The level and distribution of selected organochlorine pesticides in sediments from River Chenab,Pakistan

Organochlorine pesticides (OCPs), viz. β-hexachlorocyclohexane (β-HCH), γ-HCH, aldrin, dieldrin, endrin, heptachlor, endosulfan-I, endosulfan-II, heptachlor endoepoxide, heptachlor exoepoxide, mirex, dicofol, o,p′-dichlorodiphenyltrichloroethane (o,p′-DDT), p,p′-dichlorodiphenyltrichloroethane (p,p′-DDT), dichlorodiphenyldichloroethane (DDD), and dichlorodiphenyltrichloroethylene (DDE) and 12 other physicochemical parameters were measured in surface sediments from River Chenab during two sampling seasons (summer and winter, 2007) to evaluate spatial and temporal trends of sediment pollution. Hierarchical agglomerative cluster analysis identified three groups of sites based on spatial similarities in physicochemical parameters and OCP residual concentrations. Spatial discriminant function analysis (DFA) segregated 14 parameters, viz. dicofol, endosulfan-I, heptachlor endoepoxide, dieldrin, DDD, DDE, endosulfan-II, o,p′-DDT, p,p′-DDT, pH, electrical conductivity (EC), Cl⁻¹, total P (%), and silt, which explained 96% of total variance between spatial groups. γ-HCH was the most frequently detected (63%) pesticide, followed by DDD (56%). The ratio of DDTs to their metabolites indicated current input and anaerobic biodegradation. Temporal DFA highlighted aldrin, heptachlor endoepoxide, Cl⁻¹, total P, and EC as important variables which caused variations between summer and winter. DDTs were relatively more prevalent as compared to other OCPs in the sediments samples during both seasons. DDT metabolites were detected at greater frequencies and concentrations in winter, whereas DDT isomers were more prevalent in summer sediment samples. Factor analysis identified agricultural and industrial activities as major sources of sediment OCP contamination. Concentrations of γ-HCH, heptachlor endoepoxide, dieldrin, and DDTs (isomers and metabolites) in all sediment samples were well above interim sediment quality guidelines (ISQGs) and probable effect limits (PEL) given by Canadian Sediment Quality Guidelines (CSQGs).     

土壤中结合残留态农药的生态环境效应

土壤中农药结合残留态的形成，导致其活性暂时降低，但并未从土壤中消失，在特定的环境条件下又重新释放到环境中并表现出较高的生物有效性，从而威胁农产品质量安全与环境质量。文章论述了土壤中结合残留态农药的定义、形成过程及影响因素、老化和释放过程及机制。土壤中结合残留态农药主要通过吸附过程、化学反应及物理镶嵌等作用而形成，其形成过程受农药的结构和化学特性、土壤理化性质、环境条件和农艺措施的影响。老化是化合物和土壤组分紧密结合，减少被普通提取方法提取出来的数量，降低了化合物的生物有效性。同时老化的物质在土壤环境条件改变的情况下又重新释放到土壤溶液中或进行矿化，此过程可以通过物理一化学机制或生物化学作用而发生。重新释放到环境中的结合残留态农药又表现出较高的生物有效性，可能被植物、动物或微生物所吸收，并沿食物链富集和放大或进入水体污染水产品和造成水质恶化，从而威胁人体健康。文章还分析了土壤结合残留态农药可能带来的环境问题，提出了此问题今后研究的方向。     

太湖水体中5种有机磷农药混合物生态风险评价

有机磷农药是一类广泛分布于我国水环境中的污染物,即使在水体中的污染水平处于规定“安全标准”之下,其联合暴露产生的风险仍有可能威胁水生生态安全。采用基于浓度加和模型与生物敏感度分布曲线的混合物风险商法,评价了太湖水体中敌敌畏、对硫磷、甲基对硫磷、马拉硫磷和乐果5种有机磷农药混合物产生的生态风险。结果表明：2003～2004年期间3个不同时期太湖水体中5种有机磷农药的混合物生态风险商(RQ_m )均大于1,有机磷农药混合物在2003～2004年期间对太湖水生生物构成了一定威胁。     

Inorganic and organic contaminants in sediments from an urban playa and associated toxicity among Hyalella azteca

