6%, 45.5%, and 45.0% to the total MBC at 0–60 cm depth in BF, MF, and BLF respectively in 2005 and 47.7%, 47.5% and 44.7% in 2014. However, MBN at 0–40 cm contributed 77.7%, 77.7% and 78.0% to the total MBN at 0–60 cm in BF, MF, and BLF respectively in 2005 and 78.0%, 76.3% and 77.9% in 2014. There were no significant differences in MBC and MBN at the 40–60 cm depth among different forests between 2005 and 2014 (P > 0.05). Moso bamboo encroachment altered MBC at 0–20 depth, and altered MBN at 0–40 cm depth.Soil microbial biomass C and N also decreased with increasing soil depth for all three forests. Both were concentrated in the topsoil (0–20 cm) where MBC and MBN made up over 40% of the total amount of MBC and MBN in 0–60 cm. There were significant differences at 0–20 cm depth among different forests . Moso bamboo encroachment altered plant diversity422 concentration.Decreased soil C and N pools in response to bamboo encroachment are another example of plant invasion having a detrimental effect on ecosystems29Both C and N pools in the soil decreased along gradients of bamboo cover, suggesting that continued growth and C storage in this broadleaved forest ecosystems are likely to be constrained by moso bamboo encroachment. Despite the fact that bamboo is a fast-growing species that fixes C at a high rate, bamboo encroachment leads to a significant decline in soil C over time. If our findings also occur in other bamboo encroachment areas, widespread increases in the cover of bamboo in areas formerly dominated by evergreen broadleaved tree species will lead to a decline in regional C stocks in the southern China. Measurements taken to prevent bamboo from encroaching into evergreen broadleaved forests, could offer C retention and thus climate mitigation benefits.How to cite this article: Bai, S. et al. Effects of moso bamboo encroachment into native, broad-leaved forests on soil carbon and nitrogen pools. Sci. Rep.6, 31480; doi: 10.1038/srep31480 (2016).


The C sequestration durations were estimated as 28–73 years under CFS and 26–117 years under CFM but with high variability across climatic regions. At least 2.0 Mg ha−1 yr−1 C input is needed to maintain the SOC in ~85% cases. We highlight a great C sequestration potential of applying CF, and adopting CFS and CFM is highly important for either improving or maintaining current SOC stocks across all agro–ecosystems.Cropland soil organic carbon (SOC) is undergoing substantial alterations due to both environmental and anthropogenic changes. Although numerous case studies have been conducted, there remains a lack of quantification of the consequences of such environmental and anthropogenic changes on the SOC sequestration across global agricultural systems. Here, we conducted a global meta-analysis of SOC changes under different fertilizer managements, namely unbalanced application of chemical fertilizers (UCF), balanced application of chemical fertilizers (CF), chemical fertilizers with straw application (CFS), and chemical fertilizers with manure application (CFM). We show that topsoil organic carbon (C) increased by 0.9 ) g kg It has been reported that increasing soil carbon (C) pool can help to enhance not only crop productivity but also yield stability1Globally, agricultural soil covers a total area of ~1370 million hectares (Mha) distributed across diverse climatic and edaphic conditions, as well as complex cropping systems and management practices45The adoption of fertilizer management practices, e.g., chemical fertilization, manure application and straw retention, has been recognized to be the most efficient and effective manner to either promote SOC accumulation or reduce the rate of SOC loss. In areas with nutrient deficiency, chemical fertilizers increase the crop yield and biomass and thus the crop residue and root C input to soil14161718192315 g)125−1 yr−1, which severely hampers the crop production. In addition, complete crop residue removal for fodder and fuel is a norm in South Asia and AfricaOn one hand, the potential C sink of the world’s agricultural and degraded soils is tremendous, approximately 50–66% of the historic C loss of 42–78 Pg C , CF , and UCF  g kg−1 (B) = 1423.5, p < 0.001, Compared with the control (CK), all four of the fertilizer management practices significantly increased the SOC content at the end of the experiments . The boo) g kg−1 . The aveB = 5.47, p < 0.05), and the mean ΔSOC was 1.0, 1.4 and 2.5 g kg−1 in tropical, warm temperate and cool temperate regions, respectively. The CFS treatment followed the same trend as the CF. For the UCF group, the average ΔSOC was low in tropical and warm temperate regions (0.8 g kg−1) and high in cool temperate regions (1.1 g kg−1). For the CFM group, the average ΔSOC values were 3.0, 3.9 and 3.5 for tropical, warm temperate and cool temperate regions , 1.2–3.7 g kg−1 (1.16–1.38 times), 1.6–6.2 g kg−1 (1.19–1.76 times), 1.7–5.3 g kg−1 (1.16–1.74 times) as C input increased from 0–2 Mg to over 6 Mg ha−1 yr−1 in the 0–10 yr, 10–20 yr, 20–30 yr and 30–40 yr groups, respectively. However, this trend could not be detected in the >40 yr group due to the limited sample size (n = 13) for the 4–6 Mg ha−1 yr−1 subgroup. In addition, the sequestration efficiency of high C input  gradually declined with experiment duration (Increasing C input is one of the most efficient methods for soil C sequestration. As illustrated in duration .−1 yr−1) than under CF and UCF (0.13 and 0.07 g kg−1 yr−1) . The rates of SOC change decreased significantly with increasing fertilization duration . The RR was generally negatively correlated with initial SOC .It is well known that the loss of information caused by the exclusion of multiple results may become a more serious problem than that caused by violating the assumption of independence3131nder CFM , while Mtial SOC , which wOC et al., which c et al.31. For the et al.31. This wa et al.31, and at  et al.31, the C iet al.35et al.et al.et al.