Phenol oxidase, peroxidase and organic matter dynamics of soil

Extracellular enzymes mediate the degradation, transformation and mineralization of soil organic matter. The activity of cellulases, phosphatases and other hydrolases has received extensive study and in many cases stoichiometric relationships and responses to disturbances are well established. In contrast, phenol oxidase and peroxidase activities, which are often uncorrelated with hydrolase activities, have been measured in only a small subset of soil enzyme studies. These enzymes are expressed for a variety of purposes including ontogeny, defense and the acquisition of carbon and nitrogen. Through excretion or lysis, these enzymes enter the environment where their aggegrate activity mediates key ecosystem functions of lignin degradation, humification, carbon mineralization and dissolved organic carbon export. Phenol oxidases and peroxidases are less stable in the environment than extracellular hydrolases, especially when associated with organic particles. Activities are also affected, positively and negatively, by interaction with mineral surfaces. High spatiotemporal variation obscures their relationships with environmental variables and ecological process. Across ecosystems, phenol oxidase and peroxidase activities generally increase with soil pH, a finding not predicted from the pH optima of purified enzymes. Activities associated with plant litter and particulate organic matter often correlate with decomposition rates and potential activities generally increase with the lignin and secondary compound content of the material. At the ecosystem scale, nitrogen amendment alters the expression of phenol oxidase and peroxidase enzymes more broadly than culture studies imply and these responses correlate with positive and negative changes in litter decomposition rates and soil organic matter content. At the global scale, N amendment of basidiomycete-dominated soils of temperate and boreal forest ecoystems often leads to losses of oxidative enzyme activity, while activities in grassland soils dominated by glomeromycota and ascomycetes show little net response. Land use that leads to loss of soil organic matter tends to increase oxidative activities. Across ecosystems, soil organic matter content is not correlated with mean potential phenol oxidase and peroxidase activities. A multiple regression model that includes soil pH, mean annual temperature, mean annual precipitation and potential phenol oxidase activity accounts for 37% of the variation in soil organic matter (SOM) content across ecosystems (n = 63); a similar model for peroxidase activity describes 32% of SOM variance (n = 43). Analysis of residual variation suggest that suites of interacting factors create both positive and negative feedbacks on soil organic matter storage. Soils with high oxygen availability, pH and mineral activity tend to be substrate limited: high in situ oxidative activities limit soil organic matter accumulation. Soils with opposing characteristics are activity limited: low in situ oxidative activities promote soil organic matter storage.     

The effects of copper on microbial activity and the degradation of atrazine and indoxacarb in a New Zealand soil

Copper is present in a range of fungicides as well as in some animal manures and biosolids that are applied to agricultural soils as fertilisers. Elevated and increasing levels of copper in agricultural soils are of worldwide concern. Copper is toxic to soil microorganisms and has been reported to reduce the ability of soil microorganisms to degrade pesticides. A glasshouse study was undertaken to determine if copper inhibited the degradation of atrazine and indoxacarb in soil. A fine sandy loam agricultural soil was fortified with copper at five concentrations over a concentration range of 0–1000 mg/kg copper, then field-aged for 6 months prior to treatment with either indoxacarb or atrazine at a rate of 2 mg/kg. The soils were sampled twice at intervals based on published half-lives. The samples were analysed for a range of parameters including total and bioavailable copper, urease and phosphatase activity, ergosterol and either indoxacarb or atrazine and its degradation products. The soil microbial biomass and enzyme activities decreased with increasing copper concentration (p < 0.05). There were no significant differences in soil atrazine and indoxacarb concentrations between the copper levels. At sampling time two, the concentrations of hydroxyatrazine in treatments containing the three highest copper concentrations were significantly greater (p < 0.05) than for the control soil. Our results indicate that copper does not inhibit the first step of indoxacarb and atrazine degradation, but may affect degradation of secondary metabolites like hydroxyatrazine in soil.     