Playa wetlands are important components of the Southern High Plains (USA) landscape as they are the major aquatic surface feature. Chemical contaminants associated with playas have been documented, particularly for grassland and agricultural watersheds, but not for playas in urban settings. The objectives of this study were to determine concentrations of inorganic and organic contaminants in sediments from an urban playa within the I-20 Wildlife Preserve and Jenna Welch Nature Study Center in Midland, TX, and evaluate toxicity of these sediments to Hyalella azteca. Concentrations of most trace elements were below sediment quality guidelines with exceptions of lead, cadmium, and arsenic. Concentrations of organic contaminants, particularly PAHs, DDT, DDE, and malathion, were above sediment quality guidelines at various locations within the playa. Decreased survival was observed among H. azteca exposed to sediment from a single location when compared those exposed to reference sediments. This location also produced maximum observed concentrations for five of seven trace elements, potentially due to its location at the lowest elevation within the playa. This study documented concentrations of contaminants in sediments of an urban playa associated with past and present land uses in its urban setting, including those from automotive emissions and historical pesticide use.     

极性有机物整合采样技术在监测水环境中农药的应用

明确水环境中农残的污染状况对研究其环境行为和生态风险至关重要。传统主动采样法耗能、耗力且单次采样仅获得瞬时浓度值。被动采样节能、方便且为时间加权平均浓度,利于大规模采样。极性有机物整合采样技术(polar organic chemical integrative sampler,POCIS)是针对极性有机物的被动采样技术,近年来在水环境中农药监测的运用越来越广泛。本文概述了POCIS结构、原理和校正方法,探讨了环境因素(如水流速率、温度、p H、溶解性有机质、盐度和膜污染)、化合物性质及POCIS结构对农药采样速率(Rs)的影响。此外,综述了POCIS在监测水环境中农药的应用,展望了POCIS在该领域中的问题、解决方式及前景。     

氨基甲酸酯类农药对蛋白核小球藻联合毒性作用特点及机制

氨基甲酸酯类农药广泛应用于农业生产中,其在环境中的残留及其对非靶标生物的毒性作用引起关注。以5种氨基甲酸酯类农药包括残杀威、灭多威、抗蚜威、涕灭威和呋喃丹为研究对象,以蛋白核小球藻为受试生物,应用微板毒性分析法系统测定每种农药及其五元混合物对蛋白核小球藻在不同暴露时间(12、24、48、72和96 h)的生长抑制作用,并同步分析农药对蛋白核小球藻的生理特性如叶绿素含量、蛋白质含量、超氧化物歧化酶(SOD)活性和脂质过氧化物丙二醛(MDA)含量的影响。结果表明,5种农药对蛋白核小球藻的浓度-效应均具有明显的时间依赖性,农药抗蚜威在中低浓度促进绿藻生长,呈现非单调J型浓度-效应曲线(CRC)特征,其余4种农药的CRC呈现经典S型;以半数效应浓度的负对数(p EC50)为毒性大小指标,5种农药在96 h时毒性大小为:呋喃丹(p EC50=3.43)残杀威(p EC50=2.76)抗蚜威(p EC50=2.12)灭多威(p EC50=2.11)涕灭威(p EC50=1.89)。浓度为EC50的5种农药处理后的蛋白核小球藻中叶绿素和蛋白质含量随暴露时间的延长而减少,但不同农药处理的绿藻中叶绿素和蛋白质含量减少率随暴露时间延长变化趋势稍有不同; SOD酶活性随着暴露时间延长逐渐下降,MDA含量逐渐增加,这说明藻细胞受到破坏,脂质过氧化的损害程度超过细胞修复能力,SOD活性被抑制,细胞的抗氧化能力下降,藻细胞内的H2O2不断积累,导致MDA含量升高。五元混合物对蛋白核小球藻的毒性也具有一定的时间依赖性,并表现出刺激作用,即hormesis现象,且混合物毒性与组分浓度比具有良好的线性相关性;暴露于混合物的小球藻均在96 h出现了刺激效应,其叶绿素与蛋白质含量随暴露时间延长不断增加,SOD活性不断升高,MDA含量不断减少;五元混合物均在96 h呈现出拮抗作用,且与混合物的浓度和组分浓度比相关。     

Effects of organochlorines on the ultrastructure of the liver of the damselfish Parma microlepis from reefs in New South Wales, Australia

