In livestock, a number of pathological conditions and/or syndromes exist which may not be attributable to infection by a single organism. In many instances, these pathological conditions and/or syndromes have an effect on the equilibrium that ordinarily exists within the various bacteria comprising the normal bacterial flora. A number of these pathological conditions and/or syndromes have been clinically referred to as dysbacteriosis and bacterial overgrowth. The present invention provides methods for evaluation of the xe2x80x9cgeneral healthxe2x80x9d of livestock by analyzing the composition of microbiological flora. In one aspect, a method of the invention comprises providing a sample of microbiological flora, selectively amplifying nucleic acid in the flora sample, subjecting the amplificate to restriction digestion, and then analyzing the resulting pattern of restriction fragments.

The present invention relates to methods and means for diagnosis of pathological or other detrimental conditions affecting the general health of a group of individuals, in particular vertebrates, and more typically mammals and/or birds, such as livestock. The invention further relates to methods and means for microbiological typing of substances comprising a variety of microbiological organisms such as, but not limited to, food, food additives, waste material, soils etc. The invention is exemplified by using feces as a source of microbiological populations. However, the methods and means of the invention are equally applicable to microbiological populations from other sources.
In livestock a number of pathological conditions and/or syndromes have been noticed that do not seem attributable to, e.g., an infection with a single micro-organism. These syndromes, however, do seem to have an effect on the equilibrium which normally exists within the different bacteria constituting the bacterial flora. A number of these syndromes are referred to as dysbacteriosis and bacterial overgrowth.
Although not wishing to be bound by theory, a possible explanation for the presence of these syndromes is that these diseases may occur due to:
1. feed intake/feed type.
The feed interferes with the digestion, resulting in changes in the bacteria. Some particular bacteria or groups of bacteria are abundant and overwhelming, some particular bacteria or groups of bacteria are inhibited and cannot survive.
2. Invasion of one (or more) bacterial species.
The bacteria need not be pathogenic themselves, to disturb the balance within the flora.
3. Invasion of one (or more) viral or fungal species,
4. Immune depression,
5. Environmental effects such as temperature and humidity.
Typically, these syndromes do have an influence on the general health and performances of an animal and often even on the whole of the group of animals. In poultry, there are even a number of syndromes which show clinical symptoms directly related to the condition of the intestinal bacterial flora. Therefore, it is of importance to be capable to check the general health of a population of livestock, and/or to have a means of at least determining the presence of such an ill-defined pathological condition in a group (herd, flock) of, e.g., livestock.
Examples of syndromes related to bacterial disequilibrium are, e.g., steathorroea in calves, ileitis in pigs, and also diarrhea at weaning, diarrhea in poultry.
The present invention provides novel means (e.g., kits) and methods for determining (relative) abundance as well as (estimates) of actual amounts of different bacteria present in intestinal bacterial flora, in order to determine, e.g., the presence or absence of equilibrium in said intestinal bacterial flora, especially when compared to a set of normal compositions of intestinal bacterial flora. Until the present invention analyses of bacterial flora occurred with methods used in classical bacteriology. In classical bacteriology, which is based on cultivation and identification of different bacterial strains. Various methods are used, including aerobic and anaerobic growth, pre-enrichment, etc., Furthermore, counting the number of colonies has generally been used to obtain information about the number of bacteria present in samples.
Using these methods, only an estimated 20% of the bacteria have been isolated and characterised until now. Nearly axiomatic in these classical bacterial techniques (as well as with techniques finding other micro-organisms, such as viruses, yeast or fungi) is the thinking that one disease has one causal agent, or at least that one or a distinct few agents can be considered instrumental in causing the distinct disease. However, these classical insights to not provide any practical manner of obtaining insight into the presence or absence of a xe2x80x9chealthyxe2x80x9d equilibrium in intestinal tracts.
Thus, the present invention provides a method for analysing the composition of microbiological (e.g. viral, fungal, yeast or bacterial) flora in an intestinal tract, comprising providing a sample of said flora, selectively amplifying nucleic acid in said sample, subjecting the amplificate to restriction digestion and analysing the resulting pattern of restriction fragments, and providing detection of interrelationships between flora constituents such as known and unknown bacteria, fungi, viruses and the like, for example under distinct disease conditions without the need to identify an organism. Said nucleic acid is selectively amplified, albeit usually from as many, or the greater part of, micro-organism species as possible present in said sample, and optionally supplemented with specific amplification of known organism species or parts thereof possibly present in said sample to provide optional identification, when so desired. Typical results include the detection of a cluster or clusters of amplified fragments (which may have been subjected to restriction), said fragments localised or detected in a pattern (governed for example by molecular mass) allowing the recognition of specific (cluster) patterns. These may be identical in various samples, may be different, or may be supplemented with additional clusters (typically nearly equally sized fragments). Typically, when the sample is representative of a xe2x80x9cnormalxe2x80x9d population, a well balanced cluster pattern may be seen. In contrast, when dysbacteriosis or overgrowth has occurred, one cluster (commonly representing one micro-organism) may be over-represented, typically to the detriment of other clusters (representing others)
The sample may typically be derived from feces, or a biopsy, and in many cases can be taken post-mortem. Preferably, samples are taken under conditions which can be repeated, so that the differences in the flora are attributable to the conditions to be diagnosed and/or analyzed, and not to factors which are temporal, dietary or the like. Post-mortem samples and biopsies should be taken from the same area in the intestinal tract. When using this technique as provided by the invention, it is not per se the mere identification of a tentative causal micro-organism that matters. Rather as the recognition of distinct patters in the resulting analysis allow determinations of equilibrium with respect to a distinct or fitting condition (diseased or non-diseased) in said flora, and allow identification of similar pattern in animals from the same or different flock. Clearly, classical techniques have not paid attention to patterns in flora that, for example relate to multifactorial disease patterns, but have commonly tried to identify one or more distinct causal agents, which in general are sought after with predetermined and specific detection means, such as nucleic acid primers or probes, antibodies, and so on, for the detection of specific micro-organisms or specific components thereof. For example, U.S. Pat. No. 5,543,294; JP 05 317096; Ratcliff et al., Path. (1994) 26:477-479; Wood et al., Appl. Env. Microb. (1998) 64:3683-3689; and U.S. Pat. No. 5,571,674 all give specific and direct instructions on how to identify a specific bacterium, or a bacterium belonging to a specific genus or family, but do not pay attention to other micro-organisms that are or may be present in the test samples, clearly demonstrating that they are not interested in the whole content of the flora but only in specific micro-organisms therein. They will therefore never be able to detect interrelationships between flora constituents such as known and unknown bacteria, fungi, viruses and the like.
In the field of bacteriology, the invention, for example provides the means for quality control, identification and quantification of bacterial species of interest, as well as the detection of (presence or absence) of highly pathogenic and lethal bacterial species, or the detection of new, previously unknown, bacterial. The invention provides, for example, information on the total of bacteria in any given sample. Because of the nature of the technology, many unidentified bacteria are amplified. Thus, when testing clinical samples, new bacterial species are found which could not be identified until now from unknown bacterial populations.
Furthermore, the invention provides analysis of bacterial populations to provide a method for Quality Control. For example, in all sewage treatment plants, bacteria are used in the process of cleaning waste water. The nature of bacteria present in these systems needs to be verified for many reasons. As such, the analysis of the quality and composition of the bacteria present in these systems should be checked on a regular basis. Second, the resulting water needs to be verified as well. Both checks are currently performed using xe2x80x98classicalxe2x80x99 methods, amongst which are culturing under different conditions. The invention provides a rapid, and improved technology for Quality Control of the whole process. A further advantage of the invention is, that known pathological bacteria (e.g. legionella, salmonella spp.) can be identified in the same process of testing the sample with a method according to the invention.
In another example, the invention provides a method for testing the disease or health status of livestock, such as broilers. The bacterial flora in the intestinal tract of broilers is influenced by the composition of feed. During the growth of broilers, generally at two or more occasions, differences are provided in the composition of the feed. This is done to optimize the ratio between feed-intake and growth. Due to the change in feed, the bacterial populations in the intestinal tract are influenced as well. Analysis of the bacterial patterns according to the invention will show the differences due to the changes in feeding.
In another example, the invention provides a method comprising germ counts. Culturing of bacterial populations is performed for many purposes and in many research projects. However, it is current practice that only particular groups of bacteria are visualized or identified (e.g. all coliformes in one group). The present invention is quantitative (when so desired), and can replace the existing use of culturing bacteria. Also, on many occasions, diseases in the intestinal tract are hard to identify. The present invention provides for the analysis, which analysis of the total bacterial contents in any organ or tissue can be performed in a rapid, reliable fashion, and thus be helpful in diagnosis of disease of man and animal alike.
Yet another example comprises use of the present invention in food technology. In the process of the preparation of food, or in the quality control of raw or fermented products such as sausages or cheese, it is essential that the same product is manufactured at a constant level. The invention provides a method enabling the detection of bacterial, viral or fungal species, allowing the verification of manufacturing conditions in the same procedure.
Generally, the sample will have to be pretreated, e.g., homogenised in order to make the nucleic acid accessible for amplification. The sample may be taken from any vertebrate having an intestinal tract and a bacterial flora therein. However, mammals and birds are preferred subjects of the present invention. Especially preferred subjects are humans and domestic animals, and in particular, cattle, poultry and other livestock. Amplification of nucleic acid should be selective in that the choice of primers should be such that a representative amplificate of all or at least a representative number of micro-organisms present in the intestinal flora is obtained. If the primers are well chosen the further details of the amplification technique employed are less critical, so that any amplification method can be used, although PCR is preferred. The length of the primers and the amplification conditions can be determined by the person skilled in the art, depending on the sample, the kind of animal, the need for specificity, etc. Typically, primers will vary in length between 20 and 30 nucleotides, and typically will be directed to a nucleic acid sequence which is present in the selected micro-organisms present in the intestinal flora of the subjects to be sampled, such as 16S rRNA and 23S rRNA.
In some cases, a nested PCR may be useful.