−1 in Sub-Sharan Africa to 10.8 g kg−1 for the mean global croplands) and climatic conditions resulting from different site distributions1430−1 yr−1, respectively28−1 yr−1 29Our results suggested that, on average, the relative SOC increase under CFM was 36.2%  over 22 2 ranging from 0.08–0.29. Similar results have been reported in other studies14282930−1 yr−1, −1)141529et al.et al.et al.11et al.28We found a weak but significant correlation between the rate of SOC change and the experiment duration , with R2−1 yr−1, the percentage of C sinks increased from 72–89% (−1 in this study), at least 2.0 Mg ha−1 yr−1 C input is needed to maintain the SOC in most cases (~85%), which coincided with a recent estimation using the RothC model to maintain current SOC in global wheat systemsChanges in management practices to increase SOC must increase organic matter inputs, decrease decomposition of SOM, or use a combination of these approaches15383941m 72–89% . Under mN addition is another significant factor that influences SOC changes . Intensi1430Publication bias was evaluated using the Kendall’s tau rank and Spearman rankThe findings reported here might be useful to improve cropland surface models in terms of SOC changes subjected to chemical fertilization, straw and manure application, e.g., the C sequestration duration across climate zones should be treated differently in models. The implications were also important for the cropland management, especially for the developing areas where soils are suffering C loss. Our synthesis suggested that the RMPs should last more than decades to achieve the potential C sink and could not go back to the old routines, or the sequestered C might be lost again2452We conducted a global meta-analysis of the effects of fertilization on changes in SOC and RR, but there were a few unavoidable limitations. First, ~41% of the total experiment sites were in China, which reduced the spatial representativeness to a certain degree. We kept these sites because China covers a wide variety of climatic, edaphic and management conditions. China is a country with a long history of agriculture emphasizing manure applications, and straw return is becoming increasingly popular with the promotion of straw return and ban on straw burning by the government. Therefore, incorporating these data into the analysis was necessary and would compensate for the lack of data in other developing countries. Second, meta-analyses include some basic assumptions, such as the normal distribution of data and the independence of the effect size measures. Resampling methods relax some of these assumptions5431The study was conducted in the field and had a detailed description of the study site and the fertilization treatments.The literature reported the SOC or soil organic matter (SOM) under control  and treatment  at the end of the experiment. All other experimental conditions were identical for the control and the treatment.The literature recorded the experiment duration, and the duration was longer than one year.We searched the ISI Web of Science (http://apps.webofknowledge.com/) and China National Knowledge Infrastructure (http://www.cnki.net/) for articles with the terms “soil AND carbon AND ” in the title, abstract or keywords. The total papers found by such search were refined using the following criteria:In total, we obtained 238 published literatures comprising of 1741 comparisons at 298 sites , and the response ratio of SOC between treatment and CK . These two indexes have both been widely used as measures of experimental effect14152447We selected two indexes to estimate the effects of fertilizer managements on SOC dynamics: the mean difference between SOC under treatment and that under CK  under a treatment and CK in an experiment.where SOCIt is preferable to conduct analyses in the form of the natural logarithm of the response ratio (lnRR) for two reasons54T) among the effect sizes under fertilization was significantly larger than the expected sampling error31T was calculated as the sum of within-group heterogeneity (QW) and QBB is significant according to the Q-statistic, the ΔSOC and RRs are considered significantly different among the groups2431et al.An accurate meta-analysis requires means, standard deviations (or standard errors) and the number of replicates5548496049634964P) was calculated using The relative SOC increase in percentage :where RCR is the relative SOC change rate and C input is the quantity of C input.et al.Following Tian −1 yr−1, ~0.1 Mg C ha−1 yr−1) that is difficult to detect in field experiments, the calculated x is considered the duration of C sequestration.where RCR is the relative rate of SOC change, x is the duration of fertilization, and A and B are regressed constants. When RCR is equal to zero or a lower limit .


These urine proteins were also quantified in type 2 DKD (n = 11) before and after treatment with candesartan. The utilized immunoassays had comparable inter‐ and intra‐assay and intraindividual variation to assays used for urine albumin. Median (IQR) urine AGT concentrations were 226.0  and 13.0  μg/g creatinine in type 1 diabetes with and without DKD, respectively (P < 0.001). Median (IQR) urine gremlin‐1 concentrations were 48.6  and 3.6  μg/g, respectively (P < 0.001). Median (IQR) urine MMP‐7 concentrations were 6.0  and 1.0  μg/g creatinine, respectively (P < 0.001). Treatment with candesartan was associated with a reduction in median (IQR) urine AGT/creatinine from 23.5  to 2.0  μg/g, which did not reach statistical significance. Urine gremlin‐1 and MMP‐7 excretion did not decrease with candesartan. In conclusion, DKD is characterized by markedly elevated urine AGT, MMP‐7, and gremlin‐1. AGT decreased in response to RAS inhibition, suggesting that this marker reflects therapeutic response. Urinary components of the RAS, BMP, and WNT pathways may identify risk of DKD and aid development of novel therapeutics.The renin–angiotensin system (RAS), bone morphogenetic protein (BMP), and WNT pathways are involved in pathogenesis of diabetic kidney disease (DKD). This study characterized assays for urinary angiotensinogen (AGT), gremlin‐1, and matrix metalloproteinase 7 (MMP‐7), components of the RAS, BMP, and WNT pathways and examined their excretion in DKD. We measured urine AGT, gremlin‐1, and MMP‐7 in individuals with type 1 diabetes and prevalent DKD ( Urine angiotensinogen, matrix metalloproteinase‐7, and gremlin‐1 concentrations are markedly elevated in people with type 1 diabetes and kidney disease, compared with those with recently diagnosed type 1 diabetes or longstanding type 1 diabetes without kidney disease. Treatment with an inhibitor of the renin–angiotensin system tended to reduce urine angiotensinogen concentration, but not urine matrix metalloproteinase‐7 or gremlin‐1. Diabetic kidney disease (DKD) affects 20–40% of individuals with diabetes , a target gene highly upregulated by WNT pathway activation, is shown to correlate with renal WNT pathway activity in animal models of kidney disease , WNT, and bone morphogenetic protein (BMP) pathways. Activation of the RAS pathway has been heavily implicated in DKD pathophysiology and inhibiting it has yielded effective therapeutic interventions currently in use (RAS inhibitors). Angiotensinogen (AGT) is the sole substrate for renin, which catalyzes