Formation of [14C]cellulase-humic complexes and their stability in soil

[¹⁴C]cellulase was extracted from the culture medium of Trichoderma viride and an attempt made to complex it with humic acid by adsorption. The results showed that the humic acid extracted from soil does not form a stable complex with [¹⁴C]cellulase. In contrast, the flocculation of humic acid by 24 mM Ca²⁺ in the presence of the cellulase resulted in the formation of stable humic-cellulase complexes. These complexes showed great resistance to proteolysis and storage at high temperatures. DEAE cellulose chromatography of cellulase-humic complexes revealed that cellulase could not be separated from the humic acid. Enzyme activity was only eluted along with humic acid upon increasing gradient concentration from 1.0 to 1.5 m NaCl. Furthermore, in order to test their stability, the enzyme-humic complexes were incorporated into fresh soil for 90 days. The cellulase-humic complexes were then extracted from soil. Fractionation of the extract on DEAE cellulose and G 100 Sephadex revealed that cellulase activiiy could not be separated from humic acid and was again eluted in the form of enzyme-humic complexes. This confirmed the stability of cellulase-humic complexes in soil.     

Degradation of [14C]phosphinothricin (glufosinate) in soil under laboratory conditions: Effects of concentration and soil amendments on 14CO2 production

Summary Degradation of the herbicide phosphinothricin (L-homoalanine-4-yl-(methyl)-phosphinic acid) in a phaeozem was investigated by monitoring the ¹⁴CO₂ release from [1-¹⁴C] and [3,4-¹⁴C]phosphinothricin. The degradation was largely due to microbial activity, since the rate decreased by more than 95% when the soil was sterilized by -radiation. Data obtained with both labels suggested that decarboxylation of phosphinothricin preceded oxidation of its C-atoms 3 and 4, since a metabolite, probably 3-methylphosphinico-propanoic acid, was only labelled when [3,4-¹⁴C]phosphinothricin was used as the substrate. Maximum rates of ¹⁴CO₂ production from both the 1- and 3,4-label positions occurred without a lag phase during the breakdown of phosphinothricin as monitored for a total of 30 days at 5-day intervals. This result indicated that a phosphinothricin-degrading microbial community was already present in the soil. With low concentrations of [1-¹⁴C]phosphinothricin (10.7 mg kg^-1 soil), complete decarboxylation at 25°C was observed within 30 days of incubation, compared to 15.9% ¹⁴CO₂ release from [3,4-¹⁴C]phosphinothricin. Increasing the quantity of the herbicide in the soil (10.7–1372 mg kg^-1) resulted in increased degradation rates, irrespective of whether the herbicide was labelled in the positions 1 or 3 and 4. Addition of glucose and other carbohydrates stimulated ¹⁴CO₂ release while addition of a yeast extract had a negative effect. Glucose stimulation was reversed by ammonium nitrate, suggesting that the microorganisms were using the herbicide as a source of N.     

Degradation of [carboxyl-14C] 2,4-D and [ring-U-14C] 2,4-D in 114 agricultural soils as affected by soil organic carbon content