The amplificate itself typically will not give sufficient specific fragments so that a good analysis of the frequency/presence/absence etc. of the respective micro-organisms can be obtained. Therefore, it is important to further differentiate the nucleic acids obtained from the respective micro-organisms by subjecting them to a treatment with a number of restriction enzymes. In that manner, patterns will be obtained which contain sufficient information to enable a good analysis of flora richness, evenness, relative abundance, and amounts, revealing the state of the intestinal flora. This will certainly be very easy in the case where it is not necessary to identify particular micro-organisms, and where it suffices to compare obtained patterns with known patterns of healthy individuals (preferably obtained from an average of the patterns of many healthy individuals with certainty intervals also given). The restriction enzymes should be selected such that fragments giving relevant information are obtained. The skilled person is capable of designing a set of restriction enzymes capable of doing just that. Typically, the enzymes should recognize sequences of 4-8 basepairs, which enables selective digestion to enhance the information present in the amplificate. Typically, at least 10 different enzymes are used, but usually no more than 4 enzymes are necessary. Examples of useful restriction enzymes include, but are not limited to, CfoI and HaeIII.
The analysis of the restricted amplificate can be done in any manner capable of distinguishing between sizes of nucleic acids, in particular DNA fragments. The person skilled in the art is capable of selecting a method best fitted for his purposes. In the case of many samples having to be analyzed some kind of automated system is of course preferred. Good examples of methods for automated analysis systems require possibilities to separate and quantitate DNA fragments. For instance, for the ABI Prism (Perkin Elmer) is suitable for the present invention. In order to analyze the pattern according to the invention, it is preferred to compare the result of amplification and restriction of the sample with at least one reference sample. Some reference should be available for comparing a present state (equilibrium) of a certain animal with a healthy state(equilibrium) of the same animal, another animal, or preferably a group of animals known to be generally healthy. The more information present about the healthy state, the easier it becomes to see difference between healthy intestinal floras and floras related to pathological conditions or general health problems. Of course, the comparison is most reliable when samples are taken in the same manner under similar, if not the same, circumstances. It will often not be necessary to make direct comparisons between samples, as it will generally suffice to compare some kind of test result (numbers, barcodes, graphs, patterns and the like).
Standardizing the picture of bacterial flora can be done, e.g., in the following manner.
To enable interpretation of samples for various purposes, a baseline is established of the intestinal tract of poultry and faecal material of pigs.
The choice of animals used for the baseline is depending on
a) age,
b) housing,
c) feed(intake),
d) breeding line,
e) clinical health, and
f) sex.
The most important aspect is the comparability of the samples constituting the baseline and the sample to be tested. Thus calibration is needed for at least some of the parameters given above.
An easy way of obtaining a number of references is subjecting pure bacterial cultures (of a bacterium of which it is known that it is present in the intestinal flora) to amplification and restriction in the same manner as sample will be treated. The presence and/or abundance of these bacteria in intestinal tracts can be determined based on the presence or absence of (a number of) characteristic peaks.
The invention also provides a method of flora analysis comprising determining the relative abundance of micro-organisms included in said intestinal flora, or even determining simply whether a pattern is within a healthy variation of patterns without even knowing much about the constituting bacteria. This can be done once a collection of healthy patterns is available. Thus, one merely measures the relative abundance and presence/absence of peaks known to be present in the floras of healthy individuals, without knowing from which organisms said peaks originate.
Of course, the earlier mentioned pure bacteria cultures give the possibility to identify which peaks belong to which micro-organism if so desired. Of such peaks, the height and/or the area under the peak, especially in comparison with other known peaks, is a measure for the amount of a certain micro-organism present in a sample. Thus, the invention also provides a method, wherein the analysis involves determining the amount of at least one micro-organism present in said intestinal flora. In another embodiment, the invention provides a diagnostic testkit for performing a method according to the invention, the testkit comprising primers for amplification, restriction enzymes for digestion and optionally at least one reference sample comprising material derived from at least one pure bacterial culture. Preferably such a testkit comprises at least one reference pattern as described herein before.
Suitable primers to include in a testkit according to the invention are primers derived from aligned sequences. This sequence information enables the selection of homologues sequences from which primers can be developed. Preferably, primers are chosen from ribosomal RNA sequences. Preferably, at least one primer comprises the sequence 5xe2x80x2-AGAGTTTGATCCTGGCTCAG-3xe2x80x2 (SEQ ID NO:1), or a functional fragment and/or derivative thereof. A functional derivative or fragment is a primer having a sequence which basepairs with the sequence with which the original sequence basepairs, or with a sequence very closely in the vicinity of the original sequence, so that the same or similar nucleic acids are obtained after amplification.
Another preferred primer comprises the sequence 5xe2x80x2-CCGTCAATTCCTTTRAGTTT-3xe2x80x2(SEQ ID NO:2), or a functional fragment and/or derivative thereof.
The invention also provides the use of a testkit according to the invention for determining presence or absence of equilibrium in intestinal bacterial flora as explained hereinbefore. The invention also provides the method for determining presence or absence of equilibrium in intestinal bacterial flora.
The term xe2x80x98intestinal floraxe2x80x99 as used herein means flora present in the digestive tract, such as the intestinal tract, the stomach, the oesophagus, the mouth or beak or other parts of, or parts close in relation to, the digestive track. Thus, intestinal flora samples may be taken from any part of, or any part close in relation to, the digestive tract.
The means and methods of the invention are in general suited for the analysis of a microbiological sample suspected of containing a variety of different microbiological organisms. An example of a microbiological organism is any kind of bacterium, phage, virus, fungus or yeast. A sample of microbiological flora can be obtained from any source suspected of containing a variety of microorganisms, preferably said sample is obtained from the digestive tract, and most preferably from the intestinal tract. It is also clear to the person skilled in the art that the means and methods of the invention may also be used to determine and/or trace the kind of plant and mammal material in nucleic acid-containing foods. It is clear to the person skilled in the art that the disclosed methods and means are not limited to intestinal tract bacterial flora, but are indeed suited for the typing of microbiological flora from many different sources, such as food samples, food additives, waste material, soils etc. With the term xe2x80x98floraxe2x80x99 is meant a collection of microbiological organisms. With the term xe2x80x98bacterial floraxe2x80x99 is meant a flora present in, obtained from or derived from the digestive tract, preferably the intestinal tract.
The invention will be explained in more detail in the following experimental part.
Sampling
Part of the intestinal tract where isolated/obtained during post mortem examination.
In poultry, e.g., a well-defined part of the ileum between the exit of the pancreatic duct and the Mickel""s Diverticulun was used.
Endoscopial investigation is used to collect samples from particular parts of the intestinal tract from live subjects feces were obtained from live subjects.
Conservation and Transport
Samples were put and maintained on dry ice immediately after collection and transported to the laboratory.
Upon arrival at the laboratory, samples were stored at xe2x88x9220xc2x0 C.
Materials
Lysisbuffer DNA-extraction (pH 6,4)
142,5 g GuSCN
4,8 g TRIS
1,63 g EDTA
Aqua ad 200 ml
Wash-solution DNA-extraction (pH 6.4)
142,5 g GuSCN
4,8 g TRIS
Aqua ad 200 ml)
DE-solution DNA-extraction
0.75 ml HCL 35%
10 g Diatomaceous Earth
Aqua ad 50 ml
Sample Preparation
1. Sample handling
a. intestinal tract
Approximately 2 cm of the specified location was sliced lengthwise using a scalpel. The entire contents were transferred into a sterile tube.
b. endoscopial biopsies and fecal material
Samples were transferred into sterile tubes.
2. Homogenization
Mixing by manually shaking was performed until the substance was visually homogenous.
3. DNA-extraction
a. Quantity
Approximately 0.2 g of homogeneous material was used. In cases of watery samples, a volume of approximately 250 xcexcl was used.
b. DNA-extraction
The DNA-extraction was performed generally according to Boom""s method.
One ml of lysisbuffer was added to the homogenous sample. The suspension was vortexed for 30 seconds, and placed at room temperature for one hour.
The suspension was centrifuged at 15000-17000 g for 20 seconds.
The supernatant was transferred to a sterile tube containing 50 xcexcl of DE-solution. The suspension was vortexed for 30 seconds, and centrifugation at 15000-17000 g for 20 seconds.
After removal of the supernatant, five washing steps were performed:
1. 200 xcexcl wash-solution,
2. 200 xcexcl wash-solution,
3. 200 xcexcl 70% Ethanol,
4. 200 xcexcl 70% Ethanol,
5. 200 xcexcl Aceton p.a..
The pellet was air-dried for 15 minutes at 56xc2x0 C.
The pellet was resuspended in 75 xcexcl ddH2O (overnight 37xc2x0 C.).
Finally, the supernatant was transferred to a new eppendorf cup and stored at 4xc2x0 C.
PCR
Polymerase Chain Reaction was performed using 1,5 ul DNA in a total reaction volume of 15 xcexcl, containing amplitaq DNA polymerase (0.07 unit/xcexcl), primers (1.3 mM of forward and reverse primer), dNTPs (200 mM) and 60 mM KCl, 12 mM HCl pH 8,3 and 1,8 mM MgCl2.
Primer sequences:
Forward 5xe2x80x2-AGAGTTTGATCCTGGCTCAG-3xe2x80x2 (SEQ ID NO:1) Label FAM
Reverse 5xe2x80x2-CCGTCAATTCCTTTRAGTTT-3xe2x80x2 (SEQ ID NO:2), Label TET
PCR programme:
Both primers were labelled with fluorescent probes to enable automated analysis.
The purpose of this reaction is an amplification of as many bacterial organisms as possible. This generally results in about 50 fragments.
Restriction
Restriction digestion was performed on the PCR-products with different restriction enzymes, such as
Analysis
Size analysis of the DNA-fragments was performed on an ABI 377 DNA Sequencer using fluorescently labelled primers. The conditions of the tun on the ABI377 Prism Sequencing system were as follows: ABI Collection software version 2.1 with well-to-read distance 36 cm; running conditions 3000 V, 60 mA, 200 W, collection time 5 hours.
Size-analysis was performed using ABI-software. Lanetracking, Genotyper, Data analysis was performing using GeneScan 3.1 and Genotyper 2.0 software according to the manufacturer""s instructions.
Pure Culture Bacteria
Several bacterial strains, e.g., Clostridium perfringens, Staphylococcus aureus, Mycoplasma gallisepticum, Mycoplasma Synovium, Ornitobacter rhinotracheale, Escherichia coli, Lactobacilliae, Enterococcae and Pseudomonas aeruginosa, were used as references.
Polymerase Chain Reaction as described previously is carried out on pure cultured bacteria. The size of the PCR-product is specific for each bacterium.
Restriction enzymes can be used to identify a number of fragments.
The overall picture of fragments after the PCR and after the use of restriction enzymes results in a xe2x80x98patternxe2x80x99 of a pure culture. This pattern can be identified in any intestinal sample depending on the amount of bacteria present.
Interpretation
1. Richness
The results of the analysis are visualized in a figure, showing peaks corresponding to the size of the DNA-fragments.
These peaks generally are related to bacterial species. The number of peaks identified is a indication for the total number of bacterial species. Using information from pure cultured bacteria, this also provides information on the presence or absence of specific bacteria.
2. Evenness
Furthermore, the peaks provide information on the relative quantity of each DNA-fragment. The ratio of the DNA-fragments based on peak-height and peak-area can be used to estimate the number of certain bacteria present in the sample.
Polymerase chain reaction as described before is carried out after DNA extraction from pure cultured bacterial. Thus, samples can be interpreted to see whether some bacteria are overwhelmingly present or almost absent.

