Micromorphological and anatomical alterations caused by simulated acid rain in Restinga plants: Eugenia uniflora and Clusia hilariana

Eugenia uniflora and Clusia hilariana seedlings were submitted to simulated acid rain, pH 3, for 40 days. The degree of visible injury and the anatomical and micromorphological alterations were determined 24 hours after the last simulation. E. uniflora presented a higher degree of leaf necrosis. The adaxial epidermis and part of the palisade parenchyma of the entire leaf blade were affected and hypertrophy, hyperplasia and cicatrization tissue differentiation occurred. Erosion and morphological modification of the epicuticular wax and alterations in the epidermis were detected on the upper and lower leaf surfaces. Stomata with deformed ostioles and rupture of the stomatal ledge were observed. C. hilariana presented necrosis in leaf margin and in abaxial leaf surface, showing collapsed cells or with plasmolized aspect, cicatrization tissue, epicuticular wax alterations, occasional leaf perforations, irregular arrangement of adaxial epidermis cells and twisted subsidiary cells. The structural alterations indicated a greater susceptibility of E. uniflora to acid rain. he lower sensibility of C. hilariana can be related with the anatomical characteristics as: thick cuticle with evident cuticular flanges, three layers of hypodermis and mesophyll more thick and compact.     

Responses of restinga plant species to pollution from an iron pelletization factory

shape Byrsonima sericea DC. (Malpighiaceae), shape Cordia verbenacea DC. (Boraginaceae) and shape Psidium guineense SW. (Myrtaceae) were studied in two experimental sites: a restinga forest fragment, near an iron pelletization factory (mining area) and a restinga conservation unit “Paulo César Vinha State Park”. The aims were verify the effects of emissions from a pelletization factory on some anatomical and physiological parameters and on absorption and accumulation of particulate iron by the leaves. A protocol was adjusted using Prussian blue reaction to histochemical detection of iron. Comparing the same species in two locals, shape C. verbenacea was the greatest accumulator of metallic iron, whereas shape B. sericea presented greater content of sulphur in mining area. The greater leaf contents of metallic iron may be related to the adhesion of the particles deposited on the leaves and to the characteristics of the covering tissue and the leaf. The foliar parameters evaluated in addition to soil analysis and atmospheric precipitation data indicated that the effects of particulate deposition occurred due foliar uptake. Structural characteristics such as peltate trichomes of leaf abaxial surface in shape B. sericea and hypodermis observed in shape B. sericea (one layer) and shape P. guineense (3--4 layers), probably formed a barrier lessening the penetration of metallic iron into the mesophyll as evidenced by the lower iron leaf content and iron accumulation in trichomes observed in these two species. In leaves of shape C. verbenacea occurred a positive reaction for iron in trichomes, epidermic cells at both faces, stomata, some xylem cells, collenchyma and endodermis at the midrib, and mesophyll tissues. The obtained data indicated that the unit conservation is also under pollution impact.     

Improvement in the determination of mancozeb residues by the carbon disulfide evolution method using flow injection analysis

The sample decomposition of the carbon disulfide evolution method for the determination of dithiocarbamate residues was carried out in a closed vial in the presence of hexane. The evolved carbon disulfide was extracted by the organic solvent and injected in a flow system for its quantification as copper complex. The conditions for batch decomposition, flow injection determination, and association of both were investigated with sodium diethyldithiocarbamate as model substance. An one-channel flow system was employed where the carrier stream was the ethanolic ethylenediamine/copper solution. The determination range was of 0. 01-1.26 microg of CS(2), with a relative standard deviation of 0.06% (n = 10), with a sample throughput of 45 samples/h. The association of the batch decomposition with the flow system was carried out with the fungicide mancozeb and was applied to the analysis of its residue in potato, lettuce, cucumber, and green bean crops. The approach allowed the analysis of 11 samples in triplicate in 2 h, with recoveries between 85% and 92% and relative standard deviation about 2%.     

Tolerance of genetically characterized <Emphasis Type="Italic">Folsomia candida</Emphasis> strains to phenmedipham exposure

Background, Aims, and Scope  The springtail Folsomia candida is a commonly used model species in ecotoxicological soil testing. The species reproduces parthenogenetically and, thus, laboratories use different clonal lineages. In this study, we investigated if genetic divergence between F. candida strains impacts the reaction to chemical stress and may thus affect the outcome of toxicity tests. Methods  In two exposure assays (life-cycle reproduction test and avoidance behaviour test), three laboratory strains of F. candida from Portugal (PTG), Spain (SPN) and Denmark (DNK) were exposed to different concentrations of the reference chemical phenmedipham. Genetic divergence among strains was estimated based on mitochondrial COI sequence data. Results  No significant differences between tolerance towards phenmedipham exposure were observed in a reproduction test. In contrast, one strain (SPN) showed a decreased susceptibility to phenmedipham compared to the other strains (PTG and DNK) in the avoidance assay. Discussion  A phylogenetic tree based on mitochondrial COI sequences revealed clear genetic differentiation between both ‘reaction types’. Thus, we found a potential lineage dependent stress reaction in avoidance behaviour towards the pesticide. Conclusions  Our findings have implications for the comparability of test results among laboratories. Reproduction tests seem to be more robust towards interclonal genetic differentiation than avoidance tests. Recommendations and Perspectives  We recommend the use of molecular tools for simple and cost effective genetic characterization of F. candida strains used in chemical avoidance tests. Closer investigations concerning the relation between genetic relatedness and chemical response will provide a more detailed and comprehensive picture on the role of intraspecific genetic differentiation in stress tolerance. ESS-Submission Editor: Prof. Dr. Henner Hollert (henner.hollert@bio5.rwth-aachen.de)     