the rate‐limiting step in the RAS pathway, eventually yielding angiotensin II, which mediates most of the distal effects of the RAS pathway. The BMP pathway activation counteracts TGF‐In this study, we sought to determine if urinary excretion of AGT, gremlin‐1, and MMP‐7 increases with activation of the RAS and WNT and inhibition of BMP pathways in DKD. To do so, we characterized intra‐ and interassay and intraindividual variability in the commercially available immunoassays and used these to compare urinary concentrations of AGT, MMP‐7, BMP‐7, and gremlin‐1 in people with type 1 diabetes and DKD to those without kidney disease as well as those with new‐onset type 1 diabetes. Additionally, we examined the change in urinary concentration of these pathway components in response to RAS pathway inhibition in type 2 diabetes.2 and ACR ≥30 mg/g. People with longstanding diabetes but no evidence of DKD had ≥30 years of type 1 diabetes, estimated GFR >90 mL/min per 1.73 m2, and ACR <300 mg/g. New‐onset type 1 diabetes was defined as diagnosis of diabetes ≤12 months prior to urine sample collection with no hypertension or kidney disease. The use of these samples and data were approved by the Institutional Review Boards at the University of Washington and Benaroya Research Institute.Samples and clinical data from people with type 1 diabetes were obtained after informed consent either from the Kidney Research Institute Diabetic Kidney Disease Repository of the University of Washington or the Benaroya Research Institute Diabetes Translational Research Project. DKD was defined as either a urine albumin to creatinine ratio (ACR) ≥300 mg/g or an estimated glomerular filtration rate (GFR) <60 mL/min per 1.73 mβ1 levels, uncontrolled hypertension, creatinine clearance <30 mL/min, serum creatinine >3 mg/dL, hemoglobin A1c >10% or major comorbidities. After a 2‐week washout, the participants with DKD received candesartan doses escalating from 8 to 64 mg/day over a 12‐week follow‐up period. Use of the deidentified stored samples and participant data from this study was considered exempt from review by the Human Subjects Division of the University of Washington.Samples and clinical data from people with type 2 diabetes receiving candesartan were obtained from an open label dose escalation study of candesartan cilexetil  time from collection to −80°C storage was 5.7 (2) h. For the samples from the population with type 2 diabetes, 24‐h urine was collected at baseline and prior to administration of the daily candesartan dose at the end of each 3‐week dose escalation cycle and stored at −70°C until subsequent use  or concentrated fivefold (gremlin‐1) using Amicon 10 kDa ultrafiltration units  following manufacturer instructions. Urine AGT was measured using a quantitative solid‐phase sandwich enzyme‐Linked immunosorbent assay (ELISA) distributed by IBL‐America , with a minimum detection limit of 30 pg/mL and <0.1% cross‐reactivity with human angiotensin I, II, III, or IV, angiotensin (1–9), or angiotensin (1–7). Urine gremlin‐1 was measured using a quantitative solid‐phase sandwich ELISA , with a minimum detection limit of 60 pg/mL and no significant cross‐reactivity with Cerberus, CTGF, BDNF, or CYR61. Urine MMP‐7 was measured using a quantitative solid‐phase sandwich ELISA  with a minimum detection limit of 20 pg/mL and no significant cross‐reactivity with other human MMP or TIMPs.Inter‐ and intra‐assay variability was determined as the coefficient of variation (CV) in technical replicates of the same samples tested on the same day (intra‐assay) or different days (interassay). For intraindividual variation, CVs were calculated from measurements in spot and 24‐h urine samples, collected from the 11 individuals with type 2 diabetes and DKD over a 12‐month interval. To determine protein stability at room temperature, urine samples, collected from six individuals with type 2 diabetes and DKD, were incubated at room temperature from 1 to 48 h. To evaluate the effect of frequent freeze–thaw, urine samples obtained from six individuals with type 2 diabetes and DKD underwent one, two or five consecutive freeze–thaw cycles in liquid nitrogen prior to protein quantification.P‐values less than 0.05 were considered statistically significant. Analyses were performed using the SAS 9.3 statistical software package .Data are represented as mean (SD) for normally distributed data and median  for non‐normal data. Non‐normal data were compared using the Mann–Whitney test. We used multiple linear regression analysis to test differences in urine proteins, adjusting for age, gender, race, and diabetes duration. Gremlin‐1 concentrations in unconcentrated urine were too low to reliably detect. Urine concentration using ultrafiltration was selected from among four tested concentration methods because of a combination of ease, high yield, and low variability (data not shown). We characterized the commercially available assays for AGT, gremlin‐1, and MMP‐7 for intra‐ and interassay as well as intraindividual variation, protein stability after room temperature incubation, and repeated freeze–thaws . For the same reason, mean ACR exceeded 300 mg/g in people with DKD, but was normal (<30 mg/g) in both control populations  (TableP < 0.001). The difference in median AGT/Cr levels between people with DKD versus longstanding diabetes without DKD remained significant after additional adjustment for hemoglobin A1c (P < 0.001).Urine concentration of AGT, MMP‐7, and gremlin‐1 showed statistically significant moderate correlations with albuminuria in the combined cohort (01) Table. CreatinP < 0.001) .Creatinine‐adjusted urine MMP7 concentration was sixfold higher in people with type 1 diabetes and DKD than those with no DKD and 6.7‐fold higher than in newly diagnosed diabetes. Median urine MMP‐7/Cr  in cases (6.0 ng/mg) was significantly different from those in controls (1.0 ng/mg) and participants with new‐onset type 1 diabetes (0.9 ng/mg). These relations were unaffected by adjustment for age, race, gender, diabetes duration, and RAS inhibitor use, as well as correction for multiple testing using the Bonferroni method (01) Fig. . The difP < 0.001). The difference in median gremlin‐1/Cr levels between people with DKD versus longstanding diabetes without DKD remained significant after additional adjustment for hemoglobin A1c (P < 0.001). USAG1, another BMP pathway antagonist, was present in urine in concentrations 1000‐fold lower than gremlin‐1 and was not further pursued. Urine BMP‐7 concentration was not detectable in samples concentrated up to 200‐fold and as such was estimated to be less than 0.5 pg/mL.Creatinine‐adjusted