Detection and quantification of micro-organisms using amplification and restriction enzyme analysis


In a transmitter including a carrier source an input signal is present which contains the information to be transmitted and is composed of signal components having amplitudes less than a predetermined threshold value or equal to the threshold value and signal components whose amplitudes are less than the threshold value or equal to the threshold value. For the purpose of identification, the two signal components are sampled in different ways, for example at different frequencies and are combined into a modulation signal in such a manner that the two signal components have the same sign. In a receiver, a circuit arrangement is provided which identifies the sign of the partial signals on the basis of their different sampling frequencies, demodulates envelope curves and combines them into an output signal which corresponds to the signal.. The transmitter and receiver are advantageously components of an analog optical transmission system. They prevent the clipping effect while simultaneously increasing the degree of modulation.

The invention relates to a communications process, to a transmitting device and to a receiving device.
In connection with transmitting devices it is desirable to optimize the minimum value of the transmitting amplitude, for example, on the basis of amplitude dependent distortions and the service life of the carrier source. This may mean that, with fixed transmission parameters, a minimum maximum value for the transmitting amplitude of the transmitted communications signal is desirable and care must be taken that the lowest possible minimum value of the signal amplitude does not fall below a threshold value that is specific for the carrier source.
U.S. Pat. No. 4,789,987 discloses an optical transmitter which automatically regulates the minimum value of the signal amplitude of the communications signal by way of a carrier source specific threshold value and at the same time keeps the maximum amplitude of the communications signal at a minimum. In the prior art signal source, which is a laser diode, a so-called clipping effect occurs if the threshold value is not reached and leads to a distortion in the communications signal.
If one now considers the probability distribution curve of the signal amplitudes which serve to modulate the carrier sources, one notes that one of the two ends of this distribution curve ends below the threshold value which leads to distortion in the communications signal. In order to reduce the probability of the amplitude falling below the threshold value, the prior art circuit increases the direct current component of the injection current. The result is that the maximum value as well as the mean value of the probability distribution curve of the transmission amplitudes increases.

Communications process, transmitter and receiver for analog signals


The invention relates to a plastic shaped body with an integrated optoelectronic luminous element and to a method for the production thereof. To this end, translucent and cold-stretchable plastic film is three-dimensionally formed at least in the area of the luminous elements and subsequently sprayed with thermoplastic synthetic material. The luminous elements are screen printed onto the unshaped film in the form of luminous fields before the plastic film is formed.

The present invention is a plastic form body with graphically designed surfaces and integrated electro-luminescence elements (EL elements). For example, graphic and electro-luminescence structures are placed on transparent and cold-stretchable thermo-plastic plastic foil by means of silk screen printing (serigraphy), then subjected to a pushing isostatic high-pressure deformation, stamped and placed in an injection mold and back-injected generally on the inner wall by means of suitable thermoplastic plastic resulting in a three-dimensional plastic form component with integrated EL luminous fields.
The DE 44 30 907 A1 makes a three-dimensional electro-luminescence indicator based on an electro-luminescence lights integrated in a three-dimensional formed body. The preformed electro-luminescence lights are formed in a whole piece on a substrate. The disadvantage with this is the separate design of the aforementioned light transmitting layer and the electro-luminescence light and their exact positioning to each other that results in an expensive and costly production process.
A similar three-dimensional electro-luminescence indicator is made known with WO-A-94/14180. Here also, prefabricated, foil formed electro-luminescence lights are applied on carrier material and can be formed together. This has the disadvantage that a separate design of the light transmitting layer of the carrier material and the electro-luminescence light and an exact positioning of the EL-lights in the carrier material is necessary, which makes the production expensive and cost-intensive.
Usually the decorative foils are formed after the vacuum process or the compressed air process to a three-dimensional deep-drawing formed body. Then the graphically designed thermoplastic foils are heated to a temperature above the material""s heat distortion temperature so that they can be shaped with relatively little pressure (such as vacuum). These vacuum- and compressed air processes can be used very efficiently with graphically non-critical or neutrally designed foils and form bodies, and function with vacuum processes at a medium pressure of less than 0.95 bar and with compressed air processes at an operating medium pressure of less than 6 bar.
Form components that require exact positioning of graphically designed elements and a very precise image with patterns and/or a great contour sharpness, and which must have a precise form after the deep-drawing process, the procedures of the isostatic high pressure forming according to DE 38 40 542 C1 (also in a somewhat limited way, the so-called hydro-forming) offer advantages.
The production of these types of electro-luminescence fields in connection with the graphic design, must take the very precise positioning of the individual presses and processes together into consideration. The positioning of the nearly transparent ITO-paste graphics, especially, presents a fundamental criterion of quality. U.S. Pat. No. 5,583,394 makes a procedure of recording known that provides invisible positioning markers in a visible light, which, by means of a corresponding light source, are identified with an ultra-violet light for respective reading sensors and, thus, make an exact positioning possible. The disadvantage is that these registration marks can only be applied with additional operating steps and used only with special light sources and special positioning sensors.
Electro-luminescence screen printing pastes are generally made up of inorganic substances and, here again, are very pure ZnS, CdS, ZnxCd1xe2x88x92xS, etc. Compounds of the II and VI group of the periodic system are of importance that generally are doped or activated with Cu, Mn, Ag, etc. Normal colors are yellow, green, green-blue, blue-green and white.
Respective to the state of technology, these types of luminescence pigments can be microencapsulated with diameters of typically 15 to 60 xcexcm in order to be added to various silk screen printing inks (colors), or non-encapsulated, certainly taking the special hygroscopic characteristics of the ZnS pigments into consideration. Fixing agents are used that, on the one hand, provide good adhesion for the so-called ITO-layers, continue to insulate well, reinforce the dielectric, in order to improve the puncture or disruptive strength with high electrical field strengths and, additionally, have a good water vapor block and so additionally protect the phosphorous pigments to lengthen their life expectancy.
Generally, phosphorous pastes of these types are put on by screen printing or other coating procedures, such as brushing, rolling, coil coating, etc. on transparent plastic foils or glass, which again have a wide transparent electric conducting layer and with it the electrodes for the visual side. Finally, the dielectric and the back-side electrode are produced by printing (press) techniques and/or laminating techniques.
The usual ITO-paste layers (or also tin oxide coatings, etc.) applied by screen printing, however, have the advantage of extensive random geometrical design possibilities, but the vapor-deposited or sputter-deposited transparent and electrical-conducting layers still have the disadvantage of having less optical transparency or translucency and, furthermore, have a much lesser degree of conducting capacity, of at the most some 100 Ohm per square in comparison to some less than 10 Ohm per square with ITO-polyester foil or some less Ohm per square with ITO-coated glass. But the glass can have pastes added (for example, In2O3/SnO2), which must, however, be fired at over 500xc2x0 C. and, therefore, can already deliver with a 0.25 xcexcm film strength, an optical transparency of greater than 95% and a conducting capacity of a single layer is from 500 to 1000 Ohm per square.
The purpose of the present invention is to produce a three-dimensional graphically designed plastic form body with integrated electro-luminescence elements economically, with a longer life expectancy, increased luminous intensity and guaranteed performance regardless of the existing electric power supply.
In the framework of the present invention it has been determined that the new types of electro-luminescence screen printing inks (colors), new types of ITO (Indium-Tin-Oxide) colors, and new types of insulation and dielectric colors, together with the printing format can be applied, and then the high-pressure formation and the back-injection can be completed by means of thermoplastic plastics by following several ground rules without impairment to the function of the electro-luminescence elements.
According to the invention, the previously discussed procedure of the isostatic high-pressure formation is sufficient for forming the plastic components. So-called cold-stretchable plastic foils are provided with printing ink (colors) that, together with an operating temperature below the distortion temperature of the thermoplastic foil""s plastic, completes a push-type forming operation to a three-dimensional plastic form component with greater image precision.
Advantageously, isostatic high-pressure forming, especially the push-type stamping of the plastic components with preferably several 100 bar compress air (typically 50 to 300 bar), very short cycle times with outstanding controlled and even formation, which results in greater image accuracy and, furthermore, produces the same types of formed components with less heat (which means the time-consuming cool-down phase is discontinued), outstanding dimensionally stable formed bodies and constant contours (which remain the same), which is really essential for the final stamping process and the insertion into the injection molding die, increasing the quality.
Other processes for forming the plastic foil three-dimensionally are also possible (for example, mechanical shaping or mechanical stamping methods). A re-forming of the plastic foil placed in an injection molding die can itself result from the injection of the thermoplastic plastic material.
The present invention is also based on the cognition that for a product of this type, known screen printing pastes with phosphorous pigments, especially in the microencapsulated form (but also in the non-encapsulated form) based on respective doped compounds of the II and VI groups of the periodic system, especially in forms with Cu, Mn, Ag, etc., doped ZnS pigments in combination with special cold-formable polycarbonate substrate or the various mixtures of polycarbonate and polyethyleneterephthalate (PETP) or polyalkyleneterephthalates are very suitable. Mixtures of additional luminous colors, especially those that, through the electro-luminescence radiation for light excitation and release of respective radiation with wide ranges of discretionary spectrals (as narrow banded wave-length peaks) work very positively and effectively.
In another development of this procedure, the color printing can be imbedded sandwich-like between two layers of foil material. This results in less problems with the back-injection of thermoplastic plastics, which in this case of color printing, are better protected by the additional foil against distortion and melting in the injection area.
In another typical embodiment, a layout of a luminous field has not only two electrical connections, but many fields; for example, with various color luminous fields, such as lettering and/or symbols.
In relation to the necessary position exactness, the individual printings have been established within the framework of the present invention; that the registration of the various layers and processes can be realized manually in general by means of registering marks in visible luminous areas (and also automatically), and that each layer based on the nearly transparent ITO-paste can be controlled and registered very well concerning the preciseness of the position and the constancy of the process by means of electrical test configurations in connection with conducting paste structures. Additionally, the allowable surface resistance and/or contact or transition resistance of the ITO-paste contacts to the conductor will be evaluated and recorded.