  Parma microlepis (Günther) were collected from Malabar, an urban location close to the centre of Sydney, Australia, and from Jervis Bay, a reference location 170 km south of the city centre. At each location, fish were collected from two sites separated by 100 to 200 m. The ultrastructure of normal liver tissue is described based on 20 female fish collected from Jervis Bay, where fish are known to be exposed to low levels of organochlorine contaminants. Alterations in the endoplasmic reticulum, mitochondria, lysosomes and nuclei of hepatocytes were identified and quantified in the liver tissue of fish from this location and compared to alterations in 20 female fish collected from Malabar, where fish are exposed to higher concentrations of organochlorine pesticides such as DDT compounds. There were significant differences in the percentage of hepatocytes with swollen mitochondria (F = 124.025, df = 2, 2, P = 0.008) and atypical nuclei (F = 22.198, df = 2, 2, P = 0.043) between sites (100 to 200 m apart), but there were no clear differences between the percentage of structural alterations in the hepatocytes of P. microlepis from Jervis Bay and Malabar. Associations between liver morphology and the organochlorines aldrin, dieldrin, DDE and chlordane were examined using a Pearson correlation matrix. Significant correlations were detected between the percentage of hepatocytes with dilated endoplasmic reticulum and the concentrations of the pesticide aldrin (r = 0.600, df = 11, r _{crit(α = 0.05)} = 0.553). Significant associations were also detected between the percentage of hepatocytes with disorganised endoplasmic reticulum and the concentrations of dieldrin and DDE residues in fish (r = 0.576, r = 0.567, respectively, df = 13, r _{crit (α = 0.05)} = 0.514). However, there was little evidence that ultrastructural alterations in fish responded to increasing concentrations of these pesticides in a consistent dose-response manner. Received: 20 October 1998 / Accepted: 24 November 1999     

长三角地区池塘养殖水产品体内农药类污染与食用风险评价

为了解池塘养殖水产品体内的农药污染情况,以便为水产品质量评价及保障当地居民的饮食健康提供科学依据,本研究采用气相色谱质谱联用法(GC-MS)对长三角地区13个养殖池塘中5类(蟹类、鳖类、蚌类、虾类、鱼类)水产品体内9种有机磷农药(OPPs)、8种有机氯农药(OCPs)以及4种拟除虫菊酯类农药(SPs)进行了检测,并初步分析了其污染来源。结果表明,水产品体内3类农药均有检出,但残留水平均不高,OPPs残留总量为未检出至299 ng·g-1(dw),三唑磷、伏杀硫磷、喹硫磷和毒死蜱较易在水产品体内蓄积;OCPs残留总量为16~82 ng·g-1(dw),主要污染物为六六六(HCHs),主要来自历史残留;SPs残留总量为44~89 ng·g-1(dw),主要污染物为高效氯氟氰菊酯。水产品体内的农药污染可能与养殖池塘中的沉积物污染密切相关。5类水产品体内3类农药的总风险指数(HI)为0.0020~0.046,远小于1,因此,长三角地区由3类农药造成的健康风险处于安全水平。     

Transfer rate of pesticide residues from medicinal plants in different types of extractive solutions

Abstract

Starting from the suspicion that the medicinal herbs may contain traces of pesticides and taking into account the risks of patients being exposed to contaminated products, the aim of this research was to evaluate the pesticide residues and the degree of their transfer (%) in three types of preparations (infusion, decoctionand cold maceration), for four medicinal plant species very often used in phytotherapy (Rosmarini folium, Menthae folium, Saturejae herba and Basilica herba). For each type of plant product, four samples were purchased from different manufacturers and they have been analyzed using gas chromatography- tandem mass spectrometry. One out of four samples of the same plant species contain at least one pesticide residue above the maximum level and shows a significant transfer of these residues in aqueous extractive solutions during extraction. The highest rate of pesticide transfer from medicinal plants was identified in infusions, recommended by many manufacturers.     

