了解深部微生物群落组成和功能对探索早期生命起源和利用其独特功能均有重要意义.地热区深部碳酸盐岩岩溶-裂隙热储环境中微生物群落组成和功能特性仍然不清.为了探测冀中地热区深部热水微生物群落组成与功能特性,以浅层水和土壤为参照,利用冀中地热区地热科学钻探孔抽水试验,采集深部热水样,同时采集每个钻孔上方附近的大田农灌井浅层水和土壤样品,用于16S rRNA基因高通量测序,并使用PICRUSt软件进行功能预测.结果显示冀中地热区深部热水含有38个菌门,541个菌属,以细菌为主(97.5％),古菌很少(2.5％),特征性菌群主要是厚壁菌门Firmicutes、硝化螺旋菌门Nitrospirae、热袍菌门Thermotogae和广古菌门Euryarchaeota,优势菌属包括硫酸盐还原菌,固碳作用、发酵作用和硫酸盐还原作用可能较强,而产甲烷作用和反硝化作用可能很弱.深部热水出现好氧嗜温菌,说明在大流量抽水条件下,深部热水受到富含变形菌门Proteobacteria浅层水的补给.本次研究揭示了冀中地热区深部碳酸盐岩岩溶-裂隙热储层含有种类丰富、功能多样的微生物群落,微生物群落特征可作为深部热水与浅层水水力联系的指示剂.

The hydrogeochemical characteristics of geothermal fluids can reveal the genesis of geothermal systems and act as important references for developing and using geothermal resources. This study presents hydrogeochemical processes and thermal cycle mechanisms of typical geothermal fields in Western Sichuan. Based on the geological conditions in Western Sichuan, 29 hot springs in three geothermal fields in the Batang and Litang areas were selected for hydrochemical and isotopic ( δ D and δ 18 O) analyses. Furthermore, the temperature of the thermal reservoir was calculated and the upflow cooling process of the hot springs was analyzed. Most of the subterranean hot waters in Batang and Litang are of the HCO 3 ‐Na hydrochemical type. The ion variation in Batang is primarily affected by water‐rock interactions. There is a strong positive correlation between Na + , B − , and Cl − in Litang, suggesting that they have the same material source. The Na + and metaboric acid content is relatively high, which indicates that the groundwater runoff in both areas is relatively long‐lasting, with reduced flow velocity; moreover, the metasilicic acid content is relatively high, which supports this conclusion. Both hydrogen and oxygen isotopes plot near the atmospheric precipitation line, indicating that groundwater recharge is functionally obtained from precipitation. The calculated thermal storage temperatures in Batang and Litang were 88–199°C and 96–154°C, respectively. The proportion of cold water mixing in Batang was 64%–67%, while that in Litang was 60%–68%. According to the calculated results, the initial thermal cycle depth of the Batang area (4540–4780 m) was greater than that of the Litang area (3150–3960 m). The enthalpy of the deep parental geothermal fluid in Batang was 1550 J/g with a Cl − concentration of 37 mg/L, while that in Litang was 2100 J/g with a Cl − concentration of 48 mg/L.

[背景]地下深部存在一个生物圈,深部沉积岩、玄武岩、花岗岩和变质岩等岩性环境的微生物群落已被调查,而地下深部碳酸盐岩岩溶-裂隙热储层微生物群落特征仍然不清.硫酸盐还原菌(sulfate-reducing bacteria,SRB)是地下深部频繁检出的微生物.[目的]建立快速准确定量深部热水硫酸盐还原菌的微滴数字PCR (droplet digital PCR,ddPCR)技术.[方法]以SRB的功能基因dsrB为检测目标,优化SRB ddPCR技术的退火温度,考察其线性范围、敏感性、重复性和特异性,并将该技术用于实际样品的检测. [结果]SRB ddPCR技术的最佳退火温度为54℃,检测的线性范围为1.1×100-1.1×105 copies/μL-DNA,相关系数R2为0.996,检出限为1 copy/μL-DNA,重复性的相对标准差优于9％,对3种非SRB人工构建的质粒均没有扩增,显示该技术具有很好的线性关系、敏感性、重复性和特异性.利用该技术对冀中地热区深部热水、浅层水和土壤样品进行了检测,平均含量分别为(4.0±8.4)× 103 copies/mL、(1.6±3.5)× 102 copies/mL和(1.5±1.2)× 103 copies/g-dw.与浅层水和土壤相比,深部热水富含SRB菌.[结论]为了提高地下深部生物圈认识和合理开发利用深部热水,建立了一种快速、灵敏、准确的SRB ddPCR检测技术,同时为其他指示菌检测技术的建立提供了参考.