Plastic shaped body with an integrated optoelectronic luminous element


A solid phase extraction (SPE) device having a reservoir with an opening; a well comprising an internally tapered wall, the well having a wider interior diameter at an end closest to the opening than at an exit spout; a first filter within the well; a bed of sorbent particles within the well below the first filter, and a second filter having a diameter smaller than the first filter within the well below the bed of sorbent particles and above the exit spout is provided. A method of performing SPE using the device is also provided.

Solid phase extraction (SPE) is a chromatographic technique for preparing samples prior to performing quantitative chemical analysis, for example, via high performance liquid chromatography (HPLC), or gas chromatography (GC). The goal of SPE is to isolate target analytes from a complex sample matrix containing unwanted interferences, which would have a negative effect on the ability to perform quantitative analysis. The isolated target analytes are recovered in a solution that is compatible with quantitative analysis. This final solution containing the target compound can be directly used for analysis or evaporated and reconstituted in another solution of a lesser volume for the purpose of further concentrating the target compound, making it more amenable to detection and measurement.
Depending on the type of analysis to be performed, and detection method used, SPE may be tailored to remove specific interferences. Analysis of biological samples such as plasma and urine using high performance liquid chromatography (HPLC) generally requires SPE prior to analysis both to remove insoluble matter and soluble interferences, and also to pre-concentrate target compounds for enhanced detection sensitivity. Many sample matrices encountered in bio-separations contain buffers, salts, or surfactants, which can be particularly troublesome when mass spectrometer based detection is used. SPE can also be used to perform a simple fractionation of a sample based on differences in the chemical structure of the component parts, thereby reducing the complexity of the sample to be analyzed.
Devices designed for SPE typically include a chromatographic sorbent which allows the user to preferentially retain sample components. Once a sample is loaded onto the sorbent, a series of tailored washing and elution fluids are passed through the device to separate interferences from target sample components, and then to collect the target sample components for further analysis. SPE devices usually include a sample holding reservoir, a means for containing the sorbent, and a fluid conduit, or spout for directing the fluids exiting the device into suitable collection containers. The SPE device may be in a single well format, which is convenient and cost effective for preparing a small number of samples, or a multi-well format, which is well suited for preparing large numbers of samples in parallel. Multi-well formats are commonly used with robotic fluid dispensing systems. Typical multi-well formats include 48-, 96-, and 384-well standard plate formats. Fluids are usually forced through the SPE device and into the collection containers, either by drawing a vacuum across the device with a specially designed vacuum manifold, or by using centrifugal or gravitational force. Centrifugal force is generated by placing the SPE device, together with a suitable collection tray, into a centrifuge specifically designed for the intended purpose.
Various means have been used to contain chromatographic sorbents within SPE devices. A common method, described in U.S. Pat. No. 4,211,658, utilizes two porous filters, with chromatographic sorbent contained between the filters. In this design, the SPE device is essentially a small chromatographic column containing a packed bed of sorbent. A variation of this design is described in U.S. Pat. No. 5,395,521, where the porous filter elements are spherical in shape. In U.S. Pat. No. 4,810,381, the chromatographic sorbent is immobilized within a thin porous membrane structure. In EP Application No. 1110610 A1 a method is described for securing these filters within the SPE device by means of a sealing ring pressed around the periphery of the membrane disc. In U.S. Pat. No. 5,486,410 a fibrous structure containing immobilized functional materials is described. In U.S. Pat. No. 5,906,796 an extraction plate is described where glass fiber discs containing chromatographic sorbent are press fit into each well of the SPE device.
A number of chromatographic sorbents can be used depending on the nature of the sample matrix and target compounds. A common example is to use porous silica that has been surface derivatized with octydecyl (C18) or octyl (C8) functional groups. Porous particles that are based on organic polymers are also widely used. One such type, which is particularly well suited for SPE due to its high loading capacity and unique retention properties, is described in U.S. Pat. No. 6,254,780.
Typical SPE methods contain a sequence of steps, each with a specific purpose. The first step, referred to as the xe2x80x9cconditioningxe2x80x9d step, prepares the device for receiving the sample. For reversed-phase SPE, the conditioning step involves first flushing the SPE device with an organic solvent such as methanol or acetonitrile, which acts to wet the surfaces of both the device and the sorbent, and also rinses any residual contaminants from the device. This initial rinse is generally followed with a highly aqueous solvent rinse, often containing pH buffers or other modifiers, which will prepare the chromatographic sorbent to preferentially retain the target sample components. Once conditioned, the SPE device is ready to receive the sample.
The second step, referred to as the xe2x80x9cloadingxe2x80x9d step, involves passing the sample through the device. During loading, the sample components, along with many interferences are adsorbed onto the chromatographic sorbent. Once loading is complete, a xe2x80x9cwashingxe2x80x9d step is used to rinse away interfering sample components, while allowing the target compounds to remain retained on the sorbent. The washing step is then followed by an xe2x80x9celutionxe2x80x9d step, which typically uses a fluid containing a high percentage of an organic solvent, such as methanol or acetonitrile. The elution solvent is chosen to effectively release the target compounds from the chromatographic sorbent, and into a suitable sample container.
In many cases, elution with high concentrations of organic solvent requires that further steps be taken before analysis. In the case of chromatographic analysis (HPLC), it is highly desirable for samples to be dissolved in an aqueous-organic mixture rather than a pure organic solvent, such as methanol or acetonitrile. For this reason, SPE samples eluted in pure acetonitrile or methanol are usually evaporated to dryness (xe2x80x9cdrydownxe2x80x9d), and then reconstituted in a more aqueous mixture (xe2x80x9creconstitutionxe2x80x9d) before being injected into an HPLC system. These additional steps not only take time and effort, but can also lead to loss of valuable sample, either through target analyte loss onto collection container surfaces during drydown, or due to target analyte evaporative losses or difficulties encountered when trying to re-dissolve the dried sample in the higher percent aqueous fluid.
It can be seen then, that it is advantageous for an SPE device to have a high capacity for retaining target compounds of a wide range of chromatographic polarities, to be capable of maintaining target compound retention as sample interferences are washed to waste, and then to provide the capability to elute target compounds in as small an elution volume as possible, thereby maximizing the degree of target compound concentration obtained during SPE.
The ability to elute in very small volumes of solvent has the added benefit of minimizing the amount of time required to evaporate and reconstitute the sample before proceeding with analysis if further concentration or solvent exchange is required. If elution volume can be kept very low, then drydown and reconstitution can be entirely eliminated.
Traditional SPE device designs have attempted to address these issues, each with a limited measure of success. Packed bed devices utilize packed beds of sorbent particles contained between porous filter discs that are press fit into the SPE device. The capture efficiency of the resulting packed beds is typically quite good, especially if the sorbent properties are carefully chosen. One drawback with conventional packed bed devices is that the void volume contained within the porous filters and packed bed requires that relatively large elution volumes be used to completely elute the target compounds. Typical elution volumes required to fully elute target compounds from a packed bed type SPE device fall in the range of 200-5000 xcexcL, depending on the size of the sorbent bed.
Membrane based designs attempt to address this issue by embedding sorbent particles within a fluorocarbon based membrane, which are then placed into the SPE device. A small mass of sorbent particles is embedded into a thin membrane structure with a wide cross sectional area. Since the membrane does not require retaining filters, the volume associated with the two porous filters is eliminated. This approach reduces the total volume contained within the device, and therefore the volume of solvent required for elution. A typical elution volume required to fully elute target compounds from a particle in membrane SPE device fall in the range of 75-500 xcexcL. Designs of this type have drawbacks in other areas however. The sorbent particles are less densely packed within the membrane structure than within a packed bed, leading to poorer capture efficiency, and a greater chance that target compounds will break through the device without being well retained. In addition, the flow properties of the membrane can be highly variable, due to the poor wetting characteristics of the fluorocarbon based membrane when using highly aqueous fluids.
In U.S. Pat. No. 5,906,796 a design is described in which glass fiber based extraction discs containing chromatographic particles are press fit into each well of the SPE device. Like the membrane designs, this approach immobilizes the sorbent particles in a thin sheet, thereby minimizing device void volume and required elution volumes. Typical volumes required to fully elute target compounds from an SPE device such as this fall in the range of 75-500 xcexcL, which is comparable to particle-in-membrane devices. The sorbent particles are even less densely packed than with membranes however, so sample breakthrough tends to be higher than with either membrane or packed bed devices, and sorbent particles can often break free from the fibrous matrix and contaminate the collected sample solution. One advantage over membrane devices is that flow problems due to wetting issues are generally less common due to the more open structure of the glass fiber disc. One disadvantage of this particle embedded glass fiber disk is that it contains silanol groups that interact with basic compounds. This requires the use of more complex elution solvents, for example, the addition of 2% base or acid to the elution solvent, to maintain the 75-500 xcexcL elution volumes.
It can be seen then, that the lower elution volume capability achieved with both the membrane and glass fiber approaches is at the expense of target compound breakthrough during loading and/or poor recovery for non-polar compounds. Although the volume of fluid needed to effectively elute samples from the membrane and glass fiber formats is reduced to approximately one half of the volume required with conventional packed bed based devices, dry-down and reconstitution steps are still required before samples can be further analyzed by HPLC.
The present invention relates to an improved SPE device which has been specifically designed to contain a small packed bed of chromatographic sorbent such that the bed provides for highly efficient retention of target compounds, while the volume contained within the sorbent bed is sufficiently small as to allow for efficient elution of sample compounds in a minimal elution volume. Specifically, the solid phase extraction device of the present invention comprises a reservoir with an opening; a well comprising an internally tapered wall, the well having a wider interior diameter at an end closest to the opening than at an exit spout; a first filter within the well; a bed of sorbent particles within the well below the first filter; and a second filter having a smaller diameter than the first filter within the well below the bed of sorbent particles and above the exit spout.
The present invention provides a large bed height to top bed diameter ratio using a significantly smaller sorbent mass than is present in current state of the art devices. The large bed height to bed diameter ratio enhances the retention of target compounds and helps to prevent breakthrough of these compounds during the load and wash steps. In SPE the first filter and the top of the sorbent bed acts like a depth filter in removing insoluble sample components. The larger diameter for the upper portion of the bed and larger diameter first filter allows the device to draw through larger sample volumes than could be drawn through a device having an upper bed diameter the same as the lower bed diameter before obstructions will occur. The smaller second filter increases the bed height to bed diameter ratio for a given mass of sorbent while reducing the hold up volume of the device which minimizes required elution volumes.
Moreover, the present invention provides for conically shaped packed beds contained between spherical filters which enhance the performance of solid phase extraction devices by allowing target compounds to be both efficiently retained and eluted. The larger first spherical filter provides a surface area that is approximately two times the area of an equivalently sized cylindrical filter. For example, surface area of the top half of a sphere (xcfx80/2xc3x97d2) of a diameter of 0.1xe2x80x3 is equal to the surface area of the top of a disk of diameter 0.14xe2x80x3 (xcfx80/4xc3x97d2). The smaller second filter helps to minimize the amount of sorbent needed to create a bed length that will be free of adverse imperfections.
The present invention enables the retention of target compounds with a wide range of chromatographic polarity with elution in volumes that are much reduced from the current state of the art for solid phase extraction. This reduction in elution volume provides a solution containing the target compounds that can be diluted with an aqueous solution while still maintaining the high sample concentrations required for analysis.
According to another aspect, the present invention provides an enhanced method of performing solid phase extraction, where the volume of elution solvent is sufficiently small so as to eliminate the need for an evaporation step. The method involves elution of the target compounds in a minimal volume of organic solvent, typically 10-40 xcexcL, which is then diluted with a highly aqueous fluid to form an aqueous organic sample mixture. This mixture is suitable for direct analysis by HPLC, thereby eliminating the time, expense, and potential sample losses associated with evaporation and reconstitution steps, while still maintaining a high degree of target compound(s) concentration.
Specifically, the inventive method comprises the steps of providing the above-mentioned SPE device, and isolating target substances from interfering components in a sample medium, wherein the target substances are substantially eluted in less than 50 xcexcL volume.
In one embodiment of the present invention, the isolating step of the present invention preferably includes conditioning the SPE device with an organic solvent; equilibrating the SPE device with an aqueous solution; adding a prepared sample containing the target substances and interfering components to the SPE device; washing the SPE device with an aqueous solution to remove interfering components; and eluting the adsorbed target substances.
In a preferred embodiment of this enhanced method of the present invention, the aqueous diluent is added directly through the SPE device, while still on the processing station used to perform the SPE fluid transfers. In this way, residual elution solvent is swept through the device into the collection container, where it is diluted by the aqueous fluid and mixed by the gentle air stream that is drawn through the well at the end of the transfer. This approach has the advantage of eliminating the need for a separate pipetting operation to perform the dilution step.