This study compared the degradation of [carboxyl-¹⁴C] 2,4-dichlorophenoxyacetic acid (2,4-D) (C2,4-D) and [ring-U-¹⁴C] 2,4-D (R2,4-D) in 114 agricultural soils (0–15 cm) as affected by 2,4-D sorption and soil properties (organic carbon content, pH, clay content, carbonate content, cation exchange capacity, total microbial activity). The sample area was confined to Alberta, Canada, located 49–60° north longitude and 110–120° west latitude and soils were grouped by soil organic carbon content (SOC) (0–0.99%, 1–1.99%, 2–2.99%, 3–3.99% and >4% SOC). Degradation rates of C2,4-D and R2,4-D followed first-order kinetics in all soils. Although total microbial activity increased with increasing SOC, degradation rates and total degradation of C2,4-D and R2,4-D decreased with increasing SOC because of increased sorption of 2,4-D by soil and reduced bioavailability of 2,4-D and its metabolites. Rates of R2,4-D degradation were more limited by sorption than rates of C2,4-D degradation, possibly because of greater sorption and formation of bound residues of 2,4-D metabolites relative to the 2,4-D parent molecule. Based on the sorption and degradation parameters quantified, there were two distinct groups of soils, those with less than 1% SOC and those with greater than 1% SOC. Specifically, soils with less than 1% SOC had, on average, 2.4 times smaller soil organic carbon sorption coefficients and 1.4 times smaller 2,4-D half-lives than soils with more than 1% SOC. In regional scale model simulations of pesticide leaching to groundwater, covering many soils, input parameters for each pesticide include a single soil organic carbon sorption coefficient and single half-life value. Our results imply, however, that the approach to these regional scale assessments could be improved by adjusting the values of these two input parameters according to SOC. Specifically, this study indicates that for 2,4-D and Alberta soils containing less than 1% SOC, the 2,4-D pesticide parameters obtained from generic databases should be divided by 2.5 (soil organic carbon sorption coefficient) and 1.5 (half-life value).     

Pyrolysis of [14C]-chlorantraniliprole in tobacco

The pyrolysis of [(14)C]-chlorantraniliprole {3-bromo-1-(3-chloro-2-pyridinal)-N-[4-chloro-2-methyl-6-[(methylamino)carbonyl]phenyl]-1H-pyrazole-5-carboxamide} in tobacco was examined. Typically five commercially available cigarettes were treated separately with either [pyrazole carbonyl-(14)C] or [benzamide carbonyl-(14)C]-chlorantraniliprole at a concentration of 20 ppm (μg chlorantraniliprole equivalent/g cigarette weight; main study) to 40 ppm (for degradate identification only). All treated cigarettes were smoked using an apparatus designed to collect mainstream (MS) and sidestream (SS) smoke through a glass fiber filter and a series of liquid traps. The material balance for recovery of applied radiolabel ranged from 92.4 to 94.9%. Unchanged chlorantraniliprole was the major component found in butt and filter extracts, averaging a total of 17.4-17.9% of the applied radioactivity. A nonpolar degradation product, 2-[3-bromo-1-(3-chloro-2-pyridinyl)-1H-pyrazol-5-yl]-6-chloro-3,8-dimethyl-4(3H)-quinazolinone, designated 1, represented an average of 10.1-15.9% of the applied radioactivity in the [pyrazole carbonyl-(14)C] or [benzamide carbonyl-(14)C]-chlorantraniliprole cigarettes, respectively. (14)CO(2) was the major degradate, representing an average of 32.9 and 25.1% of the applied radioactivity in pyrazole and benzamide experiments, respectively. In the pyrazole carbonyl label a polar degradate, 5-bromo-N-methyl-1H-pyrazole-3-carboxamide (2) was present in the filter extracts at an average of 9.5% of the applied radioactivity. The most nonpolar degradate, 2,6-dichloro-4-methyl-11H-pyrido[2,1b]quinazolin-11-one (3), was present in [benzamide carbonyl-(14)C]-treated cigarettes only and represented an average of 14.7% of the applied radioactivity.     

Dissipation of the herbicide [14C]dimethenamid under anaerobic conditions in flooded soil microcosms

The objective of this research was to investigate the dissipation of the herbicide dimethenamid under anaerobic redox conditions that may develop in the soil environment. Soil-water biometers were prepared with a saturated soil and made anaerobic by either glucose pretreatment (according to the Environmental Protection Agency registration study for anaerobic fate) or N2 sparging. Treatments included glucose pretreatment, NO3- + SO42- amendment, unamended, and autoclaved. Volatile, aqueous, extractable, and bound (unextractable) 14C-residues were quantified and characterized. The redox potential decreased over time, and evidence of denitrifying, iron-reducing, sulfate-reducing, and methanogenic conditions was observed, depending on the amendments. Anaerobic degradation of 14C-dimethenamid occurred in all treatments, and the time observed for 50% disappearance (DT50) was 13-14 days for nonautoclaved treatments. 14C-metabolites accumulated to up to 20% of applied 14C. At least two major metabolites were observed in nonautoclaved treatments, whereas only one was observed in autoclaved microcosms. More than 50% of the applied 14C was eventually incorporated into soil-bound residue.     

