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Hydrolysis of carbohydrate polymers such as cellulose, xylan and starch is used to produce fermentable sugars for bio-ethanol production. Digestion of biomass material and its fermentation represents the best path forward for bio-ethanol production. The use of lignocellulosic material to produce ethanol presents a number of challenges relative to the use of starch containing sources such as corn, which can be digested with amylases. In order to improve the ethanol yield from these polymers, scientists have turned to the use of specific enzymes from a number of different sources. Xylanase enzymes digest xylan polymers, which are a major constituent of the hemicellulose, into xylose. Optimization of these enzymes in a laboratory or industrial setting requires experimentation with a number of different variables. EnzCheck Ultra Amylase assay (cat # E33651), Amplex® Red Glucose assay (cat# A13261), and EnzCheck Ultra Xylanase assay (cat# E33650) kits as well as the fluorescent cellulase substrate (cat# E33953) were obtained from Life Technologies. Xylanase activity was measured using an EnzCheck Ultra Xylanase assay kit as described previously [2]. Cellulase activity was also measured directly using an EnzCheck Cellulase substrate as described previously [2]. In order to demonstrate the utility of microplates and microplate readers, three different commonly used enzymes responsible for the digestion of polysaccharides used as feed stocks for ethanol production were investigated for activity under a number of environmental conditions. The effect of pH activity was tested on amylase enzyme isolated from three different sources. Xylanase enzyme isolated from the fungi Thermomyces lanuginosus was found to be very temperature stable. Note that the effect of pH on the fluorescence of the reacted substrate must be accounted for when comparing results at different pH levels. Enzyme titrations performed at different pH levels corroborate the fixed concentration data. These data demonstrate the utility of microplate readers to perform classic enzymology experiments on critical enzymes necessary for the digestion of polysaccharide feed stock into fermentable monosaccharides for the production of ethanol. The use of cellulosic second-generation material to generate fermentable sugars has some marked advantages over first generation starch based grains. The hydrolysis of xylan results in the pentose sugars xylose and arabinose which are not fermented by wild strains of the yeast Saccharomyces cerevisiea.
The use of ethanol as a means to replace a portion of the transportation fuel used in the United States has had legislative support for a number of years. BioTek Instruments, Inc., headquartered in Winooski, VT, USA, is a worldwide leader in the design, manufacture, and sale of microplate instrumentation and software.
This report contains the collective views of an international group of experts and does not necessarily represent the decisions or the stated policy of the United Nations Environment Programme, the International Labour Organization, or the World Health Organization.
Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organization, and the World Health Organization, and produced within the framework of the Inter-Organization Programme for the Sound Management of Chemicals. The International Programme on Chemical Safety (IPCS), established in 1980, is a joint venture of the United Nations Environment Programme (UNEP), the International Labour Organization (ILO), and the World Health Organization (WHO).
The Inter-Organization Programme for the Sound Management of Chemicals (IOMC) was established in 1995 by UNEP, ILO, the Food and Agriculture Organization of the United Nations, WHO, the United Nations Industrial Development Organization, the United Nations Institute for Training and Research, and the Organisation for Economic Co-operation and Development (Participating Organizations), following recommendations made by the 1992 UN Conference on Environment and Development to strengthen cooperation and increase coordination in the field of chemical safety. The World Health Organization welcomes requests for permission to reproduce or translate its publications, in part or in full. Publications of the World Health Organization enjoy copyright protection in accordance with the provisions of Protocol 2 of the Universal Copyright Convention. The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the Secretariat of the World Health Organization concerning the legal status of any country, territory, city, or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned.
The Federal Ministry for the Environment, Nature Conservation and Nuclear Safety, Germany, provided financial support for the printing of this publication.
Concise International Chemical Assessment Documents (CICADs) are the latest in a family of publications from the International Programme on Chemical Safety (IPCS) — a cooperative programme of the World Health Organization (WHO), the International Labour Organization (ILO), and the United Nations Environment Programme (UNEP). International Chemical Safety Cards on the relevant chemical(s) are attached at the end of the CICAD, to provide the reader with concise information on the protection of human health and on emergency action.
The primary objective of CICADs is characteri zation of hazard and dose–response from exposure to a chemical.
Risks to human health and the environment will vary considerably depending upon the type and extent of exposure. While every effort is made to ensure that CICADs represent the current status of knowledge, new informa tion is being developed constantly. The draft is then sent to an international peer review by scientists known for their particular expertise and by scientists selected from an international roster compiled by IPCS through recommendations from IPCS national Contact Points and from IPCS Participating Institutions. A consultative group may be necessary to advise on specific issues in the risk assessment document. Board members serve in their personal capacity, not as representatives of any organization, government, or industry.