Subtle reservoir exploration could not be commonly carried out by costly conventional methods especially when subtle reservoirs are becoming smaller in surfaces and reserves. The epochal genetic quantitative techniques were proved to be suitable for the subtle reservoir prospecting, but the anomaly grade classification method did not match the quantitative data qualitative dividing. And the combination of genetic quantitative techniques and geophysical exploration methods to the prospecting application for subtle reservoirs still need more studies. For the questions, 194 topsoil samples and 237.05 km continuous electromagnetic profiling (CEMP) lines were arranged in typical subtle reservoirs in the Suganhu Basin. The realtime-PCR technique was used to get methanotrophic, propanotrophic functional gene pmoA and prmA quantitative data. The results showed that the classification method using cumulative probability distribution curves with regression analyses could be used to classify the genetic quantitative anomalies by the equation of y = a(z). The genetic quantitative anomalies and source strata thickness showed positive correlation along the section lines in the east of study areas. Integrated studies between genetic quantitative anomalies and CEMP results in the stereoscopic direction indicated three promising areas in the Suganhu Basin. This research is expected to be extended to the same type of reservoirs prospecting work.

含铵化肥厂废水污灌有可能引起地下水污染.包气带土壤的吸附作用是阻滞其铵进入含水层的重要过程.选取某包气带不同深度4个典型土样,利用室内批模拟实验,以实验室配制硫酸铵溶液作对比,研究土壤对化肥厂废水中铵的吸附动力学及等温吸附特征.结果 显示:土壤颗粒极易吸附铵,废水有机物等物质能延长吸附平衡时间,并影响土壤对铵的吸附能力——在低浓度铵溶液中表现为增加作用,在高浓度铵溶液中的表现为受土壤有机质含量影响;不同质地土壤对铵具有不同的吸附行为,总体上黏性土的吸附能力大于砂土;与Freundlich和Langmuir等温式相比,BET等温式能够与实验吸附数据更好拟合,验证了BET公式用于铵等温吸附特征研究的可行性,表明铵在土壤胶体颗粒中的吸附方式为多分子层吸附.

为了揭示区域地下水不同深度微生物群落结构特征及其与地下水环境相互作用关系,选取北京琉璃河地区,采集不同深度地下水样品,用于水化学分析和微生物16S rRNA基因V4-V5区测序.水化学分析结果显示,地下水中8种主要离子浓度随深度增加均呈减小趋势,Cl-、SO42-、NO3-变化规律显著,工业较发达区NO3-浓度达155.30mg/L,SO42-浓度达321.00mg/L,部分浅层地下水受NO3-和SO42-污染.微生物分析结果显示,地下水中微生物群落多样性受深度影响显著,随深度增加微生物群落组成丰富.地下水中优势菌门为Proteobacteria(26.2％～95.2％),优势菌属为Pseudomonas(1.5％～32.2％),不同深度微生物菌属组成差异明显,浅层、中层和深层地下水特有菌属数目分别为74,60,54.NO3-、SO42-、深度是影响地下水微生物群落的主要因子,且NO3-、SO42-浓度受地下水深度影响程度大.地下水深度是影响微生物群落结构差异的重要原因.

为查明区域地下水化学特征对微生物群落结构影响,在北京琉璃河镇及周边地区采集9组地下水样.在调查地下水化学特征基础上,利用高通量测序技术,对地下水样进行16S rRNA基因V4-V5区测序,用于分析地下水化学特征与微生物群落结构间相关性.水化学结果表明,研究区地下水共有HCO3-Ca· Mg、HCO3·Cl-Ca、HCO3·Cl· SO4-Ca和HCO3-Ca水4种水型,主要水型为HCO3-Ca·Mg水,部分地下水受NO3-、SO42-污染.微生物结果表明,研究区地下水优势菌门为Proteobacteria,但无明显优势菌属.不同水型中微生物多样性呈地下水化学类型越简单,微生物群落多样性水平越低趋势.HCO3-Ca·Mg、HCO3·Cl-Ca、HCO3·Cl· SO4-Ca和HCO3-Ca水4种水型中共有菌属数量为113个,特有菌属数量分别为186、32、33和2个.影响因子对微生物群落结构影响程度为浊度＞EC(电导率)＞DO(溶解氧)＞pH,较高的EC和DO值有利于Pseudomonas、Vogesella、Hydrogenophaga菌属生长,pH适度增加有利于Gallionella、Nitrospira菌属生长.综上可知,区域地下水化学特征对微生物群落结构具有显著影响.