Decoupling of microbial glucose uptake and mineralization in soil

The rate of organic matter turnover in soil is a critical component of the terrestrial carbon cycle and is frequently estimated from measurements of respiration. For estimates to be reliable requires that isotopically labelled substrate uptake into the soil microbial biomass and its subsequent mineralization occurs almost simultaneously (i.e. no time delay). Here we investigated this paradigm using glucose added to an agricultural soil. Immediately after collection from the field, various concentrations of ¹⁴C-labeled glucose (1 μM to 10 mM) were added to soil and the depletion from the soil solution measured at 1–60 min after substrate addition. ¹⁴CO₂ production from the mineralization of glucose was simultaneously measured. The microbial uptake of glucose from soil solution was concentration-dependent and kinetic analysis suggests the operation of at least two distinct glucose transport systems of differing affinity. At glucose concentrations reflecting those naturally present in the soil solution (54±10 μM), the half-time (t_1/2) of exogenous glucose was extremely rapid at ca. 30 s. At higher glucose concentrations (100 μM to 10 mM), the t_1/2 values for the high-affinity carrier were altered little, but increasing proportions of glucose were taken up by the low affinity transport system. Glucose mineralization by the soil microbial community showed a significant delay after its uptake into the microbial biomass suggesting a decoupling of glucose uptake and subsequent respiration, possibly by dilution of glucose in labile metabolite pools. By fitting a double first order kinetic equation to the mineralization results we estimated the t_1/2 for the first rapid phase of respiration at natural soil solution glucose concentrations to be 6–8 min, but at least 87% of the added glucose was retained in the microbial biomass prior to mineralization. Our results suggest that in this soil the soil solution glucose pool turns over 100–1000 times each day, an order of magnitude faster than when determined from measurements of mineralization. These results imply that traditional isotopic based measurements of substrate turnover measured using CO₂ may vastly underestimate their rate of cycling in soil.     

Microbial community utilization of added carbon substrates in response to long-term carbon input manipulation

The chemical composition and quantity of plant inputs to soil are primary factors controlling the size and structure of the soil microbial community. Little is known about how changes in the composition of the soil microbial community affect decomposition rates and other ecosystem functions. This study examined the degradation of universally ¹³C-labeled glucose, glutamate, oxalate, and phenol in soil from an old-growth Douglas-fir (Pseudotsuga menziesii)—western hemlock (Tsuga heterophylla) forest in the Oregon Cascades that has experienced 7 y of chronic C input manipulation. The soils used in this experiment were part of a larger Detritus Input and Removal Treatment experiment and have received normal C inputs (control), doubled wood inputs, or root and litter input exclusion (no inputs). Soil from the doubled wood treatment had a higher fungal:bacterial ratio, and soil from the no inputs treatment had a lower fungal:bacterial ratio, than the control soil. Differences in the utilization of the compounds added to the field-manipulated soils were assessed by following the ¹³C tracer into microbial biomass and respiration. In addition, ¹³C-phospholipid fatty acids (PLFA) analysis was used to examine differential microbial utilization of the added substrates. Glucose and glutamate were metabolized similarly in soils of all three litter treatments. In contrast, the microbial community in the double wood soil respired more added phenol and oxalate, whereas microbes in the no inputs soil respired less added phenol and oxalate, than the control soil. Phenol was incorporated primarily into fungal PLFA, especially in soil of the double wood treatment. The addition of all four substrates led to enhanced degradation of soil organic matter (priming) in soils of all three litter treatments, and was greater following the addition of phenol and oxalate as compared to glucose and glutamate. Priming was greater in the no inputs soil as compared to the control or doubled wood soils. These results demonstrate that altering plant inputs to soil can lead to changes in microbial utilization of C compounds. It appears that many of these changes are the result of alteration in the size and composition of the microbial community.     

