No evidence for persistent natural plague reservoirs in historical and modern Europe.

Caused by Yersinia pestis, plague ravaged the world through three known pandemics: the First or the Justinianic (6th-8th century); the Second (beginning with the Black Death during c.1338-1353 and lasting until the 19th century); and the Third (which became global in 1894). It is debatable whether Y. pestis persisted in European wildlife reservoirs or was repeatedly introduced from outside Europe (as covered by European Union and the British Isles). Here, we analyze environmental data (soil characteristics and climate) from active Chinese plague reservoirs to assess whether such environmental conditions in Europe had ever supported "natural plague reservoirs". We have used new statistical methods which are validated through predicting the presence of modern plague reservoirs in the western United States. We find no support for persistent natural plague reservoirs in either historical or modern Europe. Two factors make Europe unfavorable for long-term plague reservoirs: 1) Soil texture and biochemistry and 2) low rodent diversity. By comparing rodent communities in Europe with those in China and the United States, we conclude that a lack of suitable host species might be the main reason for the absence of plague reservoirs in Europe today. These findings support the hypothesis that long-term plague reservoirs did not exist in Europe and therefore question the importance of wildlife rodent species as the primary plague hosts in Europe.

Distinct circular single-stranded DNA viruses exist in different soil types.

The potential dependence of virus populations on soil types was examined by electron microscopy, and the total abundance of virus particles in four soil types was similar to that previously observed in soil samples. The four soil types examined differed in the relative abundances of four morphological groups of viruses. Machair, a unique type of coastal soil in western Scotland and Ireland, differed from the others tested in having a higher proportion of tailed bacteriophages. The other soils examined contained predominantly spherical and thin filamentous virus particles, but the Machair soil had a more even distribution of the virus types. As the first step in looking at differences in populations in detail, virus sequences from Machair and brown earth (agricultural pasture) soils were examined by metagenomic sequencing after enriching for circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) virus genomes. Sequences from the family Microviridae (icosahedral viruses mainly infecting bacteria) of CRESS-DNA viruses were predominant in both soils. Phylogenetic analysis of Microviridae major coat protein sequences from the Machair viruses showed that they spanned most of the diversity of the subfamily Gokushovirinae, whose members mainly infect obligate intracellular parasites. The brown earth soil had a higher proportion of sequences that matched the morphologically similar family Circoviridae in BLAST searches. However, analysis of putative replicase proteins that were similar to those of viruses in the Circoviridae showed that they are a novel clade of Circoviridae-related CRESS-DNA viruses distinct from known Circoviridae genera. Different soils have substantially different taxonomic biodiversities even within ssDNA viruses, which may be driven by physicochemical factors.

Lipidic compounds found in soils surrounding human decomposing bodies and its use in forensic investigations - A narrative review.

Following decomposition of a human body, a variety of decomposition products, such as lipids, are released into the surrounding environment, e.g. soils. The long-lasting preservation in soils and their high diagnostic potential have been neglected in forensic research. Furthermore, little is known about the preservation, chemical transformation, or degradation of those human derived lipids in soils. To date, several studies identified various lipids such as long-chain free fatty acids and steroids in soils that contained decomposition fluids. Those lipids are preserved in soils over time and could serve as markers of human decomposition in forensic investigations, e.g. for estimating the post-mortem interval or identifying the burial location of a human body. Therefore, this review focuses on the current literature regarding fatty acid and steroid that have been detected in soils and associated with human body decomposition. After a short introduction about human decomposition processes, this review summarises fatty acid and steroid analysis applied in current case studies and studies related to taphonomic research. This review provides an overview of the available studies that have used fatty acids and steroids as identifiers of human decomposition fluid in soils in a forensic context and discusses the potential for developing this innovative field of research with direct application in a forensic context.

Rapid extraction of high- and low-density microplastics from soil using high-gradient magnetic separation.