实时荧光PCR实现了功能基因定量,然而定量数据在油气藏,尤其是勘探难度大的隐蔽油气藏,定性预测方面的技术优势仍具开发潜力.以勘探程度较低的苏干湖盆地隐蔽油气藏为对象,点间距1～4 km采集50 cm深土样191个,测定甲烷、丙烷氧化菌功能基因(pmoA、prmA),基于累积概率分布与回归分析,划定了微生物异常体系,中异常与物探地层解译结合,圈定了盆地油气勘探有利区.结果显示:pmoA、prmA累积概率分别符合近4X×104、2X×104 copies/g的指数分布规律,取自然数1、2、3、4分别以4、16、64、256和(2、4、8、16)×104 copies/g划分二者异常的低、中、高、超高4等级;中异常以上等级于盆地东、西、中3部均有分布,其中,东、西分布稍偏北于二叠系、侏罗系地层,中部分布于二地层之间,保守圈定以上分布为盆地油气藏勘探有利区.

Propane-oxidizing bacteria in surface soils are often used to indicate the position of oil and gas reservoirs. As a potential replacement for the laborious traditional culture-dependent counting method, we applied real-time fluorescent quantitative polymerase chain reaction detection as a quick and accurate technology for quantification of propane-oxidizing bacteria. The propane monooxygenase gene was set as the target and the assay is based on SYBR Green I dye. The detection range was from 9.75 × 10 8 to 9.75 × 10 1 gene copies/µl, with the lowest detected concentration of 9.75 copies/µl. All coefficient of variation values of the threshold cycle in the reproducibility test were better than 1%. The technique showed good sensitivity, specificity, and reproducibility. We also quantified the propane-oxidizing bacteria in soils from three vertical 250 cm profiles collected from an oil field, a gas field, and a nonoil gas field using the established technique. The results indicated that the presence of propane monooxygenase A genes in soils can indicate an oil or gas reservoir. Therefore, this technique can satisfy the requirements for microbial exploration of oil and gas.

Epidemiological studies consistently show that excessive amounts of reactive oxygen species (ROS) is an important underlying cause of the initiation of oxidative stress in various tissue injuries as well as cell death and several chronic diseases. Several compounds, such as D-Galactose (D-Gal), bromobenzene, carbon tetrachloride (CCl4), acetaminophen, and ethanol have been implicated in the etiology of liver diseases. D-Gal is commonly used as a toxin to induce reactive oxygen species and hepatic disorders, which has been extensively used in oxidative damage animal models to evaluate the therapeutic potential of drugs and dietary antioxidants. Due to the risks of synthetic antioxidants, there is a growing interest in the use of natural antioxidants to prevent ROS related liver pathologies. Therefore, during the past several decades, human nutrition and biochemistry research focused on antioxidants derived from foods that could prevent or diminish ROS-induced damage. More recently, there is growing interest in natural hydrolysed protein antioxidants from many plant and animal sources. So, we hypothesized that the administration of antioxidant peptides might be useful for preventing various types of oxidative stress in vivo. In our previous experimental studies, Fraction Ⅰ (MW <1 ku), Fraction Ⅱ (MW 1–4 ku) and Fraction Ⅲ (MW >4 ku) were isolated from the hydrolysates ofScomberomorous niphonius skin through ultrafiltration membrane with the cutoff of 1 and 4 ku, respectively. Among them, the Fraction (1–4 ku) exhibited the highest radical scavenging activity, inhibiting both lipid and protein oxidation of cooked patties during refrigerated storage, and protecting Caco-2 cells against the toxicity caused by H2O2. Therefore, the aim of this study was to evaluate the antioxidant effects of Fraction Ⅱ on D-Gal-induced liver damage in rats. Wistar rats were randomly divided into 6 groups, which were normal control, negative control, D-Gal+Low-dose of Fraction Ⅱ, D-Gal+Middle-dose of Fraction Ⅱ, D-Gal+High-dose of Fraction Ⅱ, and D-Gal +VE as positive control for seven weeks. The activities of serum asparate aminotransferase (AST), alanine aminotransferase (ALT), monoamine oxidase (MAO) and antioxidant enzyme activities including superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), total antioxidant capacity (T-AOC), nitric oxide (NO) and malondialdehyde (MDA) contents in liver were determined. The result showed that all doses of Fraction Ⅱ could significantly reduce the ALT and AST level in serum of the rat, while M-dose and H-dose of Fraction could sharply decrease the MAO level. Compared with negative control group, SOD, GSH-Px, CAT activity and T-AOC of liver of the rats were increased significantly. the MDA and NO contents in liver of the rats were reduced when treated with all doses of Fraction. Treatment with high dose Fraction (200 mg/kg) could increase the SOD, GSH-Px activity to 236.27 and 182.23 U/mg protein, which could reach the level of normal and positive control, decrease the contents of AST, ALT and MDA to 302.47 U/L, 220.43 U/L and 138.83 nmol/mg protein, which was close to the level of normal control. Moreover, Fraction Ⅱ could significantly reduce the injury score of prominent nucleus, visible central veins and necrosis in rats. These data indicate that Fraction Ⅱ can protect the liver against D-Gal induced oxidative damage.