Physiological,biochemical and growth responses of Azolla pinnata to chlorpyrifos and cypermethrin pesticides exposure: a comparative study

Cypermethrin and chlorpyrifos pesticide- (5, 15 and 30 ppm) induced effects on growth, metabolic and antioxidative behaviour of Azolla pinnata were analysed after 96 h of treatment. Growth of A. pinnata was increased by 72–76% over the initial mass under control condition. Further, the growth was decreased by 26 and 30% following treatment with 5 ppm of cypermethrin and chlorpyrifos, respectively, compared to control. Whereas, 30 ppm of pesticide treatment caused substantial decrease in biomass accumulation, even less than the initial mass. The Chlorophyll a content was declined at higher concentrations of chlorpyrifos, while carotenoids content increased significantly in the presence of both the pesticides. Photosynthetic rate showed declining trend with rising concentration of pesticides, while an increasing trend was noticed in the case of respiratory O₂ consumption. The oxidative biomarkers malondialdehyde, superoxide radicals and hydrogen peroxide increased with increasing concentration of both the pesticides. The activity of enzymatic antioxidants enzyme superoxide dismutase, glutathione-S-transferase and ascorbate peroxidase (APX) was enhanced with increasing concentrations of both the pesticides; however, a declining trend was noticed in the case of APX at 30 ppm chlorpyrifos treatment. The study concludes that chlorpyrifos appeared to be more toxic than cypermethrin; however, both the pesticides significantly reduced the growth performance.     

Characteristics and risk assessment of organochlorine pesticide residues in surface sediments collected at the Qingshitan Reservoir

ABSTRACT

An ultrasonic extraction – gas chromatography – electron capture detector analytical method was used to measure the concentration and types of organochlorine (OC) pesticides in sediment to obtain a better understanding of the characteristics and hidden ecological risks associated with OC pesticide exposure in surface sediment of the Qingshitan Reservoir. Fifteen types of OC pesticides were detected in the sediment, and the sum concentration of these chemicals was in the 149.32–490.19 ng/g range (mean value: 319.39 ng/g). The concentrations of detected OC pesticides occurred in the following order: hexachlorohexanes (HCHs) (mean value: 200.17 ng/g) > DDTs (mean value: 36.92 ng/g) > heptachlors (mean value: 32.74 ng/g) > methoxychlor (mean value: 24.13 ng/g). There was a 100% detection rate for HCH isomers, and their concentrations occurred in the following order: β-HCH > δ-HCH > γ-HCH > α-HCH. β-HCH was the main component of HCHs. Ratios between α-HCH/γ-HCH and β-/(α+γ)-HCH were used to investigate the sources of pollution. Most of the surveyed areas were polluted by lindane, which originated from past pesticide residue usage, and no new inputs of HCHs were found. DDT was the major component of the DDTs, and accounted for 52%–87% of the DDTs. The ratios of (DDE+DDD)/DDT at all sample collection points were less than 1, indicating that degradation rate of DDTs in sediment was low and there was a new input of DDTs in these surveyed areas. The ratio of DDD/DDE was less than 1 at most of the sample collection points, indicating that the degradation of DDT in the sediments primarily took place under aerobic conditions. Comparison of OC pesticide residual levels in the underwater sediment collected at the Qingshitan Reservoir to other states and countries showed the pollution level of these chemicals of the Qingshitan Reservoir was relatively high. The ecological risk was assessed based on guideline values of effects range-low (ERL) and effects range-medium (ERM). The results showed that DDD, DDE, DDTs and endrin residues in the sediment posed a moderate ecological risk, but DDT and γ-HCH showed high ecological risk. These OC pesticides might adversely affect biological systems, and need to be addressed.     

农药对秀丽隐杆线虫毒性效应及其机制的研究进展

随着大量农药被广泛的使用并最终汇聚到环境介质中,对生态环境和人体健康产生潜在影响。秀丽隐杆线虫(Caenorhabditis elegans)是土壤中最丰富的后生动物,在土壤生态系统中具有重要地位,并作为一种重要的模式生物广泛应用于环境毒理学研究。本文从秀丽隐杆线虫常用的毒理学研究方法入手,以致死率、生长发育、运动行为、繁殖、活性氧自由基(ROS)水平、细胞凋亡水平、相关基因表达量和蛋白水平等作为测试指标,归纳总结农药对秀丽隐杆线虫的衰老性、神经及生殖系统的毒性效应,分析探索其毒性机理,并展望了未来的研究重点。     

Monitoring the occurrence and distribution of selected organophosphates and carbamate pesticide residues in the ecosystem of Lake Naivasha,Kenya