Microplastics (MPs) are present in all environments, and concerns over their possible detrimental effects on flora and fauna have arisen. Density separation (DS) is commonly used to separate MPs from soils to allow MP quantification; however, it frequently fails to extract high-density MPs sufficiently, resulting in under-estimation of MP abundances. In this proof-of-concept study, a novel three-stage extraction method was developed, involving high-gradient magnetic separation and removal of magnetic soil (Stage 1), magnetic tagging of MPs using surface modified iron nanoparticles (Stage 2), and high-gradient magnetic recovery of surface-modified MPs (Stage 3). The method was optimised for four different soil types (loam, high­carbon loamy sand, sandy loam and high-clay sandy loam) spiked with different MP types (polyethylene, polyethylene terephthalate, and polytetrafluoroethylene) of different particle sizes (63 µm to 2 mm) as well as polyethylene fibres (2-4 mm). The optimised method achieved average recoveries of 96% for fibres and 92% for particles in loam, 91% for fibres and 87% for particles in high­carbon loamy sand, 96% for fibres and 89% for particles in sandy loam, and 97% for fibres and 94% for particles in high-clay sandy loam. These were significantly higher than recoveries achieved by DS, particularly for fibres and high-density MPs (p < 0.05). To demonstrate the practical application of the HGMS method, it was applied to a farm soil sample, and high-density MP particles were only recovered by HGMS. Furthermore, this study showed that HGMS can recover fibre-aggregate complexes. This improved extraction method will provide better estimates of MP quantities in future studies focused on monitoring the prevalence of MPs in soils.

Conjunctive use of synchrotron X-ray diffraction and Rietveld refinement in Fe-oxide clays for forensic applications.

Soils have distinctive chemical, physical, mineralogical and biological properties, which make it possible to differentiate them in different environments and also to test for an association of a particular questioned item with a crime scene. Mineral compositions and structures in the soil clay fraction can reflect a distinct characteristic when analyzed by powerful techniques. In this way, the aim of this study was to compare the discriminative power of Fe-oxides concentrated clay samples from the same soil class with and without differences in parent material when analyzed using the Rietveld method and two sources of X-ray diffraction: conventional and high-resolution synchrotron radiations. Clay samples were Fe concentrated (kaolinite and gibbsite removal) to clarify mineralogical composition of 12 samples from three simulated crime scenes, developed under claystone and granite. By Rietveld refinement, detailed crystallographic data were obtained to discriminate samples according their provenance. By synchrotron radiation, mineral data demonstrated the power of quantitative (crystallite size and minerals contents) and qualitative (identification of majoritarian and accessory minerals) analysis by Rietveld refinement, which provides well-resolved data able to discriminate samples from different and same geology. The techniques can be applied in other criminal investigations given their potential of discrimination.

Profiling and imaging of forensic evidence - A pan-European forensic round robin study part 1: Document forgery.

The forensic scenario, on which the round robin study was based, simulated a suspected intentional manipulation of a real estate rental agreement consisting of a total of three pages. The aims of this study were to (i) establish the amount and reliability of information extractable from a single type of evidence and to (ii) provide suggestions on the most suitable combination of compatible techniques for a multi-modal imaging approach to forgery detection. To address these aims, seventeen laboratories from sixteen countries were invited to answer the following tasks questions: (i) which printing technique was used? (ii) were the three pages printed with the same printer? (iii) were the three pages made from the same paper? (iv) were the three pages originally stapled? (v) were the headings and signatures written with the same ink? and (vi) were headings and signatures of the same age on all pages? The methods used were classified into the following categories: Optical spectroscopy, including multispectral imaging, smartphone mapping, UV-luminescence and LIBS; Infrared spectroscopy, including Raman and FTIR (micro-)spectroscopy; X-ray spectroscopy, including SEM-EDX, PIXE and XPS; Mass spectrometry, including ICPMS, SIMS, MALDI and LDIMS; Electrostatic imaging, as well as non-imaging methods, such as non-multimodal visual inspection, (micro-)spectroscopy, physical testing and thin layer chromatography. The performance of the techniques was evaluated as the proportion of discriminated sample pairs to all possible sample pairs. For the undiscriminated sample pairs, a distinction was made between undecidability and false positive claims. It was found that none of the methods used were able to solve all tasks completely and/or correctly and that certain methods were a priori judged unsuitable by the laboratories for some tasks. Correct results were generally achieved for the discrimination of printer toners, whereas incorrect results in the discrimination of inks. For the discrimination of paper, solid state analytical methods proved to be superior to mass spectrometric methods. None of the participating laboratories deemed addressing ink age feasible. It was concluded that correct forensic statements can only be achieved by the complementary application of different methods and that the classical approach of round robin studies to send standardised subsamples to the participants is not feasible for a true multimodal approach if the techniques are not available at one location.