urine gremlin‐1 concentration was 13‐fold higher in people with type 1 diabetes and DKD than those with no DKD and 27‐times higher than urine gremlin‐1 concentration at the onset of type 1 diabetes. Median urine gremlin‐1/Cr  in cases (48.6 ng/mg) was significantly different from those in controls (3.6 ng/mg) and participants with new‐onset type 1 diabetes (1.8 ng/mg)  of urine AGT/Cr concentration was 23.5  at study baseline (before candesartan) and 2.0  after 4 months of treatment with candesartan (candesartan 64 mg/day at study end). Median (IQR) urine MMP‐7 were 2.3  and 3.5  at study baseline and end. Median (IQR) urine gremlin‐1 were 2.5  and 3.0  at study baseline and end  in kidneys of several DKD animal models  after 30 or more years of diabetes avoids the potential misclassification of individuals with cross‐sectional normoalbuminuria as controls, since this latter group may harbor individuals with early or nonalbuminuric DKD. Furthermore, the finding of low urinary concentration of AGT, gremlin‐1, and MMP‐7 in new‐onset type 1 diabetes establishes a starting point for the trajectory of concentration of these proteins in the time course of DKD. This suggests that concentration of these proteins rises in urine of people with type 1 diabetes who go on to develop DKD, but not in those who do not develop DKD, possibly reflecting the known derangement of intrarenal RAS, WNT, and BMP pathways with progression of DKD. This raises the possibility that the increase in urinary concentration of these proteins may occur early enough in DKD course to be of value for detection of earlier stages of DKD. The cross‐sectional data presented here support examining association of urinary concentration of these proteins at earlier time points with subsequent development of DKD in a longitudinal study.We find that urine concentration of AGT, MMP‐7, and gremlin‐1 is low at the onset of type 1 diabetes and remains low in people who have intact kidney function after 30 or more years of type 1 diabetes. However, the concentration of each protein is markedly elevated in people with type 1 diabetes who develop DKD. Examining urinary concentration of these proteins in people with intact kidney function (GFR >90 mL/min per 1.73 mWe also find that treatment with an inhibitor of the RAS pathway shows a trend toward reducing urine AGT to creatinine concentration, as reported previously (Yamamoto et al. Strengths of this study include a first‐pass evaluation, as potential biomarkers, of novel urinary proteins representing promising candidate pathways, quantification of these urine proteins using a well characterized process from urine collection to assay, and use of a carefully ascertained extreme case–control population. The study has limitations. As DKD was not confirmed by a kidney biopsy, inclusion of other causes of kidney disease among our cases is possible. Other limitations are the cross‐sectional study design, small sample size, and lack of parallel quantification of protein concentrations in serum or kidney tissue.In conclusion, using carefully characterized assays, we find that in people with type 1 diabetes who have DKD, urine concentration of AGT, MMP‐7, and gremlin‐1 are markedly higher than in individuals with new onset of diabetes or those with longstanding diabetes without DKD. We also find a decrease in urine AGT, but not MMP‐7 or gremlin‐1, in response to RAS inhibition in people with type 2 diabetes and DKD, potentially reflecting therapeutic response. Urinary components of the RAS, BMP, and WNT pathways may identify risk of DKD as well as aid in development of novel therapies.None declared.


Drosophila suzukii, is a globally invasive pest of soft-skinned fruit. Females oviposit into ripening fruit and larvae cause direct destruction of tissues. As many plant essential oils are permitted food additives, they may provide a safe means of protecting fruit from D. suzukii infestation in both conventional and organic production systems. Twelve oils and potassium metabisulfite (KMS) were screened in the laboratory as repellents for D. suzukii flies. Most essential oils deterred D. suzukii flies from cotton wicks containing attractive raspberry juice. Peppermint oil was particularly effective, preventing almost all flies from contacting treated wicks and remaining 100% repellent for 6 d post-application. Thyme oil was unique because it caused high male mortality and reduced the number of responding flies compared to other oils. KMS was not found to be repellent to D. suzukii, but may have fumigant properties, particularly at high concentrations. Peppermint oil appears to be the best candidate for field testing to determine the effectiveness and feasibility of using essential oils as part of a push-pull management strategy against D. suzukii. This is the first time that essential oils have been evaluated and proven effective in preventing fruit-infesting flies from contacting attractive stimuli.Spotted wing drosophila,  Drosophila suzukii Matsumura (Diptera: Drosophilidae) is a globally invasive, multivoltine pest of numerous soft-skinned temperate fruit crops2D. suzukii is indigenous to Japan and is recorded from other Asian countriesD. suzukii was first noted in California and Spain in 2008; it has since spread across North America and Europe and has most recently been found in South America678D. suzukii1011D. suzukii and limited residual control afforded by insecticides (5–14 days), frequent applications may be necessary to maintain low pest levels37D. suzukii infestationD. suzukii15161718D. suzukii control, these and new tools for management need to be developed.Insecticides are available for effective control of icides 5– days, frD. suzukii locate suitable hosts, although volatile leaf odors are likely involved21242528D. suzukii available.It is not yet clear how Musca domestica L., are repelled by basil, lemongrass, lavender, peppermint, ginger, geranium, and elemol (osage orange) oils3233Haematobia irritans, L.)Cydia pomonella (L.))Resseliella oculiperda (Rübsaamen)) on apple saplingsFrankliniella occidentalis (Pergande))Drosophila auraria fliesAs crop protection agents, essential oils have been widely tested as repellents or fumigants for stored product pestsD. suzukii. KMS (K2S2O5) produces sulfur dioxide when dissolved in waterHarmonia axyridis  in a Y-tube olfactometer and reduced numbers of beetles on grape vinesBotyritis in vineyardsIn addition to essential oils, potassium metabisulfite (KMS) may be useful as a repellent for D. suzukii flies in the laboratory. A bioassay allowed male and female flies to choose between an attractant or the attractant plus