Comparison of Analytical Methods for Quantifying the Levels of Lead,Cadmium, Chromium,and Nickel in Mineral Fertilizers and Industrial By-products

The control of contaminants in fertilizers and industrial by-products is fundamental to safe food production, and it requires low-cost, reliable test methods. The aim of this study was to evaluate the performance of the USEPA 3051a and alternative analytical methods [concentrated hydrochloric acid (HCl), 10 percent HCl, 10 percent sulfuric acid (H₂SO₄), and 50 mmol L^?1 diethylenetriaminepentaacetic acid (DTPA)] for quantifying the levels of lead, cadmium, chromium, and nickel in zinc and/or copper sources. The sample digestions with concentrated HCl, H₂SO₄, and nitric acid (HNO₃) (USEPA 3051a) were heated. Extraction without heating and stirring was employed in 10 percent HCl and 50 mmol L^?1 DTPA methods. The Graybill’s modified F-test, t-test for mean error, and the linear correlation coefficient analysis were used to compare test method performance. Equivalent results to USEPA 3051a were found with the following methods: DTPA for the extraction of lead in zinc sources and concentrated HCl and H₂SO₄ for the extraction of cadmium and chromium in copper sources. However, the absolute values of cadmium, chromium, and nickel recovered by the use of H₂SO₄ were greater regardless of the source. The greater extraction of contaminants in the evaluated methods suggests that there is need to review the official method if the aim is to quantify the total levels of these elements in raw materials and mineral fertilizers.     

Effects of Iron, Ammonium and Temperature on Microcystin Content by a Natural Concentrated Microcystis Aeruginosa Population

We report results of a study conducted to evaluate effects of ammonium, iron and temperature on microcystins (MC) and proteins content in cultures of a natural Microcystis aeruginosa population, concentrated from a field sample of San Roque reservoir (Córdoba – Argentina). Based on a previous field study, we tested two temperatures (20 and 28 ^°C), two iron concentrations (1 μM and 10 μM) and two ammonium-nitrogen conditions (absence and 54 μM) in semi-continuous cultures. Total MC (TMC = MC-LR + MC-RR) and protein content per cyanobacteria cell increased when Fe concentration was enlarged. However, the ratio TMC: protein was almost the same for both iron concentrations. Thus, a high level of iron enhances both protein and MC content in the same proportion. TMC and protein content are significantly lowered in presence of 54 μM-N-ammonium. Additionally, the ratio TMC: protein is lowered by almost two folds in cultures having ammonium. Increasing the temperature does not affect the protein content or TMC per cell. However, cultures carried out at 28 ^°C maintain the same MC-LR: MC-RR ratio during all the studied period, while cultures developed at 20 ^°C show that MC-RR content is increased by 82-fold after four growth cycles (20 days total), while MC-LR remain almost constant in the same time. As a result, in our case, different temperatures produce a significant change in the pattern of MC content but not in the content of TMC per cell. The two-fold drop observed in MC content in presence of ammonium are within the range expected for physiological responses of cyanobacteria, raising questions on the probable inhibitory role of ammonium in MC production. On the contrary, the 82-fold increase of MC-RR observed at 20 ^°C exceed the effects attributable to a cell physiological response, and could be better explained by an ecological shift from the starting genotype composition. Metagenomics, or similar molecular techniques, would provide the necessary tools to elucidate the ecological effect of temperature on cyanobacteria populations.     

Chemical and microbiological parameters for the characterisation of the stability and maturity of pruning waste compost

Composting of pruning waste, leaves and grass clippings was monitored by different parameters. A windrow composting pile, having the dimensions 2.5 m (height) x 30 m (length) was establish. The maturation of pruning waste compost was accompanied by a decline in NH₄ ⁺-N concentration, water soluble C (WSC) and an increase in NO₃ ^–-N content. Both organic matter (OM) content and total N (TN) losses during composting followed a first-order kinetic equation. These results were in agreement with the microbiological activity measured either by the CO₂ respiration or dehydrogenase (DH-ase) activity during the process. Statistically significant correlations were found between DH-ase activity, easily biodegradable organic C forms, NH₄ ⁺-N and NO₃ ^–-N concentrations and organic matter content and N losses. For this reason, DH-ase activity and the CO₂ evolution could be used as good indicators of pruning waste compost maturity. In contrast, humification parameters data from the organic matter fractionation did not agree with the initially expected values and did not contribute to the assessment of compost maturity. Neither the cation exchange capacity nor the germination index showed a clear tendency during the composting time, suggesting that these parameters are not suitable for evaluating the dynamics of the process.     