In order to test the application effect of Diver automatic water level recorder in the study of hydrogeological parameters from pumping test data,a pumping test was carried out in the Liuli River area in Fangshan,Beijing.The characteristics of the original data from Diver and the manual measurement in the pumping test were compared,and the hydrogeological parameters were calculated based on Diver data.An average difference of the main well data between the Diver and the manual measurement was 0.17m,and the average difference of the auxiliary well data was 0.02m in the whole process of the pumping test.Finally,it was calculated that the value of the k was 91.51m/d,the value of the T was 1555.72m2/d and the value of the S was 0.0314 based on the Diver data.Diver has the advantages of large amounts of continuous and accurate data to ensure that the hydrogeological parameters are performed correctly.

The long-term oil exploitation in oil fields has led to pollution of surrounding soil, creating a serious ecological problem. In order to promote and improve the application of microbial remediation in oil contaminated soil, experiment is carried out in polluted area in Zhongyuan Oilfield. In the experiment, indigenous microorganisms and other physical and chemical methods are employed, ryegrass is grown, and environmental factors in soil are regulated to degrade the oil and treat the polluted soil. Results show that when the average oil content in the soil is about 523.08 mg/kg, 65 days' remediation through plants and microorganisms could help bring the oil content down to 74.61 mg/kg, achieving a degradation rate of 85.74%; through salinity treatment, salt content in soil is reduced by 62.93-82.03% to 399-823 mg/kg from previous 2.22 g/kg. Through this experiment, the bioremediation method is improved and its effectiveness and feasibility are testified. The result has been applied in Zhongyuan Oilfield and has brought fair ecological and economic benefits, providing technical support to the treatment of contaminated soil of the same kind, and offering some insights to the treatment of soil contaminated by other organic pollutants.

To evaluate the application of a DNA-based analysis of a microbial technique for oil and gas prospecting, the hydrocarbon potential predicted using the methanotrophic pmoA and propanotrophic prmA genes as targets was investigated in soil above a known oil reservoir. In total, 82 soil samples were collected at 50-cm depths above the Xiliu oil reservoir for quantitative real-time polymerase chain reaction (RT-PCR) analysis. The pmoA gene content ranged from 0 to 2.7x10(6) copies/g dw, whereas that of prmA varied from 0 to 1.6 x 10(6) copies/g dw. Microbial anomaly distribution maps of normalized values for the prmA gene as a main indicator suggested that the area with the highest potential of prospecting was an undeveloped location, and an area with a relatively large prmA background was identified at this oil-producing site. The areas of microbial anomaly exhibited different prmA/pmoA ratios (20, 1-5, 0.0n-0.25 and 0), and this ratio may be used as an indicator for predicting the properties of oil and gas reservoirs and biogenic methane or the influence of oil-producing activities. Our results suggest that a DNA-based analysis of a microbial technique is a powerful tool for oil and gas prospecting. This study provides a new microbial technique for the prospecting of oil and gas.