Effect of soil organic matter on peroxidase activity of wheat roots

Some soil organic matter fractions inhibited the peroxidase activity of wheat-root filtrates, but the effect was related to the enzyme hydrogen donor used. In the presence of o-dianisidine as the hydrogen donor, humic acid and the fractions obtained from it by water or acid refluxing inhibited the enzyme activity. The greatest effects were produced by those fractions which were insoluble after such treatments. The inhibitory effects were reversible and non-competitive, the Michaelis constant of the enzyme being only slightly affected, and independent of pH of the assay media. When guaiacol was used as the enzyme substrate the inhibitory effects of the humic acid and its fractions were variable and less marked.p-Coumaric and p-hydroxybenzoic acids inhibited peroxidase activity when o-dianisidine was used as substrate, but stimulated the enzyme in the presence of guaiacol. Polymaleic acid which is thought to have a similar structure to fulvic acid, inhibited peroxidase in the presence of both hydrogen donors. whereas fulvic acid was considerably less effective.     

大棚菜田种植年限对土壤重金属含量及酶活性的影响

为了探讨大棚种植年限对大棚菜田土壤重金属累积、土壤酶活性的影响以及二者的关系,采集不同种植年限(0、5、10、15、20、25、30 a)大棚菜田土壤样品共140份,测定土壤样品中重金属的含量以及土壤酶活性。结果表明:大棚菜田土壤中重金属Zn、Pb、Cu的含量和种植年限极显著相关;重金属Cd、Ni、Mn的含量和种植年限显著相关;重金属Cr的含量和种植年限不相关。大棚菜田土壤中过氧化物酶、多酚氧化酶、淀粉酶活性和种植年限极显著相关,磷酸酶、蔗糖酶活性和种植年限显著相关,过氧化氢酶、脲酶、蛋白酶活性和种植年限相关性不显著。随着种植年限的延长重金属Zn、Cu含量对多酚氧化酶、过氧化物酶活性有抑制作用,其敏感性顺序为:过氧化物酶对Zn敏感性>多酚氧化酶对Zn敏感性>过氧化物酶对Cu敏感性>多酚氧化酶对Cu敏感性。土壤中过氧化物酶、多酚氧化酶可以作为重金属Zn污染的指示酶,过氧化物酶可以作为重金属Cu污染的指示酶。该文为设施污染土壤环境质量评价提供依据。     

Persistenz von Atrazin und seiner Metaboliten im Boden nach einmaliger Herbizidanwendung

Persistance of atrazine and its metabolites in soil after a single herbicide application The breakdown of atrazine in soil was investigated under field conditions. In spring 1973 the soil was treated with ¹⁴C ringlabeled atrazine. In summer 1981 soil samples were collected and analysed. The investigated soil still contained about 83% from the initial ¹⁴C activity. From this only 40% could be extracted. Besides traces of atrazine were further identified six metabolites which originated from the parent compound through N-dealkylation and hydrolysis.     

农田尺度降雨入渗—重分布条件下阿特拉津在非饱和土壤中淋溶风险的评价

以农药阿特拉津为研究对象,通过在北京郊区一个面积为72 9m2 (2 7m×2 7m)的田间采集10 0个土壤样品,分别测定其主要理化特性,由土壤的机械组成和干容重测试数据,采用土壤传递函数生成了vanGenuchten型的水力学参数,并进一步间接计算得到阿特拉津运移的弥散度,同时,由实测的土壤有机碳含量估算了阿特拉津的吸附参数。在此基础上,根据柱模型假设,运用HYDRUS 1D软件,就所设计的由实际背景概化而来的降雨入渗—重分布算例,对阿特拉津在农田尺度非饱和土壤中的淋溶动态进行了数值模拟。结果表明:对一场雨量为90mm、雨强为30mmd-1的降雨,在连续3d降雨接着重分布2 0d的情况下,若忽略蒸散作用对土壤水分和阿特拉津运动的影响,则降雨入渗和降雨入渗—重分布过程结束时,通过土壤2 0cm耕层的阿特拉津的最大累积淋溶量分别占施用量的17.87%和75 .4 1% ;采样区域内阿特拉津淋溶通量的空间分布存在较大差异。所探明的阿特拉津易淋溶带,不仅为合理使用该农药、保护土壤环境提供了定量的依据,而且为预防该农药对浅层地下水的污染提供了重要的信息     