Board members, authors, reviewers, consultants, and advisers who participate in the preparation of a CICAD are required to declare any real or potential conflict of interest in relation to the subjects under discussion at any stage of the process. This CICAD on N,N-dimethylformamide (DMF) was prepared jointly by the Environmental Health Directorate of Health Canada and the Commercial Chemicals Evaluation Branch of Environment Canada based on documentation prepared concurrently as part of the Priority Substances Program under the Canadian Environmental Protection Act (CEPA). When emitted into air, most of the DMF released remains in that compartment, where it is degraded by chemical reactions with hydroxyl radicals. Since most DMF appears to be released to air in the sample country, and based on the fate of DMF in the ambient environment, biota are expected to be exposed to DMF primarily in air; little exposure to DMF from surface water, soil, or benthic organisms is expected. Consistent with the results of studies in experimental animals, available data from case reports and cross- sectional studies in occupationally exposed populations indicate that the liver is the target organ for the toxicity of DMF in humans.
Based on the limited data available, there is no convincing, consistent evidence of increases in tumours at any site associated with exposure to DMF in the occupational environment. There is also little consistent, convincing evidence of genotoxicity in populations occupationally exposed to DMF, with results of available studies of exposed workers (to DMF and other compounds) being mixed. Although the database for carcinogenicity is limited to two adequately conducted bioassays in rats and mice, there have been no increases in the incidence of tumours following chronic inhalation exposure to DMF. In studies with laboratory animals, DMF has induced adverse reproductive effects only at concentra tions greater than those associated with adverse effects on the liver, following both inhalation and oral expo sure. Available data are inadequate as a basis for assessment of the neurological or immunological effects of DMF. The focus of this CICAD and the sample risk characterization is primarily effects of indirect exposure in the general environment. Air in the vicinity of point sources appears to be the greatest potential source of exposure of the general population to DMF. The following information on analytical methods for the determination of DMF in workplace air and biological media has been derived from WHO (1991) and Environment Canada (1999a). Colorimetric methods (based on the development of a red colour after the addition of hydroxylamine chloride as alkaline solution) that have often been utilized in the past are not specific (Farhi et al., 1968).
DMF is extensively absorbed through the skin, its metabolism and kinetics are well known, and urinary metabolites exist that can be accurately measured.
Identified data on releases are restricted to the country of origin of the source document (Canada). In 1996, just over 16 tonnes of DMF were released from various industrial locations in Canada, of which 93% (15 079 kg) were emitted to the atmosphere and the remainder to water (245 kg), wastewater (204 kg), landfill sites (26 kg), or deep-well injection (669 kg) (Environment Canada, 1998). In the USA, between 23 and 47 million kilograms of DMF were produced in 1990 (US EPA, 1997).
The total consumption of DMF in Western Europe in 1989 was reported to be 55 000 tonnes (BUA, 1994).
Chemical degradation of DMF in air is likely due to reaction with hydroxyl radicals (Hayon et al., 1970). Based on experiments in chambers, reactivity for DMF relative to propane is low (Sickles et al., 1980). Although the degradation half-life of DMF in air cannot be estimated with certainty, the available evi dence therefore suggests that the half-life is at least 8 days (192 h). Once released into surface water, DMF is unlikely to transfer to sediments, biota, or the atmosphere. Biodegradation of DMF in receiving surface waters is unlikely to be affected by the inherent toxicity of DMF and its biodegradation products. Biological degradation and, to a lesser extent, chemical processes operating in surface water would also likely affect DMF contained in soil pore water (Scott, 1998). The miscibility of DMF and its low Henry’s law constant indicate limited volatilization from moist soils (BUA, 1994). Fugacity modelling was conducted to provide an overview of key reaction, intercompartment, and advection (movement out of a system) pathways for DMF and its overall distribution in the environment. Fugacity modelling also indicates that when DMF is continuously discharged into either water or soil, most of it can be expected to be present in the receiving medium. It is important to note that fugacity-based partitioning estimates are significantly influenced by input parameters such as the Henry’s law constant, which, in this case, is highly uncertain. In Canada, monitoring data are available for effluents at one southern Ontario location, which released less than ~0.03 tonnes into surface water in 1996 (Environment Canada, 1998). Although DMF was listed as a contaminant in a survey of drinking-water in the USA, quantitative data were not reported (Howard, 1993). A Health Canada-sponsored multimedia exposure study for DMF and other volatile organic compounds was conducted in 50 homes in the Greater Toronto Area in Ontario, Nova Scotia, and Alberta (Conor Pacific Environmental, 1998). Identified data on concentrations of DMF in environmental media in Canada were insufficient to allow estimates of population exposure to be developed; for water, either quantitative data on concentrations are unreliable18 or DMF has not been detected, using analytical methodology with poor sensitivity (Conor Pacific Environmental, 1998). Non-pesticidal use of DMF in Canada is small and restricted primarily to industrial applications.