Device for solid phase extraction and method for purifying samples prior to analysis


A bell crank assembly is mounted on an automotive door module of a vehicle that includes a panel. The panel supports a door latch assembly that includes a lock mechanism that is operated by a sill button and rod inside the vehicle that is located on the inner or dry side of the panel and moveable vertically with respect to the panel. The lock mechanism is operated by the sill button and rod via a generally horizontal rod that is located on the exterior or wet side of panel. The bell crank assembly connects the sill button rod to the exterior rod through an extruded hole in the panel. The bell crank assembly includes an inboard lever and an outboard lever. The inboard lever has an input end that is attached to the sill button rod and an output end. The output end has a round boss that fits in the extruded hole of the panel with a running fit to provide an axle for pivoting bell crank assembly with respect to panel. The output end has a shaped projection at an outboard end of the round boss that the fits in a matching hole of the outboard lever so that the levers pivot simultaneously. The shaped projection supports a plurality of cantilevered latch arms that have catches at their respective free ends that extend through the matching hole with the catches engaging an outer surface of the outboard lever adjacent the matching hole. The outboard lever has an input end and an output end. The input end of the outboard lever has a round flange that is spaced outwardly of the shaped hole and that engages the outer side of panel outwardly of the extruded hole to form a seal for the extruded hole. The output end is attached to the exterior rod.

This invention relates to a bell crank assembly and kit for a bell crank assembly that operates through a hole in a panel.
Automotive door modules are used in the modem construction of automotive doors. These modules generally comprise a panel that supports a door latch assembly that includes a lock mechanism and a sill button for operating the lock mechanism. The door latch usually has a lever for operating the lock mechanism that is located on the exterior or wet side of the panel while the sill button is located on the interior or dry side of the panel. The sill button usually operates the lock mechanism by a rod system that transfers the motion of the sill button to the lever that operates the lock mechanism. Although the door latch is at the rear end of the automotive door, modem automotive designs locate the sill button toward the front of the door. The forward sill button location enhances operator convenience. However, the forward location also produces routing difficulties for the rod system that is further exacerbated by the need for sealing the hole in the panel that the rod system must pass through to connect the interior sill button to the exterior latch lever that operates the lock mechanism. Thus there is a need for providing an efficient and compact mechanism for transferring motion in a rod system through a panel hole and sealing the hole in such a way as to simplify rod routing.
The invention provides a bell crank assembly and kit for an automotive door module of a vehicle that supports a door latch assembly that includes a lock mechanism on a panel. The lock mechanism is operated by a vertically moveable rod attached to a sill button located on the interior or dry side of panel via a generally horizontal rod that is located on the exterior or wet side of the panel. The bell crank assembly and kit when installed connects the sill button rod to the exterior rod through an extruded hole in the panel. It includes an inboard lever and an outboard lever. The inboard lever has an input end that attaches to the sill button and an output end that has a round boss. The round boss fits in an extruded hole of the panel with a running fit to provide an axle for pivoting the bell crank assembly with respect to the panel. The output end also has a shaped projection at an outboard end of the round boss that the fits in a matching hole at an input end of the outboard lever so that the levers pivot simultaneously. The shaped projection supports a plurality of resilient cantilevered latch arms that have catches at their respective free ends. The plurality of cantilevered latch arms extend through the matching hole with the catches engaging an exterior surface of the outboard lever adjacent the matching hole. The input end of the outboard lever has a round flange that is spaced outwardly of the shaped hole and that engages the outer side of panel outwardly of the extruded hole to form a seal for the extruded hole. The shaped projection preferably fits snugly in the matching hole of the outboard lever to prevent water leakage through the interface. The output end of the outboard lever attaches to the rod.


A dental composite material that comprises a polymerizable resin composition; a filler system comprising polyhedral oligomeric silsesquioxane fillers; and a curing system. The dental composite material is useful for a variety of dental materials, treatments, and restorative functions including crown and bridge materials, fillings, adhesives, sealants, luting agents or cements, denture base materials, orthodontic materials and sealants, and other dental restorative materials.