Effect of gallium on photosynthetic pigments and peroxidase activity of Chlorella pyrenoidosa

Green algae (Chorella pyrenoidosa) were grown in iron‐deficient and normal iron supplied nutrient solution and treated with 1 uM gallium. Treatment with gallium enhanced the growth rate and chlorophyll content of algae cultures. Significant changes were recorded in individual photosynthetic pigment composition of treated cultures. In both iron‐deficient and normal iron supplied cultures, gallium treatment enhanced peroxidase enzyme activity. Partially purified peroxidase showed different kinetic properties in the samples treated with gallium. An iron‐gallium interaction is proposed that affects chlorophyll metabolism and peroxidase activity in green algae.     

Catalytic characteristics of peroxidase from wheat grass

The crude enzyme extract of wheat grass was heated at 60 degrees C for 30 min, followed by ammonium sulfate fractionation and isoelectric chromatofocusing on Polybuffer exchanger (PBE 94) for purification. The purified peroxidase was then characterized for its catalytic characteristics. It was found that AgNO3 at a concentration of 0.25 mM and MnSO4 and EDTA at concentrations of 5 mM significantly inhibited the activity of wheat grass peroxidase. However, KCl, NaCl, CuCl2, CaCl2, ZnCl2, and MgCl2 at concentrations of 5.0 mM and HgCl2 at a concentration of 0.25 mM enhanced enzyme activity. Chemical modification significantly influenced the activity of wheat grass peroxidase. Particularly, N-bromosuccinimide (5 mM) inhibited 16% of the enzyme activity, whereas N-acetylimidazole (2.5 mM), diethyl pyrocarbonate (2.5 mM), and phenylmethanesulfonyl fluoride (2.5 mM) enhanced by 18-29% of the enzyme activity. Such results implied that tryptophan, histidine, tyrosine, and serine residues are related to enzyme activity. The pH optima for wheat grass peroxidase to catalyze the oxidation of o-phenylenediamine (OPD), catechol, pyrogallol, and guaiacol were 5.0, 4.5, 6.5, and 5.0, respectively. The apparent Km values for OPD, catechol, pyrogallol, and guaiacol were 2.9, 18.2, 2.5, and 3.8 mM, respectively. Under optimal reaction conditions, wheat grass peroxidase catalyzed the oxidation of OPD (an aromatic amine substrate) 3-11 times more rapidly than guaiacol, catechol, and pyrogallol (phenolic substrates containing one to three hydroxy groups in the benzene ring).     

Determination of Atrazine Residues in Electro-Ultrafiltration Soil Extracts by a Sensitive Enzyme Immunoassay

A sensitive enzyme immunoassay (EIA) based on polyclonal antibodies from sheep was used to screen for atrazine in electro-ultrafiltration (EUF) soil extracts without clean-up. Matrix effects were circumvented by diluting the aqueous EUF extracts. The EUF proved to be a convenient method for the extraction of atrazine residues in soil. The efficiency of EUF appeared to be equivalent to that of organic extraction methods except on weathered residues, which generally resulted in lower yields. Both the combined gas chromatography/automated Soxhlet (GC/Soxtec) and the immunochemical technique EIA/EUF yielded similar data for the 26 soil samples identified as positive (> 0.02 mg/kg) during the first screening of 479 EUF extracts by the EIA.     

Immunolabelling of Atrazine Residues in Soil

An immunolabelling procedure was developed to detect bound pesticide residues in soil using atrazine as an example. Polyclonal antibodies directed against atrazine were raised in rabbits. Antigen-binding fragments (F_ab) were prepared and coupled to the fluorescent stain Rose Bengal B. The dye conjugate produced a fluorescence signal which was related to the amount of atrazine found in native soil samples. Preincubation with unlabelled immunoglobulines (IgG) from preimmune sera proved to be essential for blocking unspecific binding of the antibodies which varied greatly between different samples of soil material. Labelled antibodies, whose specific binding sites had been blocked by free atrazine, were used as controls. Strong unspecific labelling, which also could be suppressed with IgG from preimmune sera, was observed in a soil under a barn. Changes due to the permanent dryness and the lack of aeration are discussed as possible causes.     