Occupational exposure to DMF may occur in the production of the chemical itself, other organic chemi cals, resins, fibres, coatings, inks, and adhesives (IARC, 1999).
Available data indicate that DMF is readily absorbed following oral, dermal, and inhalation expo sure in both humans and animals. The major metabolic pathway for DMF in mam malian species is oxidation by the cytochrome P-450- dependent mixed-function oxidase system to HMMF (Figure 1). Levels of parent compound and metabolites were determined in the plasma, amniotic fluid, placenta, and embryo in this investigation.
In comparative analyses of the two studies, the authors indicated that toxicokinetic differences may, in part, contribute to the observed species differences in toxicity.
In a number of early studies, the effects of co- administration of ethanol on blood concentrations of DMF, NMF, ethanol, and acetaldehyde were investigated.
Exposure in the occupational environment may occur through both the dermal and inhalation routes. Wrbitzky & Angerer (1998) noted a weak associa tion between the concentration of DMF in workplace air and urinary concentration of NMF. Following oral, dermal, inhalation, or parenteral administration, the acute toxicity of DMF in a number of species is low.
Standard tests for dermal irritation by DMF have not been identified, and data on its sensitization potential are conflicting. IARC (1999), WHO (1991), and Kennedy (1986) reviewed the effects of DMF on the skin and eyes and reported only mild to moderate effects. In a murine local lymph node assay predictive for identification of contact allergens, cell proliferation (based on [3H]thymidine incorporation in lymph nodes) was significantly increased (324 vs. While there have been a number of primarily early short-term studies, these have generally been restricted to examination of specific effects following exposure to single dose levels.
Information on the incidences of lesions in the critical medium-term exposure studies is presented in Tables 2 and 3. In a second study involving larger group sizes, a different strain (Wistar), and more comprehensive tissue examination, growth was inhibited but no tissue lesions were observed in rats administered DMF in the diet for 15 weeks (Becci et al., 1983). Information on the incidences of lesions in critical long-term studies is presented in Tables 2 and 3. The exogenous digestion of these polymers on an industrial scale can be cost prohibitive without optimization of the enzyme reaction conditions.
While starch based feedstocks have been used extensively, it is believed that lignocellulosic energy sources offer a better long-term prospect as a source for fermentable sugars. Using a combination of in vitro digestion, in conjunction with in vivo genetic manipulation of yeast and bacteria strains, the digestion efficiency to fermentable sugars cannot only be increased, but the ability to ferment monosaccharides other than glucose can be improved. The ability to measure multiple samples rapidly in small volumes offers significant advantages in terms of time, expense and effort. Carboxymethyl cellulose (cat # 419273), xylanase from Thermomyces lanuginosus (cat# X2753), from Trichoderma longibrachiatum (cat # X2629), cellulase from Aspergillus niger (cat# C1184), α-amylase from Aspergillus oryzae (cat# 10065) from Bacillus licheniformis (cat# A4551) and Bacillus subtilis (Cat#10069) were purchased from Sigma-Aldrich. In these experiments, equivalent concentrations of a reference standard were used to normalize data prior to plotting. The direct assay has similar detection limits as the glucose determination, but does not require an overnight digestion. Cellulosic materials do not compete directly with food stocks for resources such as tillable land, fertilizers and water. Its linear chains of glucose moieties closely pack with one another forming hydrogen bonds between chains resulting in a crystalline material that is insoluble in water and many organic solvents.

To address this limitation several different strains have been genetically modified in regards to xylanase and arabinose transport proteins and pentose fermentation pathways [4]. These technologies are used to aid life science research, facilitate drug discovery, provide rapid and cost-effective analysis, and enable sensitive, accurate quantification of molecules across diverse applications.
The overall objectives of the IPCS are to establish the scientific basis for assessment of the risk to human health and the environment from exposure to chemicals, through international peer review processes, as a prerequisite for the promotion of chemical safety, and to provide technical assistance in strengthening national capacities for the sound management of chemicals. The purpose of the IOMC is to promote coordination of the policies and activities pursued by the Participating Organizations, jointly or separately, to achieve the sound management of chemicals in relation to human health and the environment. Applications and enquiries should be addressed to the Office of Publications, World Health Organization, Geneva, Switzerland, which will be glad to provide the latest information on any changes made to the text, plans for new editions, and reprints and translations already available.
Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. CICADs join the Environmental Health Criteria documents (EHCs) as authoritative documents on the risk assessment of chemicals.
CICADs are not a summary of all available data on a particular chemical; rather, they include only that information considered critical for characterization of the risk posed by the chemical. Responsible authorities are strongly encour aged to characterize risk on the basis of locally measured or predicted exposure scenarios. Unless otherwise stated, CICADs are based on a search of the scientific literature to the date shown in the executive summary. Authors of the first draft are usually, but not necessarily, from the institution that developed the original review. They are selected because of their expertise in human and environmental toxicology or because of their experience in the regulation of chemicals.
Representatives of nongovernmental organizations may be invited to observe the proceedings of the Final Review Board.
The objective of assessments on Priority Substances under CEPA is to assess potential effects of indirect exposure in the general environment on human health as well as environmental effects. Indirect releases of DMF to air, such as transfers from other environmental media, play only a small role in main taining levels of DMF in the atmosphere. Based on this, and because of the low toxicity of DMF to a wide range of aquatic and soil organisms, the focus of the environmental risk characterization is terrestrial organisms exposed directly to DMF in ambient air.
Following absorption, DMF is uniformly distributed, metabolized primarily in the liver, and relatively rapidly excreted as metabolites in urine.
The profile of effects is consistent with that observed in experimental animals, with gastro intestinal disturbance, alcohol intolerance, increases in serum hepatic enzymes (aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transpeptidase, and alkaline phosphatase), and histopathological effects and ultrastructural changes (hepatocellular necrosis, enlarged Kupffer cells, microvesicular steatosis, complex lysosomes, pleomorphic mitochondria, and fatty changes with occasional lipogranuloma) being observed. Case reports of testicular cancers have not been confirmed in a cohort and case– control study.
The weight of evidence for genotoxicity is over whelmingly negative, based on extensive investigation in in vitro assays, particularly for gene mutation, and a more limited database in vivo. Similarly, in well conducted and reported primarily recent developmental studies, fetotoxic and teratogenic effects have been consistently observed only at maternally toxic concentrations or doses.
Based on the results of epidemiological studies of exposed workers and supporting data from a relatively extensive database of investigations in experimental animals, the liver is the critical target organ for the toxicity of DMF. Effect concentrations for indicators of the potential sensitivities of trees, shrubs, and other plants are high; hence, it is unlikely that terres trial plants are particularly sensitive to DMF. DMF is also a powerful solvent for a variety of organic, inorganic, and resin products (SRI Interna tional, 1994). Bobra, AMBEC Environmental Consultant, to Chemicals Evaluation Division, Commercial Chemicals Evaluation Branch, Environment Canada, 1999. Methods of choice more recently are high-performance liquid chromatography (HPLC) or gas chromatography – mass spectrometry (GCMS). As a result, biological monitoring has been extensively used in the assessment of the absorbed amounts in occupa tionally exposed populations. The production capacity was estimated to be 60 000 and 19 000 tonnes in the former Federal Republic of Germany and German Democratic Republic, respec tively, 16 000 tonnes in Belgium, 15 000 tonnes in England, and 5000 tonnes in Spain (BUA, 1994). The total quantity of DMF used in formulation of products (other than pesticides) appears to be small in comparison to its use as a manufacturing aid, cleaner, or degreaser (Environment Canada, 1998). DMF is also used in the production of polyurethane resin for synthetic leather (Fiorito et al., 1997). This is due to the fact that industrial releases of DMF into air appear to be considerably larger than releases to other environmental media (BUA, 1994; Environment Canada, 1998).
However, the degradation half-life of DMF can be roughly estimated by comparing DMF with other compounds in terms of their relative atmospheric reactivity. The mean half-life used for fugacity- based fate modelling was 170 h, as it is frequently used to represent a half-life range of 100–300 h (DMER & AEL, 1996).
As for surface water, biodegradation should therefore be the primary breakdown mechanism in soils.
A steady-state, non-equilibrium model (Level III fugacity modelling) was run using the methods developed by Mackay (1991) and Mackay & Paterson (1991). If DMF is emitted into air, fugacity modelling predicts that 61% of the chemical will be present in air, 32% in soil, and only 7% in water. For example, if it is released into water, 99% of the DMF is likely to be present in the water, and subsequent transport into sediment or bioconcentration in biota is not likely to be significant.
No infor mation was provided on proximity to sources of DMF, sediment characteristics, or hydrological regimes. Unchanged DMF initially accounted for the major proportion of radiolabelled carbon in the plasma or tissues, 61–77% for the first 4 h and 73–93% for the first 8 h after treatment on days 12 and 18, respectively.