RAPD and AFLP assessment of genetic variation in a landrace of pepper (Capsicum annuum L.), grown in North-West Italy

In several regions of Italy as well as other parts of southern Europe, the heterogeneity of the land, the climate and the soil favour the survival in cultivation of a large number of landraces specifically adapted to local conditions. Knowledge on the level and distribution of their genetic variation can help to develop appropriate strategies, in order to suistainably manage in situ these germplasm resources at risk of genetic erosion. C. annuum is an herbaceous diploid species and is considered to be self-pollinating, although different rates of out-crossing have been recorded. We used random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers to assess genetic diversity within and between five populations of a landrace of Capsicum annuum L., grown in a limited area in north-west Italy and locally known as Cuneo pepper. Partitioning the genetic variation with Shannon's diversity index revealed that 41.6% occurred between and 58.4% within populations. Analogous results were obtained when the analysis was based only on RAPD or AFLP markers. However, AFLP was more reliable, since a lower range of variation was observed among primer combinations in detecting the two components of genetic variation. Notwithstanding the rather high level of within genetic variation detected, the five populations were clearly differentiated and differed in the frequency of alleles exclusive and/or present at very low frequencies. Our results show the need for accurate estimation of allele frequencies, in order to identify populations to which priority should be given for dynamic conservation of landraces.     

Factors affecting porcine sperm mediated gene transfer

“Sperm mediated gene transfer” (SMGT) is based on the ability of sperm cells to bind exogenous DNA. The main objective of this study was to improve the production of transgenic pigs by SMGT. Taking into account that there is a lack of repeatability in studies of SMGT and that the mechanism of binding and internalization of exogenous DNA is a question that has not been solved, different factors involved in the production of transgenic animals by SMGT method were evaluated. Here we set out to: (1) evaluate the sperm capacity to bind exogenous DNA after DMSO treatment; (2) determine the location of the transgene–spermatozoa interaction; and (3) evaluate the efficiency of production of transgenic piglets by deep intrauterine artificial insemination (AI) with sperm incubated with DNA. The percentage of DNA binding was higher than 30% after 2 h of co-culture, but it was not affected by sperm treatment with DMSO (0.3% or 3%). The integrity of the sperm plasma membrane plays a critical role in DNA interaction, and altered plasma membranes facilitate interactions with exogenous DNA. DNA bound mainly to spermatozoa with reduced viability. DNA molecules were found to be mainly associated to the post-acrosomal region (61.9%). After deep intrauterine AI a total of 29 piglets were obtained, but none of them integrated the transgene. In conclusion, although it has been confirmed that DNA can associate with boar spermatozoa, the efficiency of producing transgenic pigs by AI was not confirmed by the present experiments, mainly due to a reduced DNA binding to functional spermatozoa.     

Genome organization in coffee as revealed by EST PCRRFLP,SNPs and SSR analysis

An EST-based PCR-RFLP method was employed to gain insight into genome organization in eight allopolyploid Coffea arabica cultivars and seven diploid coffee species. The PCR-amplified products at 19 EST loci were digested with 46 different restriction enzymes and size fractioned in agarose gels. Most often, the sum of the fragments length was double or more than the PCR product. In arabica, this condition could be explained by assuming the presence of duplicated loci in paralogous chromosomes and this was supported by considerable evidence of multiple loci SSR amplification. Based on the RFLP analysis, 12 EST loci were polymorphic. The level of polymorphism was higher in different species compared to the arabica varieties. Sequencing of the amplified products revealed a SNP frequency of 0.021 among diploid species and of 0.007 among arabica varieties. We propose that the involvement of two genomes in C. arabica maintains a residual level of heterozygosity in the form of paralogous chromosomes, while the self-fertilization in this species tends to drive of homozygosity. The heterozygosity of paralogous chromosomes in arabica creates valuable polymorphism essential for species diversity and survival in various ecological niches, while self-fertility tends to preserve in homozygosity many genes of functional significance.     