Spatial distribution and characterization of long-term aged 14C-labeled atrazine residues in soil

The long-term behavior of the herbicide atrazine and its metabolites in the environment is of continued interest in terms of risk assessment and soil quality monitoring. Aqueous desorption, detection, and quantification of atrazine and its metabolites from an agriculturally used soil were performed 22 years after the last atrazine application. A lysimeter soil containing long-term aged atrazine for >20 years was subdivided into 10 and 5 cm layers (at the lysimeter bottom: soil 0-50 and 50-55 cm; fine gravel 55-60 cm depth, implemented for drainage purposes) to identify the qualitative and quantitative differences of aged (14)C-labeled atrazine residues depending on the soil profile and chemico-physical conditions of the individual soil layers. Deionized water was used for nonexhaustive cold water shaking extraction of the soil. With increasing soil depth, the amount of previously applied (14)C activity decreased significantly from 8.8% to 0.7% at 55-60 cm depth whereas the percentage of desorbed (14)C residues in each soil layer increased from 2% to 6% of the total (14)C activity in the sample. The only metabolite detectable by means of LC-MS/MS was 2-hydroxyatrazine while most of the residual (14)C activity was bound to the soil and was not desorbed. The amount of desorbed 2-hydroxyatrazine decreased with increasing soil depth from 21% to 10% of the total desorbed (14)C residue fraction. The amount of (14)C residues in the soil layers correlated well with the carbon content in the soil and in the aqueous soil extracts ( p value = 0.99 and 0.97, respectively), which may provide evidence of the binding behavior of the aged atrazine residues on soil carbon. The lowest coarse layer (55-60 cm) showed increased residual (14)C activity leading to the assumption that most (14)C residues were leached from the soil column over time.     

Atrazine degradation in boreal nonagricultural subsoil and tropical agricultural soil

Purpose

The purpose of this study was to determine the natural atrazine degradation activity and the genetic potential in a soil profile spanning down to the groundwater zone, collected in Finland at a site where past use of atrazine has contaminated the groundwater, and in Indian agricultural topsoils having different histories of atrazine use.

Materials and methods

Atrazine degradation potential was assessed by quantifying the atrazine degradation genes atzA, trzN, and atzB by quantitative PCR reaction. Atrazine mineralization was studied by radiorespirometry in order to find out if these genes were expressed.

Results and discussion

Indian soils contained a large number up to 10⁴–10⁵ copies (g^?1 dry weight (dw) soil) of atrazine degradation genes after the first treatment with atrazine. These genes were also expressed, as up to 55 % of atrazine mineralized. Some unspecific binding of primers required thorough investigation and confirmation by sequencing of the qPCR products in the agricultural soil samples. The degradation capability of the nonagricultural boreal soil profile was much lower: atrazine degradation genes were present at detection limit (10² copies g^?1 soil), but mineralization studies indicated that these genes were not transcribed, since no or very little atrazine mineralization was observed.

Conclusions

Our results indicate that when atrazine was applied in agricultural practice, the soil atrazine degradation capacity was high. The organisms responsible for the degradation were effectively degrading atrazine already 3 months after the first treatment with atrazine. However, in boreal soil, decades after atrazine use had been discontinued, residual atrazine was not degraded even though a small number of degradation genes could still be detected in soil. There is a need for more specific primers for qPCR in tropical soils.     

Analysis of terbuthylazine in soil samples by two test strip immunoassay formats using reflectance and luminescence detection.