an essential oil. Essential oils tested were selected from those which may have applicability in the field . Due to its high volatility, KMS was assessed in a no-choice trapping assay. Our results are a first step towards selecting and further testing of repellents for developing a push-pull management strategy against D. suzukii.The objective of this study was to screen 12 essential oils and KMS as repellents for D. suzukii , decreasing to about 35% by 4 d post application   . Concent< 0.001) .F11,33 = 66.9, P < 0.001). Volatility was highest in tree-derived and rosemary essential oils, although volatility of Eastern white cedar oil was lower than for other tree oils , but increasing KMS concentration caused an increased percent of dead flies outside traps   nor the percent of flies outside traps that were dead , but increasing KMS concentration caused an increase in the number of trapped flies that died .In Exp. 4, KMS concentration did not affect the percent of live flies trapped (= 0.025) , even thD. suzukii male and female flies from wicks containing attractive, fresh raspberry juice. Peppermint oil was particularly effective, preventing all but a few flies from contacting treated wicks and remaining 100% repellent for up to 6 d after it was applied to wicks. Peppermint oil consists mainly of two monoterpenoids: menthol and menthoneBlattella germanica)30Musca domestica L.In these experiments most essential oils deterred Mentha pulegium L., caused high D. auraria mortalityD. suzukii adults due to peppermint oil. There were fewer responding female flies in assays with peppermint oil than with geranium oil, suggesting that peppermint oil may negatively affect flies at a greater distance from its release point than geranium oil. Peppermint oil appears to be a good candidate for testing under field conditions, where persistence of repellency and distance of repellency from ripe fruit will be important factors determining the effectiveness and feasibility of using essential oils as part of a management strategy for D. suzukii.Mint oils have also been reported as good adulticides and larvicides against insect pests2 −1 based on the wick surface area. Studies on third-instar larvae of small dipterans have found an LC50 of 0.70 mg cm2 −1 of peppermint oil for Camptomyia cortacalis (Diptera: Cecidomyiidae)50 of 2.1 and 1.1 μL of pennyroyal and spearmint oils, respectively, for Drosophila melanogasterD. auraria when eggs and adults were exposed to M. pulegium but stated only that 1–20 μL of an essential oil was used. The estimated amount of peppermint oil on the exposed part of the wick required to achieve 95% repellency in our study was 4.5–6.7 μL. It is difficult to estimate the actual dose experienced by flies as we do not know whether the wicking action and evaporation rate of raspberry juice changed and affected the oil concentration during the experiment. Distances at which oils are repellent and concentrations required to achieve repellency and deter oviposition in the field will need to be determined.Amounts of peppermint and geranium oils estimated to achieve 95% repellency of female and male flies were similar, ranging from 6.8–9.9 mg of oil per wick or 0.39–0.57 mg cmThymus vulgaris used in this experiment, have strong toxic effects on other insect pests, including southern green stink bug, Nezara viridula (L.), nymphs and adultsSpodoptera litura Fab.C. cortacalis (Diptera: Cecidomyiidae)D. melanogaster to be related to interference in tyramine receptor cascades that are involved with cAMP and calcium at the molecular levelCulex pipiens pallens Coquillet mosquitoesThyme oil was unique among oils tested because it caused higher mortality of male flies than other oils, and it reduced the number of responding males and females to 56–65% and 75–81%, respectively, of that of flies responding in arenas with citronella, lavender, and rosemary . Thyme essential oil constituents, particularly the monoterpene thymol from the chemotype of D. suzukii flies but may have fumigant properties, particularly at high concentrations. At lower concentrations (Exp. 4), there was a small but significant increase in fly mortality from less than 25% in controls to about 40% at 5 g L−1. At higher concentrations (Exp. 5), 10–15% of flies died outside traps at all concentrations, but mortality in traps reached 60% at 30 g L−1, a significant increase from no mortality in traps at 0 and 5 g L−1. KMS is highly volatile in solution, releasing half its weight in SO2 and repelling H. axyridis within 1 min after the start of assays42Platynota stultana Walshingham), providing protection to packed table grapes at low doses in combination with low temperature over long periods2 was produced to kill flies, particularly within traps where SO2 would have been more concentrated. It is also possible that once inside traps, flies died by contact with or ingestion of KMS on wicks. KMS may be more effective as a fumigant in enclosed spaces than as a repellent in open fields and could be investigated against D. suzukii larvae as a post-harvest alternative to methyl bromideOur results show that KMS is not repellent to www.aliksir.com for most of the essential oils used in these experiments) as deterrents for D. suzukii and alternatives to synthetic insecticides is desirable because they pose little or no risk to mammalian health or beneficial insectsD. suzukii infestation applicable in both conventional and organic production systems.This is the first time that essential oils have been shown to be effective in preventing fruit-infesting flies from contacting attractive host stimuli. Essential oils from the same plant species may vary in their constituent chemical composition due to production practices or plant material sources and thus affect their bioactivity. Using essential oils or their active components . Flies were kept in Plexiglas® cages (26 × 26 × 26 cm), with mesh backings and sleeves for access, at 22–23 oC, 20–30% RH and 16:8 h L:D. Flies were provided with moist cotton batting and fed and reared on diet prepared by combining: water (4 L), agar (45 g), cornmeal (125 g), white sugar (200 g) and nutritional yeast (70 g) with propionic acid (17.7 mL) and methyl paraben (3.3 g) dissolved in 95% ethanol (33.3 mL). The mixture was boiled and then cooled before pouring into Petri dishes (9 mm diameter). Flies that were 5–8 d old were used for experiments. Flies were separated by sex and held without food for 20 h and without water for the final 2 h before the start of the experiments.Pelargonium asperum or P. graveolens (L.) L’Her ex Ait., cv. Bourbon or Rosat), peppermint (Mentha x piperita