1. Field of the Invention
This invention relates to composite materials and to the methods for the manufacture of such composite materials for restorative dentistry, and more particularly to composites which are useful as crown and bridge materials, either with or without an alloy substrate, as reconstructive materials, restorative materials, filling materials, inlays, onlays, laminate veneers, dental adhesives, cements, sealants and the like.
2. Brief Description of the Related Art
In recent years, materials used for dental restorations have comprised principally acrylate or methacrylate resins. Typical acrylic resinous materials are disclosed in U.S. Pat. No. 3,066,112 to Bowen, No. 3,179,623 to Bowen, No. 3,194,784 to Bowen, No. 3,751,399 to Lee et al. and No. 3,926,906 to Lee et al. Acrylic resinous materials, however, exhibit high coefficients of thermal expansion relative to the coefficient of thermal expansion for the tooth structure, and therefore these substances by themselves proved to be less than satisfactory. The disparity in thermal expansion, coupled with high shrinkage upon polymerization, results in poor marginal adaptability, and ultimately leads to secondary decay. Furthermore, the wear and abrasion characteristics and the overall physical, mechanical, and optical properties of these unfilled acrylic resinous materials was quite poor. Composite dental restorative materials containing acrylate or methacrylate resins and fillers were thus developed, the fillers generally comprising inorganic materials based on silica, silicate glass, or quartz. Particularly suitable improved inorganic filler materials include those disclosed in commonly assigned U.S. Pat. No. 4,547,531 to Waknine, and U.S. Pat. No. 4,544,359 to Waknine. These filled compositions are useful for a variety of dental treatments and restorative functions including crown and bridge materials, fillings, adhesives, sealants, luting agents or cements, denture base materials, orthodontic materials and sealants, and other dental restorative materials. Despite their suitability for their intended purposes, however, there nonetheless remains a need in the art for dental resin materials with even more advantageous physical properties.
An improved dental composite material comprises an ethylenically unsaturated resin composition; a filler composition comprising a polyhedral oligomeric silsesquioxane filler; and a curing system. These dental composite materials are useful in a variety of dental materials, treatments, and restorative functions, including crown and bridge materials, fillings, adhesives, sealants, luting agents or cements, denture base materials, orthodontic materials and sealants, and other dental restorative materials.
A new dental restorative composition comprises a polymerizable resin composition and a polyhedral oligomeric silsesquioxane (POSS) filler. POSS-filled resins typically exhibit lower mass densities and greater stiffness, and are capable of withstanding higher temperatures, as well as higher levels of ionizing radiation. In addition, POSS-filled resins are capable of wetting fibers to desirably high degrees. The use of POSS with dental resin materials, particularly the acrylate/methacrylate resins, minimizes polymerization shrinkage and increases material toughness. The nanoscale dimensionality of the POSS fillers also allows for better aesthetic properties, including easier polishability and improved transparency.
Resin compositions suitable for use with dental restorations are well known in the art, and generally comprising ethylenically unsaturated groups that are polymerizable. Especially useful are the polymerizable acrylate or methacrylate resins such as those disclosed in U.S. Pat. No. 3,066,112 to Bowen, No. 3,179,623 to Bowen, No. 3,194,784 to Bowen, No. 3,751,399 to Lee et al., No. 3,926,906 to Lee et al., and commonly assigned U.S. Pat. No. 5,276,068 and No. 5,444,104 to Waknine, all of which are incorporated herein by reference. Other suitable resin materials include, but are not limited to, various polyurethane dimethacrylates (PUDMA), diurethane dimethacrylates (DUDMA), and other monomers and oligomers known in the art. A useful monomer disclosed in U.S. Pat. No. 5,276,068 and 5,444,104 to Waknine is polycarbonate dimethacrylate (PCDMA) which is the condensation product of two parts of a hydroxyalkylmethacrylate and 1 part of a bis(chloroformate). Another advantageous resin having lower water sorption characteristics is an ethoxylated bisphenol A dimethacrylate (EBPDMA) as disclosed in U.S. Pat. No. 6,013,694. An especially important methacrylate resin is the condensation product of bisphenol A and glycidyl methacrylate, 2,2xe2x80x2-bis [4-(3-methacryloxy-2-hydroxy propoxy)-phenyl]-propane (hereinafter abbreviated xe2x80x9cBis-GMAxe2x80x9d). Included within the scope of the resin compositions herein are the resin compositions suitable for use with glass ionomer cements, including polycarboxylic acids such as homo- and copolymers of acrylic acid and/or itaconic acid.
In addition to the aforementioned resins and oligomers, the resin compositions can further include a diluent acrylate or methacrylate monomer to increase the surface wettability of the composition and/or to decrease the viscosity of the polymerization medium. Suitable diluent monomers include those known in the art such as hydroxy alkyl methacrylates, for example 2-hydroxyethyl methacrylate and 2-hydroxypropyl methacrylate; ethylene glycol methacrylates, including ethylene glycol methacrylate, diethylene glycol methacrylate, tri(ethylene glycol) dimethacrylate and tetra(ethylene glycol) dimethacrylate; and diol dimethacrylates such as 1,4-butanedioldimethacrylate, dodecanedioldimethacryalte, or 1,6-hexanedioldimethacrylate (HDDMA). Tri(ethylene glycol) dimethacrylate (TEGDMA) is particularly preferred.
The more viscous polymerizable ethylenically unsaturated resins, i.e., PUDMA, Bis-GMA, and the like are generally present in an amount greater than or equal to about 30, preferably greater than or equal to about 50 wt % of the total composition. It is generally desirable to have the polymerizable ethylenically unsaturated resins less than or equal to about 99, preferably less than or equal to about 90, more preferably less than or equal to about 80 wt % of the total composition. Diluent monomers, when present, are incorporated into the resin composition in an amount from about 1 to about 70 wt % of the total resin composition.
In addition to the above monomers and oligomers, the resin compositions also include a curing system, which typically include polymerization initiators and polymerization accelerators; ultraviolet light absorbers; antioxidants; and other additives known in the art.
Suitable polymerization initiators are those conventional initiators known in the art. For example, visible light curable compositions employ light-sensitive compounds, including but not being limited to benzil, benzoin, benzoin methyl ether, DL-camphorquinone (CQ), and benzil diketones. Either UV-activated cure or visible light-activated cure (approximately 230 to 750 nm) is acceptable. The amount of photoinitiator is selected according to the curing rate desired. A minimal catalytically effective amount is generally about 0.01 wt % of the polymerizable resin composition, and will lead to a slower cure. Faster rates of cure are achieved with amounts of catalyst in the range from greater than about 0.01 percent to about 5 wt % of the resin composition.
Alternatively, the composition may be formulated as a self-curing system. Self-curing compositions will generally contain free radical polymerization initiators such as, for example, a peroxide in amounts ranging from about 0.01 to about 1.0 wt % of the resin composition. Particularly suitable free radical initiators are lauryl peroxide, tributyl hydroperoxide and, more particularly benzoyl peroxide.
Polymerization accelerators suitable for use are the various organic tertiary amines well known in the art. In visible light curable compositions, the tertiary amines are generally acrylate derivatives such as dimethylaminoethyl methacrylate and, particularly, diethylaminoethyl methacrylate (DEAEMA) in amounts ranging from about 0.05 to about 0.5 wt % of the resin composition. In the self-curing compositions, the tertiary amines are generally aromatic tertiary amines, preferably tertiary aromatic amines such as ethyl 4-(dimethylamino)benzoate (commonly known as xe2x80x9cEDMABxe2x80x9d), 2-[4-(dimethylamino)phenyl] ethanol, N, N-dimethyl-p-toluidine (commonly abbreviated xe2x80x9cDMPTxe2x80x9d), bis(hydroxyethyl)-p-toluidine, and triethanolamine. Such accelerators are generally present in the range from about 0.5 to about 4.0 wt % of the resin composition.