为了较系统地了解基因芯片在地下水污染研究中的应用进展,调研了基因芯片技术及其在地下水污染研究中应用的有关文献,简述了基因芯片原理、分类及实施流程,总结了系统发育寡核苷酸芯片和功能基因芯片在地下水污染研究中的最新应用进展,探讨了基因芯片检测性能、数据分析和应用等方面存在的问题和措施,在基因芯片性能改进和应用方面提出了进一步研究的方向.

Methanotrophs have long been used as an important biological indicator for prospecting of oil and gas, while the indication of propanotrophs in hydrocarbon micro-seep systems is still poorly investigated. In this study, the abundance and diversity of the methanotrophic pmoA gene and the propanotrophic prmA gene as target genes were investigated in soils above Yangxin oil reservoir and Beiguan non-petroliferous area using molecular biological techniques. A total of 14 soil samples were collected at different depths (5, 20, 50, 100, 150, 200 and 250 cm) of two 2.5-m soil profiles located separately within the oil field and the non-petroliferous area for analysis of fluorescent quantitative real-time polymerase chain reaction (RTPCR) (14 samples) and clone libraries (4 samples). The results demonstrated high presence of the propanotrophic prmA gene ranging from 7.68 x 10(5) to 2.29 x 10(7) copies/g dw (gene copies per gram soil of dry weight) in soil from the oil field relative to the non-petroliferous area for which the same measurements yielded results all below detection limit except for the 5-cm sample. On the other hand, oil field soil yielded much lower content of the methanotrophic pmoA gene (below detection limit to 5.6 x 10(2) copies/g dw) than the non-petroliferous area (1.14 x 10(3) copies/g dw to 1.26 x 10(5) copies/g dw) below 20-cm depth due to influence of biogenic methane, implying that propanotrophs may be better indicator bacteria for prospecting of oil and gas. Almost all pmoA clones of two 50-cm soil samples phylogenetically belonged to Gamma-Proteobacteria and the predominant pmoA OTUs were all uncultured bacteria. All prmA clones of two 5-cm soil samples were derived predominantly from Actinobacteridae (25.7%) and Alpha-Proteobacteria (74.3%), and all dominant prmA OTUs were also clustered with uncultured bacteria. Our results confirm that propanotrophs may be better indicator bacteria for prospecting of oil and gas and enrich the knowledge on diversity of methanotrophs and propanotrophs in the oil field and the non-petroliferous area.

Experimental study of enhanced in-situ micro-ecological remediation of petroleum contaminated loess soil was carried out in Zhongyuan oil production areas, and the enhanced in-situ micro-ecological remediation technique includes optimistic in-situ microbial communities, physical chemistry methods, alfalfa planting and regulation of soil environmental elements. Experiments showed that the oil content in the contaminated soil with oil content about 2 898.25 mg/kg can be reduced about 98.61% after in-situ micro-ecological remediation for 99 days, which demonstrated the effectiveness of in-situ micro-ecological remediation methods for petroleum contaminated soil in central plains of China, and explored the practical and feasible application of these methods.

[目的]建立快速检测甲烷氧化菌含量的SYBR Green Ⅰ实时荧光定量PCR技术,用于油气微生物勘探.[方法]以含有甲烷氧化菌功能基因pmoA片段的重组质粒为标准品,优化实验条件,建立标准曲线,进行敏感性、重复性和特异性评价,并将该技术用于实际样品的检测.[结果]该技术标准曲线的相关系数R2为0.999 9,扩增效率为99.976％,检测范围为3.897×101-3.897×109 copies/μL,检出限约为40 copies/μL,重复性实验中CT值的变异系数优于3％,对12种非甲烷氧化菌均没有扩增,显示该技术具有很好的敏感性、重复性和特异性.利用该技术对气田、油田和非油气田土样进行了检测,发现气田具有明显的异常高值.[结论]为油气田的勘探建立了一种高效、特异、灵敏、准确的甲烷氧化菌荧光定量PCR检测技术,同时为其它指示菌检测技术的建立提供了参考.