The Use of a Sequential Extraction Technique to Characterize Soil Trace Evidence Recovered from a Spade in a Murder Case in Brazil.

Soil trace evidence can be useful in criminal investigations. A homicide which had occurred in South Brazil been concluded through the courts with a guilty conviction. A spade with soil traces adhering to it was seized from the confessed killer's house, it having been established that it had been used to bury parts of the victim's body. In the context of this confession, it provided an opportunity to test a protocol of analysis and verify the potential of discriminate soil sample analysis in such case works. This allowed us to test the practice of sequential analysis which had been developed for forensic case works in Brazil, with three sequential extractions: (i) 0.2 mol/L pH 3.0 ammonium oxalate; (ii) dithionite-citrate-bicarbonate; and (iii) 0.5 mol/L NaOH. It was possible to predict the sequence of events related to the homicide by using the sequential extraction technique and to conclude that: (i) the A horizon soil from the burial location of the torso was found to be very similar to the soil samples which had been recovered from the spade, which was able to be established despite there only being a small amount of soil adhering to the spade; (ii) the location where the legs were buried contributed a low amount of soil adhering to the spade. Therefore, it is suggested that, where possible, sequential extractions should be prioritized from a questioned sample to best provide information about the likely sequence of contact places and this test likely scenarios and criminal events.

Can analysis of a small clod of soil help to solve a murder case?

Soil forensics utilizes extensive soil information to answer legal questions and test hypotheses. The main difficulty often is the determination of different variables from a small amount of soil sample collected on the suspect. We developed a sequential mineralogical and chemical analyses to assess a limited quantity of soil vestiges (0.5 g) from a suspect's vehicle (adhered to the outside rear-view mirror and to the left front fender) involved in a murder case and compared them with the surface samples found at the victim's body disposal site at the Graciosa Road, Paraná State, Brazil. All results affirm that the suspect's vehicle could have been in contact with the edge of the Graciosa Road, approximately the place where the victim's body was located. As a result of the soil analysis and comparison, the results support the likely contact of the suspect's vehicle with the crime scene.

First evidence of terrestrial ambrein formation in human adipocere.

To date, the only known occurrence of ambrein, an important perfumery organic molecule, is in coproliths found in about one in a hundred sperm whales. Jetsam ambergris coproliths from the whale are also found occasionally on beaches worldwide. Here we report on the surprising occurrence of ambrein in human adipocere. Adipocere is a waxy substance formed post-mortem during incomplete anaerobic decomposition of soft tissues. Adipocere samples obtained from grave exhumations were analysed using gas chromatography-mass spectrometry (GC-MS). In addition to the typical fatty acids of adipocere, lesser amounts of ambrein were identified in the samples, in abundances similar to those of the major accompanying faecal steroids. The distribution of these compounds suggests that ambrein was produced post-mortem during the microbial decomposition of faecal residues and tissues. It is assumed that the adipocere matrix of saturated fatty acidsaided the preservation of ambrein over extended periods of time, because adipocere is stable against degradation. The association of ambrein formation in ageing faecal material, under moist, oxygen-depleted conditions, now requires more attention in studies of other mammalian and geological samples. Indeed, ambrein and its transformation products may be useful novel chemical indicators of aged faecal matter and decomposed bodies.

Multivariate analysis of Scotch whisky by total reflection x-ray fluorescence and chemometric methods: A potential tool in the identification of counterfeits.

Most methods used in the identification of counterfeit whisky have focused on the profiling of volatile organic congeners determined by gas chromatography. We tested the use of total reflection x-ray fluorescence (TXRF) for trace element analysis of whisky and application of the data as a potential tool in the identification of counterfeit samples. Twenty five whiskies that were produced in different regions of Scotland or were blends, 5 counterfeit whiskies, 1 unmatured grain whisky, and 1 matured grain whisky were analysed for 11 elements (P, S, Cl, K, Ca, Mn, Fe, Cu, Zn, Br and Rb). The effect of cold plasma ashing with oxygen on whisky residues evaporated on the TXRF reflector on the instrument performance was investigated. Cold plasma ashing with oxygen reduced beam scatter and improved the limits of detection but was ultimately deemed unnecessary. The element concentration data for whisky obtained by TXRF (after log transformation) was compared with the values obtained by inductively coupled plasma spectroscopy and showed correlation values (R2) ≥ 0.942 for K, Mn and Cu: ≥ 0.800 for Ca, Fe and Rb; and ≥0.535 for P, S and Zn. The range of concentration values for individual elements was variable and principal components analysis of the elemental concentrations partially differentiated the whiskies by region or type but showed clear separation of the counterfeit samples from the other samples. Using the principal component scores of the elemental concentration data, linear discriminant analysis also distinguished the counterfeits from the other samples.