Results of several of these earlier studies were also suggestive that at very high concentrations, DMF inhibits its own biotransformation. Although there were variations in results depending on dose, time interval between administration of DMF and ethanol, and routes of exposure, there were increases in concentrations of DMF, NMF, ethanol, or acetaldehyde in blood upon co-exposure. Results of these investigations indicated that DMF was rapidly excreted (the majority in 24 h), primarily as HMMF. With the exception of more recent studies involving personal air sampling (Wrbitzky & Angerer, 1998),21 few provide reliable quantitative data on rela tionship with exposure, though still not accounting for additional dermal exposure.
Hence, only limited conclusions can be drawn concerning the potential of DMF to induce these effects. 193 decompositions per minute per lymph node in exposed and control groups, respectively) in mice (strain not specified) receiving a daily topical application of 25 µl on the dorsum of both ears for 3 consecutive days (Montelius et al., 1996). They are not additionally informative concerning the toxicity of DMF but confirm a range of effects in the liver, which, when considered collectively across studies, are consistent with a profile in rats of alterations in hepatic enzymes and increases in liver weight at lowest concentrations and degenerative histopathological changes, cell death, and increases in serum hepatic enzymes at higher concentrations.
Relative liver weight was significantly increased in both sexes at all levels of exposure, although the dose–response was not clear.
Microscopic examination of an extensive range of organ tissues revealed only mild effects on the liver in the majority of high-dose males and females.
The protocol included measurement of food consumption, measurement of body weight gain, hearing tests, ophthalmoscopic examination, clinical laboratory investigations, measurement of organ weights, and histopathological observations.
We have previously shown the ability to quantitate these enzymes; here we describe the optimization of some of the reaction conditions for the enzymes that catalyze the digestive hydrolysis of polysaccharide polymers into monomeric constituents. Lignocellulosic feedstocks, such as wood chips, corn stover or switchgrasses do not directly compete with food sources for land or consumption. The chain structure forms a stiff extended conformation that results in extensive hydrogen bonding with adjacent chains.
Termites and ruminant herbivores digest cellulose through the action of symbiotic bacteria located in their intestines and ruminating chambers respectively. The use of microplates is one way that large numbers of samples can be handled with minimal amounts of reagents and time.
Amylase enzyme isolated from different organisms were compared for their activity at different pH levels. Enzyme concentration titration curves indicate that higher temperatures result in slightly more hydrolysis than ambient temperatures (Figure 7).
Using the direct substrate assay, the effect of pH on Aspergillus niger cellulase enzyme activity was investigated. Grain based feed stock rich in starch requires amylase enzymatic activity, while plant and wood material needs cellulase and xylanase activity to break down the cellulose and xylan material contained in the plant fibers.
It can be chemically digested with a combination of high temperature and concentrated acids [3].
Ethanol is currently used as an additive to gasoline in the US to replace MBTE as a means to increase gasoline oxygen content. BioTek espouses a "Think Possible" approach that sets the tone for fresh ideas, unsurpassed customer service and original innovations. They may be complemented by information from IPCS Poison Information Monographs (PIM), similarly produced separately from the CICAD process. Before acceptance for publication as CICADs by IPCS, these documents undergo extensive peer review by internationally selected experts to ensure their complete ness, accuracy in the way in which the original data are represented, and the validity of the conclusions drawn. The critical studies are, however, presented in sufficient detail to support the conclusions drawn. To assist the reader, examples of exposure estimation and risk characteriza tion are provided in CICADs, whenever possible. In the event that a reader becomes aware of new informa tion that would change the conclusions drawn in a CICAD, the reader is requested to contact IPCS to inform it of the new information.
The IPCS Risk Assess ment Steering Group advises the Co-ordinator, IPCS, on the selection of chemicals for an IPCS risk assessment, whether a CICAD or an EHC is produced, and which institution bears the responsibility of the document production, as well as on the type and extent of the international peer review.
Authors are required to take reviewers’ comments into account and revise their draft, if necessary. Boards are chosen according to the range of expertise required for a meeting and the need for balanced geographic representation.
Observers may participate in Board discussions only at the invitation of the Chairperson, and they may not participate in the final decision-making process. The major pathway involves the hydroxylation of methyl moieties, resulting in N-(hydroxymethyl)-N- methylformamide (HMMF), which is the major urinary metabolite in humans and animals. There have been no consistent increases in tumours at other sites associated with exposure to DMF. However, in view of the positive dose–response relationship observed in the one study in which it was investigated, this area may be worthy of additional work, although available data on genotoxicity in experimental systems are overwhelmingly negative. In acute and repeated-dose toxicity studies, DMF has been consistently hepatotoxic, inducing effects on the liver at lowest concentrations or doses. At temperatures below 100 °C, DMF remains stable in relation to light and oxygen (BUA, 1994). The metabolite most often analysed is N-methylformamide (NMF), and several GC methods exist (Ikeda, 1996). Both are commonly occurring natural substances and are also used in industrial applications (European Chemicals Bureau, 1996a, 1996b).