L.), ginger , eucalyptus , citronella (Cymbopogon winterianus Jowitt), lavender (Lavandula angustifolia Mill.), rosemary , thyme (Thymus vulgaris L. (thymol chemotype)), eastern white cedar , balsam fir  Mill.), white spruce (Picea glauca (Moench) Voss), and white pine (Pinus strobus L.); all obtained from Aliksir Inc. . Essential oil density was determined by weighing three samples of 10 μL (−1 were prepared with acetone as the solvent. Varying concentrations (0.3–30 g L−1) were prepared for experiments with geranium and peppermint oils.Essential oils tested in repellency bioassays were geranium , air-dried and wiped or rinsed with hexane.Choice bioassays were conducted in arenas consisting of clear, plastic containers (33.3 × 20.3 × 12.1 cm) with tight fitting lids . Fine white mesh covered ventilation holes (2.2 cm diam.) made singly on each vertical side (7.0 cm from the bottom) and three holes in the lid. In each arena, two glass specimen vials (12 mL) were placed 18.7 cm apart and 7.3 cm from the ends of the container. Vials were secured to the bottom of containers with small pieces of mounting putty . A cotton dental wick  and 10 mL of raspberry juice was added to each vial. A second dental wick was treated with either an essential oil  or acetone only (2 mL) and placed in a fumehood for one hour to allow acetone to volatilize. Raspberry juice (2 mL) was applied to each wick before it was put in a vial; the wick rested on the wick already in the vial and protruded 2 cm above the rim of the vial. Raspberry juice was prepared by heating fresh, store-purchased red raspberries (400 g) mixed with distilled water (250 mL) and pectinase  at 45 Arenas were arranged in a completely randomized design on a table covered with white paper. Three 60 W incandescent bulbs were hung 1 m above the table at 16:8 h L:D with photophase beginning at 0600 h. Trials began at 1100 h by placing plastic vials (70 mL) containing approximately 20 flies upright in the center of the container and removing lids so that flies could walk or fly from vials. The number of responding flies (those on treated or control wicks) and the number alive or dead in the arena and release vial were counted at 1, 6, and 24 h after flies were introduced.Essential oils were tested in three trial periods. The first trial tested peppermint, geranium, ginger, eucalyptus, the second trial tested eastern white cedar, balsam fir, white spruce, white pine, and the third trial tested citronella, lavender, rosemary, thyme with female and male flies separately. Each oil was replicated six times for each sex, except peppermint, geranium and group 2 oils that were replicated five times for males due to lower than expected numbers of available flies.Persistence of repellency to female flies was assessed by treating wicks 0, 1, 2, 4 d or 0, 1, 2, 4, and 6 d before the start of the experiment with geranium and peppermint oil, respectively. Wicks were held on aluminum foil in a fumehood at 22 °C ± 1 °C for aging. There were four geranium and five peppermint oil replicates.The effect of geranium and peppermint oil concentrations on repellency to male and female flies was evaluated by treating wicks with 0, 0.6, 1.2, 6, and 30 mg of peppermint and geranium oil; 18 mg of geranium oil was also included in assays with females. Each peppermint and geranium concentration was replicated five times, except geranium concentrations were replicated four times for females because of the extra concentration and a shortage of female flies.Release rates of essential oils were determined by measuring weight loss from 2 mL polypropylene vials , each with a 3 mm hole in the lid. Vials were loaded with 400 μL of an undiluted essential oil and placed in a fume hood at 22 °C ± 1 °C. Vials (three per oil) were weighed 0, 2, 6, 12, 24, 48, 72, and 96 h after loading.Solutions of 1.0, 1.5, 2.5, 5.0, 15.0, and 30.0 g KMS  per litre of distilled water were prepared 1 h prior to the start of the experiments.® cylinders (31 × 8.8 cm) with ends covered by fine white mesh held in place at the top of the cylinder by an elastic band and glued to the base of the cylinder. There were two ventilation holes (5 cm) covered with fine white mesh 20.3 cm from the bottom of each cylinder. Traps were plastic jars (7.3 × 4.8 cm) with aluminum foil secured by an elastic band over the jar opening. A cut microcentrifuge tube (1.5 mL) was inserted through the middle of the foil. Two store-purchased red raspberries (6.9 ± 1.4 g) and a cotton wick that was treated with KMS solution (1.5 mL) or untreated (1.5 mL water) were placed in each trap. One trap was placed in each cylinder. Cylinders were arranged in a completely randomized design on a table under the same conditions as described above for choice bioassays.A no-choice trapping bioassay was conducted in arenas consisting of upright clear Plexiglas−1 with five replicates (Exp. 4). Because there was no repellent effect of KMS at these concentrations, Exp. 5 was conducted using concentrations of 5, 15, and 30 g L−1 with six replicates. For each experiment, 25–35 flies (approximately 1:1 sex ratio) were released into each cylinder. After 24 h, the number of trapped flies and the number of dead flies inside and outside traps were counted.KMS was tested at four concentrations between 1–5 g LD. suzukii on treated and untreated wicks were compared using goodness-of-fit G-tests. The pooled G-test statistic is presented as results of each essential oil for either male or female flies were consistent between replicates For Exp. 1, numbers of flies on treated wicks − flies on untreated wicks)/(flies on both wicks)RI = Y was the repellency index and X was the age of the treated wicks in days. For male and female flies, probit analysis was used to generate concentration–repellency regressions for peppermint and geranium oils. Concentrations that achieved 95% repellency (EC 95), confidence limits, slopes, and goodness-of-fit χ2 vales were determined.Linear regression was used to evaluate geranium oil persistence, where Y was the percent trapped flies or percent dead flies inside and outside traps and X was the KMS concentration (g L−1). A quadratic regression was also fit to the percent dead flies inside traps in experiment five. Significance of slopes (deviation from zero) and coefficients of determination (R2) are reported for regressions.For Exps. 4 and 5, linear regression was used to assess repellency, where JMP softwareHow to cite this article: Renkema, J. M. et al. Plant essential oils and potassium metabisulfite as repellents for Drosophila suzukii (Diptera: Drosophilidae). Sci. Rep.6, 21432; doi: 10.1038/srep21432 (2016).