Two different immunoassay (IA) formats for terbuthylazine analysis were developed and optimized using the same monoclonal antibodies. The measuring range for the dipstick IA (using an alkaline phosphatase tracer, a 5-bromo-4-chloroindolylphosphate/nitro blue tetrazolium substrate and a portable reflectometer) was from 3 to 300 microg/kg. The teststrip IA (investigating a horseradish peroxidase tracer, a luminol substrate, and a portable luminometer prototype developed by Immunotek, Moscow, Russia) had a measuring range from 0.05 to 10 microg/kg. From 24 soil samples collected in the Veneto area, Italy, 18 samples contained different amounts of terbuthylazine (but <1 microg/kg atrazine) as was analyzed by gas chromatography with mass selective detection. Eight soil samples (six positive, two negative controls) were analyzed according to the two IA formats. Whereas the dipstick with reflectance detection yielded satisfying results, the test strip IA using luminescence detection has failed so far for soil samples.     

典型相关分析在湿地土壤特征研究中的应用——以黄河三角洲冲积平原湿地土壤为例

 为了更好地评价和管理湿地,对黄河三角洲冲积平原湿地土壤养分特征和酶活性进行研究,同时运用典型相关分析法研究土壤养分和土壤酶2组变量的关系。结果表明,随着远离海岸线的方向,土壤有机质及各养分表现出有规律的变化,土壤脲酶、过氧化氢酶活性沿湿地演替的方向活性升高,过氧化物酶活性降低。运用"土壤综合养分因子"和"土壤综合酶因子",结合典型变量排序图对湿地土壤性质进行分析评价,得出:极大典型相关变量系数为0.9988,特征值占总特征值的99.36%,达极显著水平;极大典型相关变量的"土壤综合养分因子"和"土壤综合酶因子"的典型变量排序图显示很好的线性特征,可以作为冲积平原湿地土壤酶活性和土壤养分的指示因子。     

Auswirkungen von Cu,Cd und Atrazin auf den Stoffwechsel der einzelligen Grünalge Scenedesmus subspicatus

Influence of Cu, Cd and atrazine on the metabolism of the unicellular green alga Scenedesmus subspicatus The effect of Cu, Cd and atrazine on biomass and cell number was studied in 24h old cell cultures of untreated Scenedesmus subspicatus. Furthermore, the effect of some enzymes of the N-metabolism, on peroxidase and protein content was investigated in long term experiments in which the Scenedesmus strains were adapted to high concentrations of Cu, Cd and atrazine.

• In the 24h experiments, atrazine was more toxic than copper according to biomass production and cell number.
• The determination of the enzyme activities from the adapted strains showed positive and negative effects on enzymes of the N-metabolism. Under high copper concentrations, nitrate reductase activity in the copper strain was reduced and nitrite reductase significantly increased. The effects on the activity of the other enzymes of the copper strain were not significant. The protein content, however, was significantly decreased. Cd and atrazine had no significant effects on the activities of nitrate and nitrite reductase and glutamine synthetase or on protein content. Only the glutamate dehydrogenase showed a significant increase in activity under high concentrations of atrazine.
• All substances tested increased the activity of peroxidase, but only the effect of Cd was significant.
• All adapted strains secreted nitrite into the culture medium. The secretion was significantly increased under the influence of copper.

     

阿特拉津对我国东北半干旱区黑钙土微生物活性影响研究

按照0.5～800 μg·g^-1 soil 施入量添加阿特拉津到黑钙土中培养54 d进行实验室培养试验,研究了阿特拉津对我国东北半干旱区黑钙土微生物量碳、土壤碳及氮矿化量、脲酶和脱氢酶活性影响。结果表明,阿特拉津添加到土壤中后,显著提高了（P〈0.05）土壤微生物量碳,增加了土壤碳及氮矿化量,提高土壤脱氢酶活性;多数情况下,各培养时间添加阿特拉津各处理间土壤脲酶活性的差异未达到显著水平（P〈0.05）。由此得出结论：土壤脱氢酶活性、土壤微生物量碳和土壤碳矿化量及氮矿化量是对阿特拉津处理土壤较敏感的生物学指标,适合作为半干旱区黑钙土微生物活性对阿特拉津响应的参数;而土壤脲酶活性不适合作为半干旱区黑钙土土壤微生物活性的指标,因为它不能敏感地反映阿特拉津作用下土壤脲酶活性差异。     