The petrochemical sector was responsible for 84% (12.7 tonnes) of the reported atmospheric releases.
Other chemical degradation processes — for example, reaction with nitrate radicals — are not known to significantly affect the fate of DMF in air.
This Kow also suggests that DMF does not concentrate in aquatic organisms (BUA, 1994); indeed, no bioaccumulation was observed in carp during an 8- week bioaccumulation test (Sasaki, 1978). The photooxidation half-life of DMF in water was estimated experimentally at 50 days and would be even longer in the natural environment where other compounds compete for reaction with hydroxyl radicals (Hayon et al., 1970). Inter mediate biodegradation products include formic acid and dimethylamine, which further degrade to ammonia, carbon dioxide, and water (Dojlido, 1979; Scott, 1998). However, even with continuous releases, such high concentrations of DMF are not anticipated in natural waters. A soil bacterial culture acclimated to small amounts of petroleum and petroleum products degraded DMF under aerobic conditions within 18 h (Romadina, 1975), indicating a soil biodegradation half-life similar to the one observed in water.
Assumptions, input parameters, and results are summarized in Environment Canada (1999a) and presented in detail in DMER & AEL (1996) and by Beauchamp12 and Bobra13.
These results suggest that most of the DMF released into air will remain in that compartment, where it will be degraded by chemical reactions. When releases are into soil, 94% of the material remains in the soil — presumably in soil pore water (Scott, 1998). In addition, because information on sampling and analyti cal methods was not provided, the quality of these data cannot be assessed.
DMF is metabolized primarily in the liver and is relatively rapidly excreted as metabolites in urine, primarily as N-(hydroxymethyl)-N-methylformamide (HMMF).
However, there was no corresponding decrease in NMF levels; rather, they increased proportionally with increases in exposure concentrations. In a separate protocol, three volunteers ingested 20 mg AMCC dissolved in water, and metabolites were determined for a period of 8 h after exposure.
Results of such studies have confirmed, however, the presence of AMCC (the product of the putatively toxic metabolic pathway) in the urine of workers. The metabolism of DMF to HMMF by human liver micro somes in vitro has also been demonstrated. Clinical signs following acute exposure include general depression, anaesthesia, loss of appetite, loss of body weight, tremors, laboured breathing, convulsions, haemorrhage at nose and mouth, liver injury, and coma preceding death. In subsequent assays, thymidine incorpora tion in DMF-exposed mice was up to 3-fold higher than in naive mice.

Although results of a short-term study in monkeys also indicate that this species is less sensitive to the effects of DMF than rats, the protocol had only one exposure concentration, and there were only two monkeys in the experiment (Hurtt et al., 1991).
Absolute liver weight was significantly increased in females at all dose levels, although the dose–response was not clear.
Two males were maintained for a further 13-week observation period after exposure had ceased.
Although there was an apparent increase in serum cholesterol in both sexes at the highest dose, statistical analyses were not presented. There was a dose-related increase in relative liver weight at all dose levels, although this was statistically significant only in the mid- and high-dose females and in the high-dose males. Regardless of the source of feed stock, current production costs make them unpalatable as a fuel.
Primary and secondary walls contain cellulose, hemicellulose and pectin, albeit in different proportions.
The result is the formation of microfibrils with high tensile strength and poor water solubility. Xylose is a five carbon monosaccharide that can form either furanose (5-member) or pyranose (6-member) ring structures.
Here we describe the use of microplates to assess physical assay characteristics, such as pH, temperature and enzyme concentration, of some of the enzymes that catalyze the hydrolysis of plant polysaccharides.
Correction for the influence of pH on fluorescent signal data was made by normalization each well of the initial unreacted read to a common value.
Interestingly, temperatures very near ambient (25° C) had the greatest reduction in enzyme activity (Figure 4) when assessed using the fluorescent amylase substrate. The fluorescence was plotted after 30 minute incubation from assays run at different temperatures.
As seen in Figure 10, Aspergillus niger cellulase exhibits significant activity in acidic pH levels. Regardless of the enzymatic requirements it is important that the conditions for maximal enzymatic activity be optimized. However, this technology is currently unfeasible from both an economic and hazardous waste standpoint.
For additional information, the reader should consult the identified source documents upon which the CICAD has been based. These examples cannot be considered as representing all pos sible exposure situations, but are provided as guidance only.