Regulation of Contaminated Soils in Spain - A New Legal Instrument (4 pp)

Background, Aims and Scope   This paper presents the new Spanish regulation on contaminated soils and its scientific basis. The regulation offers a regulatory framework for establishing industrial activities which may result in soil contamination, and presents the methodology for setting the generic reference levels of organic pollutants.Legal Framework   The Spanish regulation on contaminated soils is derived from the waste legislation and covers 101 industrial activities, as well as facilities handling significant amounts of hazardous chemicals (over 10,000 kg per year) or fuel (300,000 l fuel per year or storing 50,000 l fuel at any time). The regulatory framework includes initial declarations and a tiered system for selecting those soils requiring a proper site-specific risk assessment.Scientific Basis   The regulation is risk based, and covers human and environmental risks. The human health risk assessment focuses on chemical analysis; the selection of relevant exposure routes is associated to the soil uses. The environmental risk assessment includes chemical analysis and direct toxicity testing, and covers three main ecological receptors: Soil organisms, associated aquatic systems and terrestrial vertebrates. Low-risk threshold concentrations are established as generic reference levels; if exceeded, a site-specific risk assessment is required. The detection of a very high level of acute toxicity of soil or leachates led to the declaration of the soil as contaminated due to the capacity for contaminating the adjacent areas.Conclusion   Overall, the Spanish regulation offers a balance for combining regulatory needs, proper scientific basis and practicability. The use of European risk assessment protocols and the European legal framework would facilitate the pan-European extrapolation of this approach. The inclusion of direct toxicity testing as a legal method for classifying a soil as contaminated is considered a key element.     

Quantitation of nine organic acids in wild mushrooms

The organic acids composition of six wild edible mushroom species (Amanita caesarea, Boletus edulis, Gyroporus castaneus, Lactarius deliciosus, Suillus collinitus, and Xerocomus chrysenteron) was determined by an HPLC-UV detector method. The results showed that all of the samples presented a profile composed of at least five organic acids: citric, ketoglutaric, malic, succinic, and fumaric acids. Several samples also contained oxalic, ascorbic, quinic, and shikimic acids. In a general way, the quantitation of the identified compounds indicated that malic acid, followed by the pair citric plus ketoglutaric acids, were the main compounds in the analyzed species, with the exception of A. caesarea, in which malic and ascorbic acids were the most abundant compounds. The relative amounts and the presence/absence of each identified compound may be useful for the differentiation of the species.     

Comparison of urease inhibitor N‐(n‐butyl) thiophosphoric triamide and oxidized charcoal for conserving urea‐N in soil

Charcoal‐based amendments have a potential use in controlling NH₃ volatilization from urea fertilization, owing to a high cation‐exchange capacity (CEC) that enhances the retention of NH . An incubation study was conducted to evaluate the potential of oxidized charcoal (OCh) for controlling soil transformations of urea‐N, in comparison to urease inhibition by N‐(n‐butyl) thiophosphoric triamide (NBPT). Four soils, ranging widely in texture and CEC, were incubated aerobically for 0, 1, 3, 7, and 14 d after application of ¹⁵N‐labeled urea with or without OCh (150 g kg^?1 fertilizer) or NBPT (0.5 g kg^?1 fertilizer), and analyses were performed to determine residual urea and ¹⁵N recovery as volatilized NH₃, mineral N (as exchangeable NH , NO , and NO ), and immobilized organic N. The OCh amendment reduced NH₃ volatilization by up to 12% but had no effect on urea hydrolysis, NH and NO concentrations, NO accumulation, or immobilization. In contrast, the use of NBPT to inhibit urea hydrolysis was markedly effective for moderating the accumulation of NH , which reduced immobilization and also controlled NH₃ toxicity to nitrifying microorganisms that otherwise caused the accumulation of NO instead of NO . Oxidized charcoal is not a viable alternative to NBPT for increasing the efficiency of urea fertilization.     

Novel protein from Labramia bojeri A. DC. seeds homologue to Kunitz-type trypsin inhibitor with lectin-like properties

This study starts by isolating and characterizing the first protein from Labramia bojeri seeds, which belong to the Sapotaceae family. The purified lectin analyzed by SDS-PAGE with and without beta-mercaptoethanol shows two protein bands (M(r) = 19 and 20 kDa), which cannot be resolved. Protein bands have shown similar characteristics as molecular masses, determined by gel filtration and native gel; N-terminal sequences presented a difference in their isoelectric points. We have suggested that those protein bands might be variants of the protein named Labramin. The sequence database search has shown that the N-terminal sequence of Labramin presented a high degree of homology to Kunitz-type trypsin inhibitor (82-52%) despite no trypsin inhibition activity detection. The lectin-like form from Labramin was better inhibited by glycoproteins and has also presented growth inhibition of the fungus Colletotrichum lindemuthianum and the yeast Saccharomyces cerevisiae, but it has not presented an apparent effect on Fusarium oxysporum.