Although use of pesticides is critical in agricultural production, their residues present a potential risk to non-target organisms and lower the quality of surface water. In Kenya for instance, widespread use of pesticides in the catchment of Lake Naivasha, has raised concern over the years due to possible pollution of the lake through discharge of runoff from agricultural fields. In this study, sediment, water, and fish samples were analyzed for selected pesticide residue contamination. Chlorpyrifos-ethyl (CPF) was detected in the range of 2.6–24.9 ng/ml and 6.8–35.8 ng/g dry weight (dw) in water and sediment, respectively. Meanwhile, diazinon was detected in the range of below detection limit (bdl) to 33.3 ng/ml and bdl to 9.3 ng/g dw in water and sediment, respectively. CPF was detected in fish tissues (Niloticus leucosticus) in the range of bdl to 8.9 ng/g dw with diazinon and carbofuran not detected in any fish sample. A significant difference was observed between different seasons with wet season recording higher levels. Concentrations detected varied seasonally and on average exceeded the maximum criterion set by European Union. Therefore, data generated in this study are useful in environmental risk assessment and as a baseline in formulation of mitigation measures to protect the lake from pesticides residues pollution.     

Effect of commercial pesticides on plant growth-promoting activities of Burkholderia sp. strain L2 isolated from rhizosphere of Lycopersicon esculentum cultivated in agricultural soil

The in vitro study was carried out to examine the toxicity of four commercially used pesticides (phorate, mancozeb, chlorpyrifos, and endosulfan) at the recommended dose (1X) and higher field doses (1.5X, 2X, 2.5X, and 3X) on plant growth-promoting activities of Burkholderia sp. strain L2, isolated from pesticide-treated rhizosphere of Lycopersicon esculentum. At the recommended dose, level of toxicity of all the pesticides to phosphate solubilization activity and indole-3-acetic acid (IAA) production was less detrimental as compared to exceeding doses. At maximum dose rate of each pesticide, the percentage decrease in phosphate solubilization and IAA production over control was found in order of chlorpyrifos < mancozeb < endosulfan < phorate. Present study proves that Burkholderia sp. strain L2 could be used as a potential microbial inoculant in the agricultural soil having persistent pesticide residues.     

Acute toxicity evaluation of triazophos,deltamethrin and their combination on earthworm,Eudrilus eugeniae and its impact on AChE activity

The acute toxicity of three formula grade pesticides namely, triazophos (an organophosphate, OP), deltamethrin (a pyrethroid) and combined pesticide (triazophos?+?deltamethrin) was determined in earthworm, Eudrilus eugeniae. They were exposed to different concentrations of these pesticides for 48 h by paper contact toxicity method. The LC₅₀ values for triazophos, deltamethrin and of combination were determined as 0.076, 0.031 and 0.065?μg/cm², respectively. To study the sublethal effect of these pesticides, E. eugeniae were exposed to 5% and 10% of LC₅₀ of pesticides for 48 h. Morphological alterations such as coiling, clitellar swelling, mucus release and bleeding followed by body segmentation were observed in exposed earthworm. Acetylcholinesterase (AChE) activity assayed in different regions of body segment exhibited a significant (p?<?0.05) decrease in its activity particularly in the pre-clitellar region as compared to other regions. The altered behavioural responses in pesticides exposed earthworms would have been due to decline in AChE activity of the nervous system.     

Hematological and biochemical alterations in sprayers occupationally exposed to mixture of pesticides at a mango plantation in Lucknow,India

The aim of this study was to examine the hematological and cholinesterase (ChE) activity levels of pesticide sprayers employed in mango plantations at Lucknow. Fifty-two sprayers from mango plantations who regularly spray mixtures of pesticides including organochlorines (OC), organophosphates (OP), and carbamates were included as exposed group. Twenty residents living close to mango plantations, but not involved in pesticide spraying were taken as controls. Pesticide residues for OC were analyzed in blood by gas chromatography with electron capture detector. There was a significant decrease in acetylcholinesterase and butyrylcholinesterase activities in pesticide sprayers (n?=?52) relative to the control group (n?=?20). A significant difference in the frequency distribution of hematological variations in red blood cell, white blood cell, monocytes, neutrophils, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and platelet count was observed. Residues of OC pesticides including α-hexachlorocyclohexane (HCH) and total HCH were found to be significantly higher in the blood of sprayers compared to controls. Hematological variations were quantitatively more frequent among the sprayers with 5 years of pesticide exposure compared to the controls and to subjects with less than 5 years of exposure group. Hematological abnormalities in sprayers were correlated with pesticide exposure as evidenced by lower ChE activity as well as the presence of OC pesticides in blood. The poorly controlled use of pesticides in the plantation appeared to have produced sub-clinical intoxication in the sprayers and indicated the need for training and implementation of proper, safer hygiene practices.     