Influence of the N and P status of plant material and of added N and P on the mineralization of C from 14C-labelled ryegrass in soil

Ryegrass was grown under conditions of low N, low P, or high N and P nutrient supply in an atmosphere containing ¹⁴CO₂ and then incubated in soil supplemented with or without N or P fertilizer. Determined in fresh plant tissue, the persistency of residual labelled C after 6 months was in the order low-N plants>low-P plants>high-N and-P plants. The addition of N conserved C, particularly when there was additional P present. Hydrolysable labelled C (12M/0.5M H₂SO₄) showed similar trends. In analyses of freeze-dried plant tissue, the main effect was also the increased persistency of C from low-N plants compared to high-N plants. The addition of N fertilizer increased the persistence of plant residue C, but only with grass containing low P. The addition of P fertilizer had no effect. In freeze-dried low-P plant tissue, sampled after 1.5, 6, and 12 months, the conserving effect of adding fertilizer N was confirmed. The addition of P, in contrast, enhanced the rate of decomposition. After 6 months, about a third of the C remained, and after 12 months, about one-quarter. It is concluded that P, whether intrinsic or added, can increase the rate of decomposition of organic residues in soil, but there is a strong interaction with N, which has a predominant influence. The effects of N depend on the form it is in. Increased intrinsic tissue N can increase the rate of C loss, whereas added inorganic N can decrease the rate of C loss during decomposition.     

Influence of soil phosphorus status and nitrogen addition on carbon mineralization from 14C-labelled glucose in pasture soils

 This study examines the effect of soil P status and N addition on the decomposition of ¹⁴C-labelled glucose to assess the consequences of reduced fertilizer inputs on the functioning of pastoral systems. A contrast in soil P fertility was obtained by selecting two hill pasture soils with different fertilizer history. At the two selected sites, representing low (LF) and high (HF) fertility status, total P concentrations were 640 and 820 mg kg^–1 and annual pasture production was 4,868 and 14,120 kg DM ha^–1 respectively. Soils were amended with ¹⁴C-labelled glucose (2,076 mg C kg^–1 soil), with and without the addition of N (207 mg kg^–1 soil), and incubated for 168 days. During incubation, the amounts of ¹⁴CO₂ respired, microbial biomass C and ¹⁴C, microbial biomass P, extractable inorganic P (P_i) and net N mineralization were determined periodically. Carbon turnover was greatly influenced by nutrient P availability. The amount of glucose-derived ¹⁴CO₂ production was high (72%) in the HF and low (67%) in the LF soil, as were microbial biomass C and P concentrations. The ¹⁴C that remained in the microbial biomass at the end of the 6-month incubation was higher in the LF soil (15%) than in the HF soil (11%). Fluctuations in P_i in the LF soil during incubation were small compared with those in HF soil, suggesting that P was cycling through microbial biomass. The concentrations of P_i were significantly greater in the HF samples throughout the incubation than in the LF samples. Net N mineralization and nitrification rates were also low in the LF soils, indicating a slow turnover of microorganisms under limited nutrient supply. Addition of N had little effect on biomass ¹⁴C and glucose utilization. This suggests that, at limiting P fertility, C turnover is retarded because microbial biomass becomes less efficient in the utilization of substrates. Received: 18 October 1999     

Nitrogen fertilizer–induced mineralization of soil organic C and N in six contrasting soils of Bangladesh