Microbial soil community analyses for forensic science: Application to a blind test.

Soil complexity, heterogeneity and transferability make it valuable in forensic investigations to help obtain clues as to the origin of an unknown sample, or to compare samples from a suspect or object with samples collected at a crime scene. In a few countries, soil analysis is used in matters from site verification to estimates of time after death. However, up to date the application or use of soil information in criminal investigations has been limited. In particular, comparing bacterial communities in soil samples could be a useful tool for forensic science. To evaluate the relevance of this approach, a blind test was performed to determine the origin of two questioned samples (one from the mock crime scene and the other from a 50:50 mixture of the crime scene and the alibi site) compared to three control samples (soil samples from the crime scene, from a context site 25m away from the crime scene and from the alibi site which was the suspect's home). Two biological methods were used, Ribosomal Intergenic Spacer Analysis (RISA), and 16S rRNA gene sequencing with Illumina Miseq, to evaluate the discriminating power of soil bacterial communities. Both techniques discriminated well between soils from a single source, but a combination of both techniques was necessary to show that the origin was a mixture of soils. This study illustrates the potential of applying microbial ecology methodologies in soil as an evaluative forensic tool.

Using oral fluids samples for indirect influenza A virus surveillance in farmed UK pigs.

Influenza A virus (IAV) is economically important in pig production and has broad public health implications. In Europe, active IAV surveillance includes demonstration of antigen in nasal swabs and/or demonstration of antibodies in serum (SER) samples; however, collecting appropriate numbers of individual pig samples can be costly and labour-intensive. The objective of this study was to compare the probability of detecting IAV antibody positive populations using SER versus oral fluid (OF) samples. Paired pen samples, one OF and 5-14 SER samples, were collected cross-sectional or longitudinally. A commercial nucleoprotein (NP)-based blocking ELISA was used to test 244 OF and 1004 SER samples from 123 pens each containing 20-540 pigs located in 27 UK herds. Overall, the IAV antibody detection rate was higher in SER samples compared to OFs under the study conditions. Pig age had a significant effect on the probability of detecting positive pens. For 3-9-week-old pigs the probability of detecting IAV antibody positive samples in a pen with 95% confidence intervals was 40% (23-60) for OF and 61% (0.37-0.80) for SER (P = 0.04), for 10-14-week-old pigs it was 19% (8-40) for OF and 93% (0.71-0.99) for SER (P < 0.01), and for 18-20-week-old pigs it was 67% (41-85) for OF and 81% (0.63-0.91) for SER (P = 0.05). Collecting more than one OF sample in pens with more than 25 less than 18-week-old pigs should be further investigated in the future to elucidate the suitability of OF for IAV surveillance in herds with large pen sizes.

Explosive detonation causes an increase in soil porosity leading to increased TNT transformation.

Explosives are a common soil contaminant at a range of sites, including explosives manufacturing plants and areas associated with landmine detonations. As many explosives are toxic and may cause adverse environmental effects, a large body of research has targeted the remediation of explosives residues in soil. Studies in this area have largely involved spiking 'pristine' soils using explosives solutions. Here we investigate the fate of explosives present in soils following an actual detonation process and compare this to the fate of explosives spiked into 'pristine' undetonated soils. We also assess the effects of the detonations on the physical properties of the soils. Our scanning electron microscopy analyses reveal that detonations result in newly-fractured planes within the soil aggregates, and novel micro Computed Tomography analyses of the soils reveal, for the first time, the effect of the detonations on the internal architecture of the soils. We demonstrate that detonations cause an increase in soil porosity, and this correlates to an increased rate of TNT transformation and loss within the detonated soils, compared to spiked pristine soils. We propose that this increased TNT transformation is due to an increased bioavailability of the TNT within the now more porous post-detonation soils, making the TNT more easily accessible by soil-borne bacteria for potential biodegradation. This new discovery potentially exposes novel remediation methods for explosive contaminated soils where actual detonation of the soil significantly promotes subsequent TNT degradation. This work also suggests previously unexplored ramifications associated with high energy soil disruption.