Measurement of residues of benomyl and mancozeb pesticides in shrub and nonshrub cucumbers sampled from different regions of Mazandaran province (Iran)

Farmers use mancozeb against cucumber pests, such as Perono spoxa, in large amounts without consideration of potential chronic human health hazards resulting from pesticide accumulation in food and fruits. Considering the necessity of food for humans and importance of fruit in daily diet, as well as the determination of pesticide residues in food and fruits as mandated by WHO and FAO, the residues benomyl and mancozeb, the most prominently used pesticides in Iran, were measured. In this study cucumbers from 76 sample regions were used. Data showed that the amounts of benomyl was related to the type of cucumber in the regions, but there was no significant relationship between the amount of mancozeb and type of cucumber.     

Quantification of pesticide residues on plastic mulching films in typical farmlands of the North China

• Pesticide residuals on mulching film of Shandong, Tianjin and Hebei. • Detected 29 pesticides in soil and 30 in mulching film. • Pesticides on plastic films: 86.4‒22213.2 ng/g and in soil: 9.3‒535.3 ng/g. • Pesticides on plastic films 20 times higher than in soil. Plastic debris as new pollutants attracts much attention in the recent years. The plastic mulching films is one of the most important plastic debirs source in the environment. The aim of this work was to investigate the current status of pesticide residues on the plastic mulching films. Based on the QuEChERS method, multi-residue methods for detection of pesticide residues with gas chromatography tandem mass spectrum (GC-MS) and high performance liquid chromatography tandem mass spectrum (HPLC-MS) were developed for the analysis of the pesticides residues in plastic film and soil samples from Tianjin, Hebei and Shandong. The total concentrations of pesticide residues were in the range of 86.4‒22213.2 ng/g in plastic film debris, which was about 20 times higher than that in soil (9.3‒535.3 ng/g). Residual level of pesticides varied greatly in different samples. The historical usage and recent application of pesticides were the main sources for pesticide residues on plastic films and soil. In short, plastic mulching films could act as a sink for pesticides in farmland and the ubiquitous pesticide residues on plastic films should draw more attention.     

Visual disturbances with cholinesterase depletion due to exposure of agricultural pesticides among farm workers

In this study, risk assessment for visual disturbances among farm workers spraying agricultural pesticides in farms have been assessed. A cross-sectional study for the assessment of visual disturbances was undertaken using questionnaire survey and clinical examination among agricultural workers exposed to pesticides. Two hundred and thirty nine pesticide sprayers participated in the study. The study was compared to 110 controls not occupationally exposed to pesticides with similar socio-economic status. The prevalence of ocular morbidity was found to be 40% among pesticide sprayers which was significantly higher (p?p?相似文献   

A study of neurotoxicity of BHC in relation to residual accumulation on the brain tissue of Heteropneustes fossilis (Bloch)

Neurotoxic effect of BHC, the organochlorine pesticide in Heteropneustes fossilis has been studied exposing at the dose concentrations of 1 ppm, 5 ppm and 10 ppm in lab aquarium for 96 hours over a period of one year. The results showed the behavioural abnormalities in different exposure concentrations such as dysfunction of endocrine gland, excretion of mucus, dispigmentation, sign of restlessness, erratic swimming with rapid jurkey movement, spiralling and convolution showing severe effect in central nervous system. Therefore an attempt has been made for monitoring of BHC residues viz. alpha, beta, gamma isomers in the brain tissue exposed to different sublethal concentrations using Gas liquid chromatography. The mean values of isomers were found to be 1.587 microg/gm for 1 ppm, 2.993 microg/gm for 5 ppm and 3.78 microg/gm for 10 ppm test group. Severe behavioural abnormalities were recorded at high dose concentration of pesticides with higher accumulation of pesticide residues in brain tissue.