Vitis vinifera L.) is the most widely cultivated and economically important horticultural crop in the world. As a one of the origin area, Anatolia played an important role in the diversification and spread of the cultivated form V. vinifera ssp. vinifera cultivars and also the wild form V. vinifera ssp. sylvestris ecotypes. Although several biodiversity studies have been conducted with local cultivars in different regions of Anatolia, no information has been reported so far on the biochemical  and antioxidant diversity of local historical table V. vinifera cultivars grown in Igdir province. In this work, we studied these traits in nine local table grape cultivars viz. ‘Beyaz Kismis’ (synonym name of Sultanina or Thompson seedless), ‘Askeri’, ‘El Hakki’, ‘Kirmizi Kismis’, ‘Inek Emcegi’, ‘Hacabas’, ‘Kerim Gandi’, ‘Yazen Dayi’, and ‘Miskali’ spread in the Igdir province of Eastern part of Turkey.The Eurasian grapevine  assay. ‘Hacabas’ cultivar had the highest vitamin C content of 35.74 mg/100 g.Variability of all studied parameters is strongly influenced by cultivars (P < 0.01). Among the cultivars investigated, ‘Miskali’ showed the highest citric acid content (0.959 g/l) while ‘Kirmizi Kismis’ produced predominant contents in tartaric acid (12.71 g/l). The highest glucose (16.47 g/100 g) and fructose (15.55 g/100 g) contents were provided with ‘Beyaz Kismis’. ‘Kirmizi Kismis’ cultivar had also the highest quercetin (0.55 mg/l), o-coumaric acid (1.90 mg/l), and caffeic acid (2.73 mg/l) content. The highest ferulic acid (0.94 mg/l), and syringic acid (2.00 mg/l) contents were observed with ‘Beyaz Kismis’ cultivar. The highest antioxidant capacity was obtained as 9.09 μmol TE gPresent results illustrated that the historical table grape cultivars grown in Igdir province of Eastern part of Turkey contained diverse and valuable sugars, organic acids, phenolic acids, Vitamin C values and demonstrated important antioxidant capacity for human health benefits. Further preservation and use of this gene pool will be helpful to avoid genetic erosion and to promote continued agriculture in the region. More than a dozen classes of these biologically active plant chemicals, now known as ‘phytochemicals’ , have been identified. The majority of naturally occurring health-enhancing substances appear to be of horticultural plant origin [Overwhelming evidence from t origin –5.The grape, cultivated since ancient times is one of the most significant edible and processing for wine crop due to its beneficial influences on human health and economic significance on a large scale , and TurV. vinifera throughout grape growing areas in the world. Despite the existence of thousands of cultivars of V. vinifera in grape producer countries, only a few dozen cultivars account for the vast majority of world-wide production. Numerous local cultivars have regional importance and historically played a significant role in many viticulture regions [There are vast germplasm resources available within the genus  regions . In fact regions , 8, 9. TGrape berry ‘quality’ is one of the cardinal variables that determine wine quality. Berry ‘quality’ , however, is a generic term that refers to levels of a diverse range of berry chemical constituents including organic acids, sugars, phenolics, acidity etc. .The presence of adequate levels of organic acids in the grape berries is one of the key factors to determine the quality of berries and wines. Conde et al.  underlinVitis vinifera, Vitis labrusca, and Vitis hybrid. More comprehensively, Orak [Vitis spp.) cultivars, depending on the various stages of the grape berry development, were scrutinized.Several variables viz. total soluble sugar, titratable acidity, nitrogen and phenolic compounds balance provide a major contribution in the description of the grape quality , which aly, Orak  investigly, Orak , variatiConsidering all those former studies together, it is indispensible to conduct further studies on the identification of biochemical composition and phenolic contents that can influence the quality of the grape and its products, and notably, on the determination of the major factors  linked to those contents under different climate conditions.In Turkey, the native table grape cultivars are extensively cultivated in order of local usage fresh grape, raisin and grape juice. Therefore, the cultivation of some varieties has been recognized after the observations of plant and phenological characteristics in the arable soil of Igdır province located in the Eastern Anatolia region of Turkey. Igdir province includes well-known Agri Dagi - the same Ararat Mountain - that is recognized as a location where the Hoah’s Ark stopped and the birthplace of viticulture, when Noah after the Flooding planted vineyard and made the wine according to Bible. In spite of the examination, the detection of organic acid, phenolic compounds, and antioxidant compositions of the regional grape cultivars from Igdir province have not been yet documented which may gain more significance in improving the grape industry of the province in the future. Hence, an attempt was made in the current investigation to identify the sugars, organic acids and phenolic compounds for the local nine grape cultivars from Igdır province. The region cultivates only those local grape 2cultivars and there are no well-known international cultivars in the region – in opposite: we have to take in consideration, that the local cultivar ‘Sultanina’ of Turkish origin is widely spread in America, Australia, Mediterranean, Northern Africa, Europe and other countries ; as wellSamples of nine native grape cultivars available in the single collection vineyard together in Necefali village of Igdır province have been evaluated to detect organic acids including citric, tartaric, malic, succinic, and fumaric acids at the harvest time. The statistical analysis results for organic acids are summarized in Table In citric acid, the averages of the evaluated cultivars had a great range between 0.254 to 0.959 g/l. Among the available cultivars, ‘Miskali’ produced the highest average in citric acid (0.959 g/l) and was found significantly different from the others, statistically (P < 0.01).A very desirable coefficient of variation with 1.09 (%) was found for citric acid .The results and statistical evaluations for the Trolox Equivalent Antioxidant Capacity (TEAC), vitamin C (ascorbic acid), and sugars (fructose and glucose) described in the present investigation are presented in the Table In the study, vitamin C varied between 11.21 and 35.74 mg per 100 g  cultivars and selections had a very wide range of 1.8 to 209.1 μg/g. The present results demonstrated that the rutin content of the