Soil characterisation by bacterial community analysis for forensic applications: A quantitative comparison of environmental technologies.

The ubiquity and transferability of soil makes it a resource for the forensic investigator, as it can provide a link between agents and scenes. However, the information contained in soils, such as chemical compounds, physical particles or biological entities, is seldom used in forensic investigations; due mainly to the associated costs, lack of available expertise, and the lack of soil databases. The microbial DNA in soil is relatively easy to access and analyse, having thus the potential to provide a powerful means for discriminating soil samples or linking them to a common origin. We compared the effectiveness and reliability of multiple methods and genes for bacterial characterisation in the differentiation of soil samples: ribosomal intergenic spacer analysis (RISA), terminal restriction fragment length polymorphism (TRFLP) of the rpoB gene, and five methods using the 16S rRNA gene: phylogenetic microarrays, TRFLP, and high throughput sequencing with Roche 454, Illumina MiSeq and IonTorrent PGM platforms. All these methods were also compared to long-chain hydrocarbons (n-alkanes) and fatty alcohol profiling of the same soil samples. RISA, 16S TRFLP and MiSeq performed best, reliably and significantly discriminating between adjacent, similar soil types. As TRFLP employs the same capillary electrophoresis equipment and procedures used to analyse human DNA, it is readily available for use in most forensic laboratories. TRFLP was optimized for forensic usage in five parameters: choice of primer pair, fluorescent tagging, concentrating DNA after digestion, number of PCR amplifications per sample and number of capillary electrophoresis runs per PCR amplification. This study shows that molecular microbial ecology methodologies are robust in discriminating between soil samples, illustrating their potential usage as an evaluative forensic tool.

RisaAligner software for aligning fluorescence data between Agilent 2100 Bioanalyzer chips: Application to soil microbial community analysis.

Ribosomal Intergenic Spacer Analysis (RISA) is a high-resolution and highly reproducible fingerprinting technique for discriminating between microbial communities. The community profiles can be visualized using the Agilent 2100 Bioanalyzer. Comparison between fingerprints relies upon precise estimation of all amplified DNA fragment lengths; however, size standard computation can vary between gel runs. For complex samples such as soil microbial communities, discrimination by fragment size is not always sufficient. In such cases, the comparison of whole fluorescence data as a function of time (electrophoregrams) is more appropriate. When electrophoregrams [fluorescence = f (time)] are used, and more than one chip is involved, electrophoregram comparisons are challenging due to experimental variations between chips and the lack of correction by the Agilent software in such situations. Here we present RisaAligner software for analyzing and comparing electrophoregrams from Agilent chips using a nonlinear ladder-alignment algorithm. We demonstrate the robustness and substantial improvement of data analysis by analyzing soil microbial profiles obtained with Agilent DNA 1000 and High Sensitivity chips.

Effect of DTPA on Cd solubility in soil--accumulation and subsequent toxicity to lettuce.

In a controlled environment experiment, using Cd spiked soil, lettuce plants were grown under a range of DTPA levels and were subsequently harvested to determine levels of phytoaccumulation. Cadmium phytoaccumulation significantly increased with increasing soil Cd level (P<0.05) but unexpectedly decreased with increasing DTPA levels, despite the fact that solubility of Cd was increased in the soil. Cadmium translocation (from root to shoot) increased after DTPA application. Lettuce growth was inhibited by both Cd and DTPA (at and above 10 and 500 mg kg(-1) respectively), as a result of higher Cd mobility and subsequent toxicity which was caused by DTPA higher dosages. Metal solubility in the soil (ranged between 2.8 and 26.5 mg kg(-1)) was found to be significantly higher (P<0.01) as compared to control with increasing DTPA levels even after 3 months of DTPA application. Cadmium tissue concentration in all DTPA treatments was less than in the corresponding control treatment, indicating a negative effect of DTPA application on Cd uptake. In conclusion, lettuce was an unsuitable plant species for Cd accumulation, at least when associated with a DTPA chelator.

A comparison of enhancement techniques for footwear impressions on dark and patterned fabrics.