黄河三角洲冲积平原湿地土壤酶活性与养分相关性研究

对黄河三角洲冲积平原湿地酶活性与土壤养分相关性做了研究.结果表明:随着离海距离的增加,土壤有机质及各养分表现出有规律的变化,土壤脲酶、过氧化氢酶活性沿湿地演替的方向活性升高,过氧化物酶活性降低.土壤有机质和各养分与酶活性相关性达极显著和显著水平,不同土壤酶活性之间也有显著的相关性.3种酶可以作为评价黄河三角洲冲积平原湿地土壤肥力的指标.     

Interference with soil phosphatase activity by maize herbicidal treatment and incorporation of maize residues

Summary This study is concerned with the way alachlor, atrazine and metolachlor interfered with phosphatase activity in a clay loam soil unenriched and enriched with maize residues. Enrichment caused an increase in all phosphatase activities (acid and alkaline phosphomonoesterase, phosphodiesterase, phosphotriesterase) tested. Interference with phosphatase activity following herbicidal treatment was found in both unenriched and enriched soil samples. Statistically significant interference was dependent on soil enrichment, the type of herbicide and its rate of application and the time elapsed since the herbicidal treatment. The observed changes in phosphatase activities are attributed to herbicidal action on phosphatase-producing microorganisms. Among the herbicides tested, the acetanilide derivatives exerted a lesser inhibiting effect than atrazine. Nevertheless, all the altered phosphatase activities showed a tendency, more or less rapid, to reach the levels in the corresponding untreated soil samples.     

A quick and sensitive method for the quantification of peroxidase activity of organic surface soil from forests

The purpose of the present study was to test the non-mutagenic compound 3,3′,5,5′-tetramethylbenzidine (TMB) as a model substrate for peroxidase in forest topsoil, as an alternative to the conventional substrate l-3,4-dihydroxyphenylalanine (l-DOPA). TMB was highly sensitive; linear absorbance changes of 0.6 were achieved within 20 min for 1000-fold diluted soil. Brief heating (denaturation) of the soil suspension gave a 34-fold reduction of TMB oxidation, indicating that the reaction measured by TMB was indeed an enzymatic reaction. TMB oxidation showed a narrow peak at pH 4.4. A proportional decrease in peroxidase activity, when the soil suspension was diluted, demonstrated that TMB estimates of peroxidase activity are directly comparable when corrected for differences in sample size. Oxidation of TMB was slow in the absence of H₂O₂ suggesting that TMB is a poor substrate for phenol oxidases. TMB oxidation was tested in nine different forest topsoils. The peroxidase activity, when normalised to the amount of soil organic matter, ranged from 1.4±0.1 Δabs₄₅₀ h^?1 mg^?1 to 34.9±4.3 Δabs₄₅₀ h^?1 mg^?1. In comparison, l-DOPA oxidation by soil peroxidases and commercial peroxidases gave inconsistent results, suggesting that one should be cautious when using l-DOPA as a soil peroxidase substrate. The high sensitivity of TMB, compared to l-DOPA, and the low interference from phenol oxidase and humic substances suggest that TMB is a better substrate than l-DOPA for estimation of peroxidase activity of forest topsoil.     

黄河三角洲冲积平原湿地土壤酶活性与养分相关性研究

对黄河三角洲冲积平原湿地酶活性与土壤养分相关性做了研究。结果表明,随着离海距离的增加,土壤有机质及各养分表现出有规律的变化,土壤脲酶、过氧化氢酶活性沿湿地演替的方向活性升高,过氧化物酶活性降低。土壤有机质和各养分与酶活性相关性达极显著和显著水平,不同土壤酶活性之间也有显著的相关性。三种酶可以作为评价黄河三角洲冲积平原湿地土壤肥力的指标。