It is furthermore preferred to employ an ultraviolet absorber in amounts ranging from about 0.05 to about 5.0 wt % of the total composition. Such UV absorbers are particularly desirable in the visible light curable compositions in order to avoid discoloration of the resin from any incident ultraviolet light. Suitable UV absorbers are the various benzophenones, particularly Uv-5411 available from American Cyanamid Company.
The filler composition comprises at least one polyhedral oligomeric silsesquioxane (POSS) of the generic formula (RSiO1.5)n, wherein R is a hydrocarbon and n is 6, 8, 10, 12, or higher. These molecules have rigid, thermally stable silicon-oxygen frameworks with an oxygen to silicon ratio of 1.5, and covalently-bound hydrocarbon groups that provide an organic outer layer comprising, for example, phenyl, isooctyl, cyclohexyl, cyclopentyl, isobutyl, or other groups. Such silsesquioxanes include, for example, dodecaphenyl-POSS, octaisooctyl-POSS, octacyclohexyl-POSS, octacyclopentyl-POSS, octaisobutyl-POSS and the like. POSS typically have surface areas greater than 400 square meters per gram (m2/gm).
Functionalized POSS (also known as xe2x80x9cPOSS monomersxe2x80x9d) are particularly preferred, wherein one, two, or more of the covalently bound organic groups are reactive with at least one component of the resin composition. In some cases, it is possible to have all of the covalently bound organic groups be reactive groups. POSS monomers may be prepared, for example, by corner-capping an incompletely condensed POSS containing trisilanol groups with a substituted trichlorosilane. For example, the trisilanol functionality of R7T4D3(OH)3 (wherein R is a hydrocarbon group) can be reacted with Cl3Si-Y to produce the fully condensed POSS monomer R7T8Y. Through variation of the Y group on the silane, a variety of functional groups can be placed off the corner of the POSS framework, including but not limited to halide, alcohol, amine, isocyanate, acid, acid chloride, silanols, silane, acrylate, methacrylate, olefin, and epoxide.
Preferred functional groups are acrylate (xe2x80x94Xxe2x80x94OC(O)CHxe2x95x90CH2) and methacrylate (xe2x80x94Xxe2x80x94OC(O)CH(CH3)xe2x95x90CH2) groups, wherein X is a divalent linking group having 1 to about 36 carbons, such as methylene, ethylene, propylene, isopropylene, butylene, isobutylene, phenylene, and the like. X may also be substituted with functional groups such as ether (e.g., xe2x80x94CH2CH2OCH2CH2xe2x80x94), as long as such functional groups do not interfere with formation or use of the POSS. X is preferably propylene, isobutylene, or xe2x80x94OSi(CH3)2CH2CH2CH2xe2x80x94. One, all, or an intermediate number of the covalently bound groups may be acrylate or methacrylate groups. Such functionalized POSS are available from Gelest, Inc. (Tullytown, Pa.) and Hybrid Plastics. A methacryloxypropyl-substituted T8 POSS (wherein all positions of the polyhedron are methacryloxypropyl-substituted) is available under the trade designation MA0735 from Hybrid Plastics Corp.). Another methacryloxypropyl-substituted T8 POSS (wherein one position is methacryloxypropyl-substituted and the remaining positions are isobutyl-substituted) is available under the trade designation MA0702 from Hybrid Plastics Corp (Fountain Valley, Calif.).
Of course, the linking groups X are also suitable for use with other functional groups. Other POSS fillers include, for example T6, T8, T10, or T12 structures functionalized with alkoxysilanes such as diethoxymethylsilylethyl, diethoxymethylsilylpropyl, ethoxydimethylsilylethyl, ethoxydimethylsilylpropyl, triethoxysilylethyl, and the like; with styrene, such as styrenyl (C6H5CHxe2x95x90CHxe2x80x94), styryl (xe2x80x94C6H4CHxe2x95x90CH2) and the like; with olefins such as allyl, xe2x80x94OSi(CH3)2CH2CH2xe2x95x90CH2, cyclohexenylethyl, xe2x80x94OSi(CH3)2CHxe2x95x90CH2 and the like; with epoxies, such as 4-propyl-1,2-epoxycyclohexyl, 3-propoxy, glycidyl (xe2x80x94CH2CH2CH2OCH2CH(O)CH2), and the like; with chlorosilanes such as chlorosilylethyl, dichlorosilylethyl, trichlorosilylethyl, and the like; with amines such as aminopropyl, aminoethylaminopropyl, and the like; with alcohols and phenols such as xe2x80x94OSi(CH3)2CH2CH2CH2OC(CH2CH3)2(CH2CH2OH), 4-propylene-trans-1,2-cyclohexanediol, xe2x80x94CH2CH2CH2OCH2C(CH2OH)(OH), and the like; with phosphines such as diphenylphosphinoethyl, diphenylphosphinopropyl, and the like; with norbomenyls such as norbomenylethyl; with nitrites such as cyanoethyl, cyanopropyl, xe2x80x94OSi(CH3)2CH2CH2CH2CN, and the like; with isocyanates such as isocyanatopropyl, xe2x80x94OSi(CH3)2CH2CH2CH2NCO, and the like, with halides such as 3-chloropropyl, chlorobenzyl (xe2x80x94C6H4CH2Cl), chlorobenzylethyl, 4-chlorophenyl, trifluoropropyl (including a T8 cube with eight trifluoropropyl substitutions) and the like; and with esters, such as ethyl undecanoat-1-yl and methyl propionat-1-yl, and the like. Certain polymers such as poly(dimethyl-comethylhydrido-co-methylpropyl polymers, poly(dimethyl-comethylvinyl-co-methylethylsiloxy, poly(ethylnorbonenyl-co-norbonene) and poly(ethylsilsesquioxan) may also be used to functionalize POSS. Many of these substitutions are commercially available on T8 POSS from Hybrid Plastics.
Without being bound by theory, it is hypothesized that the functionalization of the cubes allow for better dispersion in and reactivity with the matrix resin, which reduces moisture uptake, as well as volumetric shrinkage. Bonding of the T8 cubes with the matrix resin may be achieved by co-polymerization with the resin monomers or oligomers in the presence of a cure system, for example by treatment with radiation such as UV light.
In addition to the POSS-based filler, the filler composition may further comprise one or more of the inorganic fillers currently used in dental restorative materials. Preferred additional fillers include those that are capable of being covalently bonded to the resin matrix itself or to a coupling agent that is covalently bonded to both. Examples of suitable filling materials include but are not limited to, silica, quartz, strontium silicate, strontium borosilicate, lithium silicate, lithium alumina silicate, amorphous silica, ammoniated or deammoniated calcium phosphate, tricalcium phosphate alumina, zirconia, tin oxide, and titania. Some of the aforementioned inorganic filling materials and methods of preparation thereof are disclosed in U.S. Pat. No. 4,544,359 and No. 4,547,531, pertinent portions of which are incorporated herein by reference. Suitable high refractive index filler materials such as high refractive index silica glass fillers and calcium silicate based fillers such as apatites, hydroxyapatites or modified hydroxyapatite compositions may also be used. Alternatively, inert, non-toxic radiopaque materials such as bismuth oxide (Bi2O3), barium sulfate, and bismuth subcarbonate may be used. Suitable fillers have a particle size in the range from about 0.1 to about 5.0 microns, and may further comprise unbound silicate colloids of about 0.001 to about 0.07 microns. These additional fillers may also be silanized.
The amount of total filler composition in the dental composite can vary widely, being in the range from about 1 to about 90 wt % of the total composition. The amount used is determined by the requirements of the particular application. Thus, for example, crown and bridge materials generally comprise from about 60 to about 90 wt % filler; luting cements comprise from about 20 to about 80 wt % filler; sealants generally comprise from about 1 to about 20 wt % filler; adhesives generally comprise from about 1 to about 30 wt % filler; and restorative materials comprise from about 50 to about 90 wt % filler, with the remainder in all cases being the resin composition and curing system.
The amount of POSS in the filler composition relative to other filler may also vary widely, depending on the requirements of the particular application. The POSS may accordingly comprise from less than about 1 to 100 wt % of the total filler composition, preferably from about 1 to about 100 wt % of the total filler composition for sealers and adhesives, and from about 2 to about 30 wt % of the total filler composition for crown and bridge materials and dental restorative materials.
In a preferred embodiment, in one manner of proceeding the polymerizable resin composition (including any desired diluent monomers), the POSS filler, and any additional fillers are mixed. The curing system is added to the mixture, which is again stirred and stored or used, as appropriate. The cure may be initiated through the use of UV light or by raising the temperature of the mixture. The dental restorative resin thus obtained is then placed in the tooth to be repaired after it is appropriately prepared. Methods for use of the above-described compositions are well known in the art. The invention is further illustrated by the following non-limiting examples.