grape cultivars had a narrow range of 1.09 to 3.34 mg/l. The cultivar of the highest rutin content among the analyzed grape cultivars was verified to be ‘Hacabas’, numerically and statistically (Table Vitis vinifera L.) cultivars were detected.As antioxidants, plant flavonoids are the most important in free radical scavenging. In a previous review on nutritional benefits of flavonoids, it was reported that catechin among those flavonoids had the highest antioxidant activity . Among tAs to the statistical evaluation performed in quercetin, ‘Kırmızı Kismis’ was the grape cultivar that contained the utmost content in comparison to others (P < 0.01), whereas the lowest contents were established for the cultivars with letter “e”, on the basis of Tukey test, as also depicted from Table Vitis vinifera L.) cultivars and selections were identified as 6.9 and 24.5 μg/g, respectively. In terms of the grape characterization, analyzing phenolic and aromatic compounds which can be varied to cultivar and Terroir of the grape is necessary for producing very high qualified wines.Of the probed local grapes, ‘Kırmızı Kismis’ had much more content in o-coumaric acid than the others, but ‘Kerim Gandi’ with the letter “f” produced much lower content as compared to the other cultivars as also understood from Table The obtained results reflected that the historical nine table grape cultivars grown in the Igdir province of Eastern Turkey contained valuable sugars, organic acids, and phenolic compounds in health benefits. In the development of the grape industry of Igdır province, the present study will provide industrialists at first and then breeders to get baseline information for selection of grape cultivars for cultivation or breeding programs, which may be gained great importance with the support of the further studies to be carried out more comprehensively in future years.The present investigation was conducted on nine historical local table grape cultivars ‘Beyaz Kismis’ , ‘Askeri’ , ‘El Hakki’ , ‘Kirmizi Kismis’ , ‘Inek Emcegi’ , ‘Hacabas’ , ‘Kerim Gandi’ , ‘Yazen Dayi’ , and ‘Miskali’ grown in a single collection vineyard of Igdir province - located in the Eastern Anatolia Region of Turkey. The aim is to identify their biochemical content  and antioxidant capacity. In fact the prime name of ‘Beyaz Kismis’ is ‘Sultanina’ or Thompson seedless . ‘Hacaba°10′N latitude, 44°4′E longitude on the 865 meters above sea level) at a distance of 13 km to the city Igdir - the administrative center of the province.The collection site is located in the village of Necefali (40Fruits (berries) for analyses were collected from bunch of 8 grapevine plants ensured good representative of the cultivars to make average sampling, and the berries of those grape cultivars were taken during commercial ripe stage of the grapes in the dates varying between 1 to 20 August of the year 2013. Thus the sampling structured according to differential ripening dates across cultivars.The plants were established with planting scheme of 2.5 m × 2.5 m in distance. In winter months, plants were covered by soil to avoid winter injury. Pruning is applying in February and 3–4 buds left on shoots.2SO4 was pured on the samples and homogenized . Then, the samples were mixed for 1 hour on a shaker  and centrifuged at 15000 g for 15 min. Their watery part extracted from the centrifuge was initially distilled from filter paper and then distilled twice from a membrane filter of 0.45 μm, and lastly, it was distilled from the cartridge SEP-PAK C18. Organic acids were exposed to a HPLC device on the basis of the method identified by Bevilacqua and Califano [Samples were stored at -20°C until processed. In the present paper succinic, citric, malic, fumaric and tartaric acids as organic acids were determined. Organic acids were extracted after the method suggested by Bevilacqua and Califano  was modiCalifano .In the HPLC system, Aminex HPX - 87 H, 300 mm × 7.8 mm column , was used and the HPLC device was commanded by Agilent package program. In the system, DAD detector  was adjusted to the wavelengths of 214 and 280 nm.In the present paper, gallic acid, catechin, chlorogenic acid, caffeic acid, p-cumaric acid, o-cumaric acid, ferulic acid, vanillic acid, rutin, syringic acid and quercetin as phenolic acids were identified. The phenolic compounds were identified with HPLC using the modified method of Rodriguez-Delgado et al. .The samples were mixed with distilled water in a ratio of 1:1, and centrifuged for 15 min at 15000 rpm. Afterwards, the upper part was filtered with 0.45 μm millipore filters, and enjected to HPLC. Chromatographic analysis was performed by Agilent 1100 (Agilent) HPLC system using DAD dedector (Agilent. USA) and 250*4.6 mm, 4 μm ODS column . As a mobile phase, A methanol:acetic acid:water (10:2:28) and B methanol:acetic acid.water (90:2:8) were used. The extraction was made at 254 and 280 nm, 1 mL/min flow rate, and 20 μL injection volume.2 column with 85% acetonitrile liquid phase in HPLC that has a refractive index detector. Calculation of the concentrations was done based on fruit juice standards.The analysis of sugars was performed using the modified method of Melgarejo et al. . The sug2SO4. The DAD detector with 254 nm wavelength was used for the readings. For determination of ascorbic acid different concentration levels of L-ascorbic acid (SigmaA5960)  were used [The sample of 5 g from the whole fruits was transferred to test tubes and then 5 ml 2.5% M-phosphoric acid solution was poured on it. The mixture was centrifuged with 6500 g for 10 min at 4°C. From the clear part in the centrifuge tube 0.5 ml was taken and 2.5% M-phosphoric solution was poured until reaching 10 ml. The new mixture was filtered by 0.45 μm teflon filter and injected to HPLC. Ascorbic Acid was detected by the C18 column  in the HPLC. Ultra distilled water was used as a mobile phase with 1 ml/min flow rate and pH of 2.2 adjusted with Here used .+ solution was mixed with 20 μl fruit extract, and incubated for 10 min. Absorbance values obtained for 734 nm.For a standard TEAC measurement, ABTS acetate was solved in a buffer and potassium persulphate was prepared . To keepEight grapevine plants for each cultivar were used for sampling. Fruits (berries) were collected from different bunch in these 8 plants per cultivar. The descriptive statistics for all quantitative characteristics under investigation were expressed as Mean ± SE. The data were analyzed through One-Way ANOVA with four replications and mean separation was done by using Tukey multiple comparison test. In the present paper, a significance level of 1% was considered. All statistical computations were performed using SPSS 20 program.