Crop management based on field observations: Case studies in sugarcane and coffee

For millennia farmers have continually improved their crop management and production practices through their observations and experience. More recently modern science and research methods based on controlled experiments became the most visible instrument of technological change in agriculture, nevertheless farmers continued to develop and implement new technologies based on their own observations made under commercial conditions. Modern information technology and social organization of producers make it possible to use operational research, which is based on the observation and analysis of operations so as to improve them, to manage crops better. The article describes two cases, coffee and sugarcane, in which observation of the results obtained by farmers, with the natural variation in the environment and the distinct management practices they apply can be used to determine site specific crop management practices. The basis of the methodology is to (a) obtain data from a series of cropping events that characterizes the conditions under which each crop is grown, how it is managed and how it performs under commercial conditions (data capture), (b) to manage and analyze the data in centralized databases (data management and analysis) and (c) make the information derived from the data analysis available to growers so that they can use it to make better informed decisions (interpretation). All aspects of the methodology depend on the social organization of the growers and the supply chain of which they form a part, and hence social organization is an integral part of the methodology.The processes of characterization of the growing conditions, including both environmental and management parameters, the establishment of databases, the data analysis and interpretation, and mechanisms of interacting with producers are described with emphasis on the importance of social organization and farmers’ groups. Examples are given of how this approach can be used to better understand the crop response to variation in the environment and management, and how this can be used by farmers to improve productivity and quality in two contrasting crops. The paper demonstrates that operational research can be used to evaluate farmers’ experiences and to share that knowledge amongst them so as to improve their production practices in the context of their particular environment. It is suggested that the operation research approach is particularly effective in heterogeneous landscapes with perennial crops that have not been the subject of intense research. Furthermore operational research is effective in determining the crop response to variables that are not readily studied in small plots and in determining optimal combinations of multiple variables. Producers believe in the results obtained as there are none of the problems of scaling up from experimental plots to commercial conditions. It is proposed that the approaches described can readily be applied to other crop species.     

Retrospective study of pestivirus infection in Pyrenean chamois (Rupicapra pyrenaica) and other ungulates in the Pyrenees (NE Spain)

In 2001 a new Pestivirus (Family Flaviviridae) was associated with an outbreak of a previously unreported disease in Pyrenean chamois (Rupicapra pyrenaica) in the Pyrenees (NE Spain). Molecular characterization assigned this virus to the Border Disease Virus (BDV) cluster, BDV-4 genotype. A retrospective study was performed in archived sera and spleen of 74 Pyrenean chamois and in archived sera of 28 mouflon (Ovis ammon), 56 red deer (Cervus elaphus), 43 roe deer (Capreolus capreolus) and 29 fallow deer (Dama dama) from the Pyrenees between the years 1990 and 2000. Thirty six of 74 (48.6%) sera of Pyrenean chamois, one of mouflon and one of red deer were positive by an ELISA antibody test. Comparative virus neutralization tests were performed on 26 seropositive chamois, one mouflon and one red deer, using five pestivirus strains. An ELISA antigen test was performed on 37 seronegative chamois and yielded positive results in one chamois and inconclusive result in two. RT-PCR and virus isolation performed on spleen samples from these three animals gave positive results in the positive and one inconclusive animal. Sequence analysis in the 5' unstranslated region revealed that they were grouped into the BDV-4 genotype. Virological and serological data of the present study indicate that BDV infection has been present in the chamois population since at least 1990, 11 years before the first outbreak of disease. Therefore, the emergence of the disease in 2001 is apparently due to other factors rather than the introduction of a new virus in the chamois population.     

Fate of iprovalicarb, indoxacarb, and boscalid residues in grapes and wine by GC-ITMS analysis

The behavior in field and the transfer from grapes to wine during winemaking of iprovalicarb, indoxacarb, and boscalid was studied. The residue levels found in grapes were far below the MRLs set for grapes in EU, accounting at harvest time 0.81, 0.43, and 4.23 mg/kg for iprovalicarb, indoxacarb, and boscalid, respectively. The residue levels in the samples treated with boscalid may have residual problems (due to an accumulation effect) if repeated field treatments will be performed. Winemaking experiments showed a complete transfer of all pesticide from grapes to the must, while in wine the residues were low or negligible due to the adsorbing effect of lees and pomace. The clarification experiments showed a good removal of pesticide residues from the wine media, for all pesticides. The GC-ITMS method showed good performance with adequate recoveries ranging from 75 to 115%, and good method limits of quantitation (LOQs) and of determination (LODs) far below MRLs.