The use of chemical enhancement techniques on porous substrates, such as fabrics, poses several challenges predominantly due to the occurrence of background staining and diffusion as well as visualization difficulties. A range of readily available chemical and lighting techniques were utilized to enhance footwear impressions made in blood, soil, and urine on dark and patterned fabrics. Footwear impressions were all prepared at a set force using a specifically built footwear rig. In most cases, results demonstrated that fluorescent chemical techniques were required for visualization as nonfluorescent techniques provided little or no contrast with the background. Occasionally, this contrast was improved by oblique lighting. Successful results were obtained for the enhancement of footwear impressions in blood; however, the enhancement of footwear impressions in urine and soil on dark and patterned fabrics was much more limited. The results demonstrate that visualization and fluorescent enhancement on porous substrates such as fabrics is possible.

Investigation of sterols as potential biomarkers for the detection of pig (S. s. domesticus) decomposition fluid in soils.

This study was carried out to evaluate the potential of using cholesterol and coprostanol, as indicators for the detection of decomposition fluid of buried pigs (S. s. domesticus) in soils. In May 2007, four pig carcasses (∼35kg) were buried in shallow graves (∼40 cm depth) at the University of Ontario Institute of Technology in Canada. Two pigs were exhumed after three months (Pig 1, Pig 2) and six months (Pig 3, Pig 4) post burial. Soil samples were collected beneath the pig carcasses (∼40cm depth) and from grave walls (∼15-20 cm depth) as well as from a parallel control site. Coprostanol and cholesterol were extracted from soils, purified with solid phase extraction (SPE) and analysed with gas chromatography/mass spectrometry (GC/MS). A significant increase in cholesterol concentrations (p<0.05) and amounts of coprostanol were detected in soil located beneath the pig carcasses after three months of burial. It is assumed that during the putrefaction and liquefaction stages of decomposition pig fluid which contains cholesterol and coprostanol is released into the underlying soil. Therefore, cholesterol and coprostanol could be used as potential biomarkers to detect the presence of decomposition fluid three months after burial under comparable soil and environmental conditions. Further research is suggested for additional soil sampling before and after three months to investigate the abundance of these and other sterols.

Organic matter characterization of sediments in two river beaches from northern Portugal for forensic application.

In a forensic investigation, the analysis of earth materials such as sediments and soils have been used as evidence at a court of law, relying on the study of properties such as color, particle size distribution and mineral identification, among others. In addition, the analysis of the organic composition of sediments and soils is of particular value, since these can be used as complementary independent evidence to the inorganic component. To investigate the usefulness of organic indicators in sediment characterization and discrimination, seventy-seven samples were collected during a period of one year in two river beaches located at the southern bank of the Douro River estuary in the North of Portugal. Isotopes of total carbon, pollen and plant wax-marker analyses were performed. In both beaches, an increase of the organic matter concentrations was noticeable, moving landward, related with the higher cover of associated plant material. The results obtained showed that the combination of all the techniques adopted showed a clear discrimination between samples from the two beaches, and also showed a differentiation of samples in relation to distance from the river in both beaches. The results also show that seasonality in these beaches was not a determining factor for discrimination, at the times considered. In addition, the effects of time was not marked.

Chemical enhancement of soil based footwear impressions on fabric.

This study investigates the enhancement of footwear impressions prepared with soils from different locations on a variety of fabric surfaces with different morphology. Preliminary experiments using seventeen techniques were carried out and the best responding reagents were evaluated further. Results indicated that the soils investigated (a cross-section of soils from Scotland) are more likely to respond to reagents that target iron ions rather than calcium, aluminium or phosphorus ions. Furthermore, the concentration of iron and soil pH did not appear to have an effect on the performance of the enhancement techniques. For the techniques tested, colour enhancement was observed on all light coloured substrates while enhancement on dark coloured fabrics, denim and leatherette was limited due to poor contrast with the background. Of the chemical enhancement reagents tested, 2,2'-dipyridil was a suitable replacement for the more common enhancement technique using potassium thiocyanate. The main advantages are the use of less toxic and flammable solvents and improved clarity and sharpness of the enhanced impression. The surface morphology of the fabrics did not have a significant effect on the enhancement ability of the reagents apart from a slight tendency for diffusion to occur on less porous fabrics such as polyester and nylon/lycra blends.