A 90‐day laboratory incubation study was carried out using six contrasting subtropical soils (calcareous, peat, saline, noncalcareous, terrace, and acid sulfate) from Bangladesh. A control treatment without nitrogen (N) application was compared with treatments where urea, ammonium sulfate (AS), and ammonium nitrate (AN) were applied at a rate of 100 mg N (kg soil)^–1. To study the effect of N fertilizers on soil carbon (C) turnover, the CO₂‐C flux was determined at nine sampling dates during the incubation, and the total loss of soil carbon (TC) was calculated. Nitrogen turnover was characterized by measuring net nitrogen mineralization (NNM) and net nitrification (NN). Simple and stepwise multiple regressions were calculated between CO₂‐C flux, TC, NNM, and NN on the one hand and selected soil properties (organic C, total N, C : N ratio, CEC, pH, clay and sand content) on the other hand. In general, CO₂‐C fluxes were clearly higher during the first 2 weeks of the incubation compared to the later phases. Soils with high pH and/or indigenous C displayed the highest CO₂‐C flux. However, soils having low C levels (i.e., calcareous and terrace soils) displayed a large relative TC loss (up to 22.3%) and the added N–induced TC loss from these soils reached a maximum of 10.6%. Loss of TC differed depending on the N treatments (urea > AS > AN >> control). Significantly higher NNM was found in the acidic soils (terrace and acid sulfate). On average, NNM after urea application was higher than for AS and AN (80.3 vs. 71.9 and 70.9 N (kg soil)^–1, respectively). However, specific interactions between N‐fertilizer form and soil type have to be taken into consideration. High pH soils displayed larger NN (75.9–98.1 mg N (kg soil)^–1) than low pH soils. Averaged over the six soils, NN after application of urea and AS (83.3 and 82.2 mg N (kg soil)^–1, respectively) was significantly higher than after application of AN (60.6 mg N (kg soil)^–1). Significant relationships were found between total CO₂ flux and certain soil properties (organic C, total N, CEC, clay and sand content). The most important soil property for NNM as well as NN was soil pH, showing a correlation coefficient of –0.33** and 0.45***, respectively. The results indicate that application of urea to acidic soils and AS to high‐pH soils could be an effective measure to improve the availability of added N for crop uptake.     

Distribution of total 14C residue in egg yolk, albumen, and tissues following oral [14C]sulfamethazine administration to hens

The distribution of total 14C residues was studied in egg yolk and albumen after administration of either single or multiple oral dosages of [14C]sulfamethazine (SMZ). One day after a single dose of [14C]SMZ (121 mg of sulfamethazine, 2.42 x 10(7) dpm), the 14C residue concentration peaked in egg albumen and egg yolk with the concentration in the former >4-fold greater than in the latter. Three days postdose, the 14C residue concentration in the yolk was approximately 7-fold higher than in the egg albumen. A multiple dose of [14C]SMZ containing sulfamethazine mass equivalent of an average therapeutic dose (282 mg, 2.9 x 10(7) dpm) for chickens was also administered orally for six consecutive days to hens. A significantly reduced level of egg production was observed during the medication, and most of the hens stopped laying eggs after the last dose. The 14C residue concentrations peaked on the last day (sixth) of medication in egg albumen and yolk. The 14C residue concentrations were also measured in liver, muscle, blood, and plasma of chickens sacrificed at 1, 24, 48, and 72 h after the last dose. Highest concentrations of 14C residue were accumulated in liver followed by, in decreasing order, blood, plasma, and muscle.     

Microbial biomass, enzyme and mineralization activity in relation to soil organic C, N and P turnover influenced by acid metal stress

This study focused on the potential of using soil microbial biomass, enzyme and mineralization activities involved in organic C, N and P turnover, to evaluate the quality of a subtropical agricultural soil affected by long-term acid metal stress. Fractions of C, N and P involved in soil organic matter, microbial biomass and mineralization processes were estimated. Total enzyme activity (FDA) and eight hydrolase activities (xylanase, amylase, β-glucosidase, invertase, N-acteyl-glucosaminidase, urease, alkaline and acid phosphatases) in different decomposition stages of organic C, N and P were selected to characterize the soil functional diversity. These biological datasets were compared with soil metal variables (total contents and free and ligand-complexed ions of Cu, Pb, Zn, Cd, Al and Mn), using principal component analyses, co-inertia and discriminant analyses. The multiple statistics indicate that the metal variables were significantly related with not only general biological factors, but also respective datasets of biomass, enzyme activities and mineralization rates (all P < 0.001). In general, metal variables were inversely related to parameters and indices of microbial biomass C, N and P, FDA and C-related polysaccharidase and heterosidase activities, and P mineralization. As comparison, metal variables exhibited positive relationships with parameters and indices of N-related N-acteyl-glucosaminidase, urease, ammonification, total N mineralization and metabolic quotient, compared with inhibited nitrification. Specifically, free and complexed metal cations showed higher bioavailability than total contents in most cases. Cu, Pb, Al and Mn had different ecotoxicological impacts than Cd and Zn did. Stepwise regression models demonstrated that metal variables are key stress factors, but most of them excluded soil pH. Furthermore, spatial distribution in land uses and of sampling sites clearly separated the soil samples in these models (P < 0.001). We conclude that such a statistical analysis of microbiological and biochemical indices can provide a reliable and comprehensive indication of changes in soil quality and organic nutrient cycling, after exposure to long-term acid metal stress.     