The first draft undergoes primary review by IPCS and one or more experienced authors of criteria documents in order to ensure that it meets the specified criteria for CICADs. The resulting second draft is submitted to a Final Review Board together with the reviewers’ comments. Data identified as of the end of September 1999 (environmental effects) and February 2000 (human health effects) were considered in this review. However, some atmospheric DMF can reach the aquatic and terrestrial environment, presumably during rain events.
The profile of effects includes alterations in hepatic enzymes charac teristic of toxicity, increases in liver weight, progressive degenerative histopathological changes and eventually cell death, and increases in serum hepatic enzymes. Comparison of this value with a conserva tive estimated exposure value indicates that it is unlikely that DMF causes adverse effects on terrestrial organisms in the sample country.
Because DMF is a miscible compound, it is preferable to determine the Henry’s law constant experimentally. Releases from the pharmaceutical industry accounted for 87% (0.212 tonnes) of total releases to water. The industrial DMF deposited directly in landfill sites consists only of residues remaining after incineration (Environment Canada, 1998). Modelling predictions do not reflect actual expected concentrations in the environ ment but rather indicate the broad characteristics of the fate of the substance in the environment and its general distribution among the media. They also indicate that some atmospheric DMF can reach the aquatic and ter restrial environment — presumably in rain and runoff (Scott, 1998).14 However, the quantity of DMF available for entrainment in rain and runoff is limited by degra dation in the atmosphere. Therefore, indirect releases of DMF to air, such as transfers from other environmental media, play only a small role in maintaining levels of DMF in the atmosphere.
Therefore, the greatest potential for exposure of the general population to DMF from non-pesticidal sources is in air in the vicinity of industrial point sources. For each concentration, AUC values, peak plasma concentration, and plasma half-lives were consistent throughout the duration of exposure. Contrary to the results in animals, there were no signi ficant differences in the blood levels of ethanol and acetaldehyde upon co-exposure, which the authors attributed to the relatively low concentrations of DMF (Eben & Kimmerle, 1976).
Where protocols included histopath ological examination, damage was observed primarily in the liver (WHO, 1991). However, statistical analyses were not presented, and the increase was not considered to be significant (Montelius et al., 1998). The protocol included microscopic examination of a comprehensive range of organ tissues in all animals. The secondary walls of woody tissue and grasses are composed predominantly of cellulose, lignin, and hemicellulose. In order to correct for the influence of pH on the fluorescent signal data was normalized using a fluorescence reference supplied by the assay kit at each pH.
The effect of temperature was measured by running separate assays at defined temperatures using the Synergy H4 Hybrid Multi- Mode Microplate Reader to maintain temperature. Subsequent data was normalized using the same factor and expressed as the change in signal of the initial unreacted fluorescence. Because microplates provide the ability to test large numbers of samples they are an ideal tool to test multiple experimental conditions.
Towards that end, efforts have focused on ways to reduce the amount of acid used as a treatment by using a combination of dilute acids in conjunction with enzymatic hydrolysis.
Because it is currently being used in large quantities the infrastructure to deliver the fuel already exists. The reader is referred to EHC 1701 for advice on the derivation of health-based tolerable intakes and guidance values.
Information on the nature of the peer review and availability of the source document is presented in Appendix 1. It is completely miscible with water and most organic solvents and has a rela tively low vapour pressure.
In turn, enzymatic N-methyl oxidation of NMF can produce N- (hydroxymethyl)formamide (HMF), which further degenerates to formamide. A dose–response has been observed for these effects in rats and mice following inhalation and oral exposure.
DMF sold commer cially contains trace amounts of methanol, water, formic acid, and dimethylamine (BUA, 1994). Extended adaptation under specific experimental conditions may also account for negative degradation results observed in a few studies with incubation times ? 14 days (Kawasaki, 1980; CITI, 1992).
DMER & AEL (1996) recommend a half-life in sedi ment of 170 h based on the assumption that reactivity in sediment is slower than in soil. HMMF was the main urinary metabolite (56–95%), regardless of exposure level or duration of exposure. The half-times of excretion for these various metabolites were approximately 2, 4, 7, and 23 h, respectively.
The naive (non- treated) mice were included in the protocol to measure the magnitude of vehicle (DMF)-induced proliferation.
Serum cholesterol was increased at all levels of expo sure; again, there was no clear dose–response. There were some changes in clinical chemistry and haematological parameters at the highest doses. The effect of temperature was measured by running separate assays at defined temperature settings using the Synergy™ H4 Hybrid Multi-Mode Microplate Reader to control temperature.