以某废弃采油井井场做为研究对象,对油井不同方位、距离和深度剖面土壤石油含量和物理化学性质进行测试,并利用实时定量PCR技术对场地细菌和真菌生物量展开调查,以期能够为修复工作提供数据基础.结果显示,该场地遭受了严重石油污染和盐碱污染.土壤细菌16S rDNA和真菌ITS rDNA定量调查结果显示,细菌和真菌的生物量均高于普通土壤,尤其石油含量与细菌16S rDNA基因具有一定的正相关性,土壤的生态系统的健康和稳定性变弱.通过土壤三氮的测定,结合石油和微生物量的对比得出,嗜油微生物大量生长导致了土壤硝化作用的减弱和NH4+的积累、土壤N素的缺失和土壤健康及稳定性的减弱.因此,有必要对该污染场地进行治理.基于分析数据,提出了一些该污染场地的修复建议.

To assess whether the anomalies of methanotrophic pmoA and propanotrophic prmA genes as target genes can reflect the existence of underlying oil and gas reservoirs, natural abundance and distribution of target genes were investigated in soil above known gas fields, oil fields and non-oil and gas fields, China. A total of 45 soil samples were collected from different depths of 2.5-m soil profiles in Xiaoquan gas field, Xiliu oil field and Beiguan non-oil and gas field and from 50cm depth of the old oil area, the new developed area, the oil producing area and the undeveloped area of Xiliu oil field, respectively for analysis of target genes by SRBR Green-based fluorescent quantitative real-time polymerase chain reaction (RT-PCR) technique. Distributions of target gene contents in 2.5-m soil profiles of the three fields show that 50cm was considered to be optimal sampling depth for microbial prospecting of oil and gas reservoirs. At 50cm depth of 2.5-m soil profiles, the gas field had very high contents of methanotrophic pmoA gene (3.8×105copies/gdw) and propanotrophic prmA gene (5.92×105copies/gdw), the oil field had medium contents of methanotrophic pmoA gene (2.8×104copies/gdw) and propanotrophic prmA gene (4.1×104copies/gdw), and the non-oil and gas field had low contents of propanotrophic prmA gene (below detection limit) and relatively high contents of methanotrophic pmoA gene (1.26×105copies/gdw) due to the influence of biogenic methane. The anomalies of methanotrophic pmoA gene and propanotrophic prmA gene can reflect the existence of underlying gas deposits. Different areas of the oil field had different target gene contents in the new developed area ((1.7–2.3)×106copies/gdw for the prmA gene, 1.0×103–1.2×104copies/gdw for the pmoA gene), the old oil area (below detection limit −2.0×106copies/gdw for the prmA gene, below detection limit −7.1×104copies/gdw for the pmoA gene), the undeveloped area (1.8×105–1.5×106copies/gdw for the prmA gene, 5.3×104−3.3×105copies/gdw for the pmoA gene) and the oil producing area (below detection limit −1.0×106copies/gdw for the prmA gene, 1.4×105–4.8×105copies/gdw for the pmoA gene). Comparisons of target gene contents in different areas of Xiliu oil field indicate that oil productive activities had a large impact on contents of target genes in soil above the oil field. The anomalies of propanotrophic prmA gene with low contents of methanotrophic pmoA gene can reflect the existence of underlying oil reservoirs. Analysis of the relationships between contents of target genes may be used for identification of gas fields, oil fields and the influence of biogenic methane. Our results suggest that quantitative RT-PCR assay-based methanotrophic pmoA gene and propanotrophic prmA gene anomalies can reflect the existence of underlying oil and gas reservoirs.

Various analytical techniques have been built based on physical and chemical properties of ammonium ion.The conventional NH+4-15N Isotopic analytical technique include the processes of NH+4-15N collection,transforming NH+4 to N2 and analyzing with 15N isotope mass spectrometric.The methods of NH+4 collection are various including distillation,diffusion,ion exchange column method,zeolite extraction and so on.The methods of transformation of gaseous N2 which are suitable for mass spectrometry analysis include chemical wet and dry combustion.The methods of ammonium collection,transformation,and the test of 15N were summarized in this article.The advantage,disadvantages and applicable ranges of each method were also introduced.The latest methods of chrom-mass spectrometry and azide were discussed.Finally,in order to make more widespread applications in the field of environment and hydrogeology,the development of testing techniques of NH+4-15N isotopes in the groundwater was prospected.