Uptake and metabolic fate of [14C]-2,4-dichlorophenol and [14C]-2,4-dichloroaniline in wheat (Triticum aestivum) and soybean (Glycine max)

The uptake and metabolism of [14C]-2,4-dichlorophenol (DCP) and [14C]-2,4-dichloroaniline (DCA) were investigated in wheat and soybean. Seeds were exposed to a nutrient solution containing 50 microM of one of two radiolabeled compounds, and plant organs were harvested separately after 18 days of growth. In wheat, uptake of [14C]-2,4-DCP was 16.67 +/- 2.65 and 15.50 +/- 2.60% of [14C]-2,4-DCA. In soybean, uptake of [14C]-2,4-DCP was significantly higher than [14C]-2,4-DCA uptake, 38.39 +/- 2.56 and 18.98 +/- 1.64%, respectively. In the case of [14C]-2,4-DCP, the radioactivity absorbed by both species was found mainly associated with roots, whereas [14C]-2,4-DCA and related metabolites were associated with aerial parts, especially in soybean. In wheat, nonextractable residues represented 7.8 and 8.7% of the applied radioactivity in the case of [14C]-2,4-DCP and [14C]-2,4-DCA, respectively. In soybean, nonextractable residues amounted to 11.8 and 5.8% of the total radioactivity for [14C]-2,4-DCP and [14C]-2,4-DCA, respectively. In wheat, nonextractable residues were nearly equivalent to extractable residues for [14C]-2,4-DCP, whereas they were greater for [14C]-2,4-DCA. In soybean, the amount of extractable residues was significantly greater for both chemicals. However, in both species, nonextractable residues were mainly associated with roots. Isolation of soluble residues was next undertaken using excised shoots (wheat) or excised fully expanded leaves including petioles (soybean). Identification of metabolite structures was made by comparison with authentic standards, by enzymatic hydrolyses, and by electrospray ionization-mass spectrometric analyses. Both plant species shared a common metabolism for [14C]-2,4-DCP and [14C]-2,4-DCA since the malonylated glucoside conjugates were found as the final major metabolites.     

[噻唑基-2-14C]-毒氟磷在产蛋鸡体内的分布

为阐明毒氟磷在动物体内的分布代谢特征,并深入认识毒氟磷的安全性和膳食风险,本研究选择白羽产蛋鸡为试验对象,[噻唑基-2-¹⁴C]-毒氟磷为同位素示踪剂,研究了毒氟磷在产蛋鸡体内的排泄分布特征。结果表明,毒氟磷在产蛋鸡体内排泄水平高,首次给药24 h后即排泄出当日给药量的82.04%,连续给药7 d后的累计排泄率为82.24%。毒氟磷在组织中的总残留仅占引入量的3.81%,其中胃中残留量占比相对最高,占引入量的2.14%,而肺、肾、脂肪、胰腺中的放射性残留量均不超过引入量的0.01%,膳食评估结果表明在上述内脏组织中的毒氟磷残留无膳食风险。蛋、肌肉、心、脑、脾、卵巢等组织中未检测到放射性残留。本研究为科学评价毒氟磷在家禽中的安全性提供了试验依据。