Fluorescence for all experiments was measured kinetically with a Synergy H4 Hybrid Multi-Mode Microplate Reader.
Enzymes that can be shown to work well in high temperatures in an acidic environment would provide a distinct advantage over those that are labile under the same conditions. When releases are into soil, most of the DMF remains in the soil — presumably in soil pore water — until it is degraded by biological and chemical reaction. An alternative pathway for the metabolism of NMF is oxidation of the formyl group, resulting in N-acetyl-S-(N-methylcarbamoyl) cysteine (AMCC), which has been identified as a urinary metabolite in rodents and humans.
Limited degradation was reported in seawater (range 1–42%) (Ursin, 1985), and no degradation was found after 8 weeks’ incubation under anaerobic conditions (Shelton & Tiedje, 1981). In rats exposed on day 18 of gestation, fetal tissues accounted for 6% of the administered dose.
DMF was not readily excreted in the urine, and NMF was more prevalent in plasma than in urine, suggesting that it was metabolized to compounds not determined in the study. In contrast to this slow elimina tion after exposure to DMF, AMCC was rapidly eliminated after ingestion of AMCC, with a half-time of 1 h. Kafferlein21 reported that urinary NMF concentrations were highest in post-shift samples, with a median half- time of 5.1 h. In rats exposed to the highest dose, excretion of DMF metabolites (including AMCC) was delayed. In contrast, Kimber & Weisenberger (1989) detected no difference in proliferation in a lymph node assay in which lymph node cells from DMF (the solvent)- exposed mice were compared with those from naive mice. Cross-linking of this network is believed to result in the elimination of water from the wall and the formation of a hydrophobic composite that limits accessibility of hydrolytic enzymes and is a major contributor to the structural characteristics of secondary walls.
As demonstrated in Figure 6, enzymes from these different sources have markedly different responses to differing pH levels. Likewise the xylanase enzymes that work effectively under similar conditions are desirable as xylan is a significant component of most cellulosic feed stocks.
Releases to water or soil are expected to be followed by relatively rapid biodegradation (half-life 18–36 h). A reactive interme diate, the structure of which has not yet been determined (possibly methyl isocyanate), is formed in this pathway; while direct supporting experimental evidence was not identified, this intermediate is suggested to be the putatively toxic metabolite. Less than 1 tonne of DMF was released from wastewater treatment facilities and in landfills (Envi ronment Canada, 1998). HPLC analysis performed at intervals from 1 to 24 h indicated that unchanged DMF and metabolites were readily transferred to the embryonic and fetal tissues, where levels were generally equal to those in maternal plasma. These results were considered to be consistent with rate-limiting reversible protein binding of a reactive meta bolic intermediate of DMF, possibly methyl isocyanate. Concentrations of urinary AMCC reached a steady state 2 days after the beginning of exposure, with a half-time greater than 16 h. There was no clear dose-related variation in proportion of the metabolites determined excreted as AMCC in the animal species. Xylan, which accounts for up to 30% of the mass of the secondary walls in wood and grasses, contributes to the recalcitrance of these walls to enzymatic degradation. This CICAD was approved as an international assessment at a meeting of the Final Review Board, held in Helsinki, Finland, on 26–29 June 2000. Available data indicate that a greater proportion of DMF may be metabolized by the putatively toxic pathway in humans than in experimental animals.
With a few exceptions, most industries reported little to no seasonal variation in releases (Environment Canada, 1998).
The parent compound accounted for most of the radioactivity until 4–8 h and then decreased. In humans, a greater proportion of the absorbed dose (14.5%) following inhalation was present as AMCC in the urine.
In females, relative liver weight was signifi cantly increased at all levels of exposure, with the weight declining at the highest dose.
The background fluorescence from an unreacted sample (no enzyme) at that pH raw was first subtracted and then the blanked data normalized by a known fluorescence reference at the same pH.
The use pattern of DMF is such that exposure of the general population is probably very low. There is metabolic interaction between DMF and alcohol, which, though not well understood, may be due, at least in part, to its inhibitory effect on alcohol dehydrogenase.
Although quantitative data were not presented, urinary elimination 16 h following the fifth exposure was approximately 14% HMMF, 32% HMF, and 54% AMCC.
Serum cholesterol was significantly increased at all levels of exposure in females, with no clear dose–response. In contrast to males, serum AP was increased in a dose-related manner (significant at the two highest concentrations). The International Chemical Safety Card (ICSC 0457) for N,N-dimethylformamide, produced by the International Programme on Chemical Safety (IPCS, 1999), has also been reproduced in this document. A NIOSH (1994) gas chromatographic (GC) method has an estimated detection limit of 0.05 mg per sample.

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