Dental composite materials and method of manufacture thereof


A method is providing for treating waters, such as standing and flowing waters and waste waters, soils, sediments and/or silts. At least one alkaline earth metal peroxide is brought into contact with such waters or the like, especially to effect immobilization of phosphates and heavy metals except manganese and iron therein.

The present invention relates to a process for treating waters, such as standing and flowing waters and waste waters, soils, sediments and/or silts.
Water bodies, soils, sediments and/or silts always comprise a certain proportion of organic matter. Especially in the case of waters, that is to say both water bodies and waste waters, the organic compound content, inter alia, determines the water quality. The organic matter firstly increases the COD and BOD values and secondly leads in the course of time to the formation of detritus, that is to say to silting and, in the extreme case, to accretion.
High COD and BOD values mean that material is present which has a high oxygen demand and leads to oxygen deficit for the microorganisms and other forms of life, and finally to their dying off. The degraded material is present in the water as additional organic matter, for example as suspended particles, which is accompanied by a further decrease in water quality.
To prevent accretion in water bodies, in particular ponds, the silts formed must be mechanically removed at regular intervals. However, this mechanical removal of silts requires an adequate particle size. Suspended particles generally cannot be removed.
Also, the fouling of waters, soils, sediments and/or silts impairs the activity of the microorganisms living therein, which in turn leads to an interference in the natural equilibrium.
A further problem, especially in the case of inland water bodies, is the phosphate contents, which are responsible for eutrophication of water bodies. The reduction in phosphate concentration in eutrophied water bodies is generally the key to successful remediation, that is to say to improving the water quality and possibilities for use. Therefore, for more than two decades, attempts have been and are being made with varying degrees of success to decrease the phosphate concentration in particular in lakes and sewage treatment plant effluents. Established processes employ iron salts and aluminum salts as precipitants. Recent developments relate to the optimization of biological phosphate elimination and technical control of biogenic calcite precipitation and flushing lacustrine chalk deposits as potential methods of ecotechnological water body remediation.
The known processes for phosphate removal, however, have the disadvantage that the precipitation of phosphate proceeds only incompletely and is also sometimes technically complex. By adding Al compounds or Fe compounds, furthermore, aluminum ions and iron ions are also introduced into the water, which does not always have beneficial consequences on water quality.
From the prior art it is known to treat fouled water with a mixture of Ca(OH)2 and H2O2. In processes of this type the existing phosphate is precipitated as calcium hydrogen phosphate, whose solubility product is highly dependent on the pH. An equilibrium state forms with the components present in the aqueous environment. In some cases, the hydrogen phosphate even redissolves. Such a process does not permit permanent mineral phosphate binding in the treated systems and does not ensure a balanced oxygen environment which can be tolerated by water organisms and sediment dwellers.
The object underlying the present invention is thus to provide a process for treating waters, soils, sediments and/or silts which enables organic matter which is present as silt or suspended matter to be degraded and thus to reduce the content of oxygen-consuming substances and simultaneously to improve the nitrification and enzyme activity of the microorganisms present.
A further object was to provide a process which enables phosphate to be removed from waters, soils, sediments and/or silts such that the phosphate content is decreased below the ecologically required maximum content and the phosphate is bound in such a manner that it is not re-released by the water by dissolution or leaching. A further object is to reduce the content of other pollutants present in such systems simultaneously with the phosphate elimination.
The present invention relates to a process for treating waters, such as standing and flowing waters and waste waters, soils, sediments and/or silts, characterized in that the waters or sediments are treated with alkaline earth metal peroxides.
Surprisingly, it has been found that by using alkaline earth metal peroxides for treating waters, soils, sediments and/or silts, the content of organic, matter which is present as silt or suspended matter, can be greatly reduced and the microbial degradation performance can also be promoted. It is assumed that when alkaline earth metal peroxides are added to waters, soils, sediments and/or silts a mineralization of the organic matter takes place, that is to say conversion of organic matter to inorganic matter. At the same time other harmful substances can be immobilized, for example if these are precipitated together with other solids. Experiments have found that odor and flavor substances are also degraded and nitrification is promoted.
In the treatment of phosphate-containing waters, soils, sediments and/or silts with alkaline earth metal peroxides, phosphate can be eliminated so excellently that the phosphate concentration is far below 30 xcexcg of P/L after treatment. The currently available experimental results have found a decrease in orthophosphate concentration of 3.26 and 0.33 mg of P/L to 6 and 3 xcexcg of P/L, respectively, within a very short time.
It has further been found that the pH of the system treated according to the invention increases in the alkaline region and elemental oxygen is released. This action must generally be assessed as positive in aquatic systems, since it counteracts the oxygen deficit due to oxygen-consuming degradation processes.
The advantage over biological phosphate elimination is the reliable elimination of the phosphate. The advantage over other chemical phosphate elimination processes is the high efficacy, that is to say the great reduction in orthophosphate concentration with relatively low use of alkaline earth metal peroxide used according to the invention. The inventive process can prevent development of algal mass and thus avoid the associated water body problems. The alkaline earth metal peroxide, used in equimolar amounts, can remove phosphate from the waters. In addition, the precipitation product is suitable as mineral fertilizer raw material and thus capable of being recycled.
After use of the inventive process, it is not necessary to remove any precipitated material from the waters or soils, sediments and/or silts, it can remain in the water or in the sediment. The precipitated material essentially consists of mineralized substances to which substances impairing water quality are also bound by absorption or else can be incorporated into the solid. The present process is therefore to be used advantageously, in particular in what are termed passive water treatment systems.
Alkaline earth metal peroxides have been previously used, inter alia, as additives in bread, in toothpastes, in the cosmetics industry and as oxygen donors in composting. Alkaline earth metal peroxides are obtained from aqueous alkaline earth metal hydroxide solutions and H2O2 in a specific process. It is known that simple mixing of the hydroxide solutions and H2O2 does not lead to alkaline earth metal peroxides, but only to the decomposition of the H2O2 used.
When this process is used for waste water clean-up, there is additionally the advantage that, depending on the properties of the waste water, this may be cleaned up in a short time in a very simple single-stage process. This involves comparatively less outlay on equipment and is thus less costly.
It has been found that the inventive use of alkaline earth metal peroxides for treating water body sediments, silts and soils removes the phosphates as sparingly soluble compounds and permanently binds them, so that remobilization or leaching of the phosphates on contact with water is effectively suppressed.
The observations to date imply that, without verifying this, phosphate is precipitated and immobilized in mineral form as hydroxylapatite by the process.
It has further been found that the inventive treatment of the waters or soils, sediments and/or silts, due to the precipitation of the phosphates, also reduces the contents of other pollutants which impair water quality, such as heavy metal compounds and organic compounds.
It is assumed that synergies occur which result from the oxidative action of the peroxides used according to the invention, the increase in pH and the associated formation of sparingly soluble heavy metal species.
For the purposes of the present invention, waters is taken to mean all waters, for example waters in ponds, lakes, rivers, seawater and freshwater aquaria, centers for breeding fish and other marine animals, water from sewage treatment plants and other water treatment plants etc. and any waste waters including industrial waste waters, the resultant waters also being able to comprise soils, sediments and/or silts and sink and float matter. For the purposes of the present invention sediments is taken to mean solids present in waters, for example in ponds, lakes and rivers. The silts can originate, for example, from sewage treatment plants, waste water filters etc.
Suitable alkaline earth metal peroxides are, for example, the peroxides of magnesium, calcium, barium, strontium and their mixtures, with calcium and magnesium or their mixtures preferably being used. Particular preference is given to calcium peroxides, with calcium being able to be replaced by magnesium peroxide, strontium peroxide or barium peroxide, in amounts of 0.02% by weight to 50% by weight, preferably up to 30% by weight, based on CaO2. In commercial products the alkaline earth metal peroxide is present as a mixture with the corresponding carbonate and hydroxide.
Particularly good results have been obtained when the alkaline earth metal peroxides are used in a mixture with alkali metal carbonate-peroxide hydrates. A microbicidal action of alkali metal carbonate-peroxide hydrates is known when used in water.
The alkali metal carbonate-peroxide hydrates are adducts of alkali metal carbonates and H2O2, Me2CO3.xH2O2, for example 2Me2CO3.3H2O2, and preferably 2Na2CO3.3H2O2. They are also termed alkali metal percarbonates and are commercially available. From economic and ecological aspects, sodium carbonate-peroxide hydrate has proved to be particularly suitable.
The alkaline earth metal peroxides and alkali metal carbonate-peroxide hydrates are preferably used in ratios of 1:1 to 1:0.03.
The precipitation of phosphate can be increased if seed crystals of apatite or moderately water-soluble phosphate compounds are added to the system to be treated.
To carry out the inventive process, the substances used, that is to say alkaline earth metal peroxides and if appropriate alkali metal carbonate-peroxide hydrates and other optional constituents, are added in an amount from 30 to 300 g/m2 of water. In the treatment of sediment, silt or soil, because of the usually greater amount of oxidizable matter, the addition of the substances used according to the invention can be several times the amount to be added to waters.
Furthermore, using the novel process it is possible to decrease heavy metal contents and the contents of interfering anions, for example PO43xe2x88x92, NO2xe2x88x92, SO32xe2x88x92, S2xe2x88x92. When used to treat industrial waste waters, in some cases even the currently applicable official limit values (ordinance on indirect introduction) can be achieved, which makes direct introduction of the waste waters into the receiving water body possible.
The inventively used peroxides and other possibly used components can be added to the systems to be treated either as individual substances or in a mixture with other substances as solids, aqueous solutions or suspensions. Solids of this type which can be used are especially silicates, such as sheet silicates or framework silicates, preferably selected from the group consisting of zeolites and bentonites. For practical reasons it is particularly expedient to compact the materials present in solid form and use them for example as granules, pellets or tablets.
In the elimination of phosphate from waters it has proved to be advantageous to pass the waters through an apparatus which comprises the inventive components, possibly applied to support materials. Passing the waters over a fixed bed which comprises the inventively used components is also possible, in which case a contact time must be ensured such that the reaction between the phosphate and the peroxide can take place.
Depending on the water quality and sediment quality, such as carbonate content, pH, etc., it can be appropriate to add other compounds which increase the water quality or sediment quality. Examples of such compounds which may be mentioned are Ca(OH)2, CaO, CaCO3, CaCl2, Ca(NO3)2, CaSO4, Ca2SiO4, CaF2, Cal2, CaBr2, Ca3(PO4)2, Ca4H(PO4)3, Ca2P2O7, Ca4P2O9, CaHPO4, Ca(PO3)3, Ca(H2PO4)2 and the apatites of calcium and mixtures of the above. Depending on the properties of the untreated water, it can be necessary to treat the water to increase the pH using alkali metal salts or alkaline earth metal salts, in particular oxides, hydroxides, carbonates or hydrogencarbonates.
In a preferred embodiment, the inventively used compounds will be present in combination with a mixture of CaCO3, CaCl2 and/or Ca(NO3)2 and possibly magnesium salts, together with NaHCO3 and possibly KHCO3, with CaCO3 and CaCl2 and/or Ca(NO3)2 and possibly magnesium salts being present in a quantitative ratio of 0.01:1 to 2:1 and CaCl2 and/or Ca(NO3)2, and possibly magnesium salts, and NaHCO3 and possibly KHCO3 being present in a quantitative ratio of 1:3 to 2:1. Such a mixture and its suitability for treating waters and sediments is described in European patent application 737 169.
The presence of salts, for example of iron and aluminum, and of oxides, hydroxides, hydrogencarbonates, carbonates, sulfates, nitrates, chloride and fluoride can promote the efficacy of the process: especially by dosing the metals iron and aluminum and fluoride, for example in the form of NaF, KF or MgF2, or other ions having a small ionic radius, these compounds being used in an amount such that the water quality is not impaired.
The inventive process can be used to treat soils, water sediments, silts and water/waste water in open and closed water-containing systems, bodies of water such as seawater, brackish water and freshwater, for example in dams, artificial or natural lakes, bathing waters or fishery waters, ornamental ponds and aquaria. In addition in process waters, for example sewage treatment plants, waste water treatment plants, recycling plants, cooling water plants and heat-exchange plants, waste waters of chemical production plants, or in the treatment of water which results from decomposition and condensation processes (for example landfill leachate water or condensate from thermal waste utilization plants) or by leaching processes (for example water which percolates through contaminated soils, water body sediments or silts).
The inventive process can be carried out by direct dosage of the solid components by hand or by technical devices or aids such as metering systems. The material can be metered directly into the water to be treated or by the water to be treated passing through vessels which comprise the material in solid form, for example filter cartridges, fixed-bed or fluidized-bed reactors.
Thus experiments with highly polluted waste waters from plastics recycling operations have found that, after separating off the precipitation products, the quality of the process water had improved so greatly that in some cases introduction of the treated water into the receiving body of water was possible according to the criteria of the ordinance on indirect or direct introduction. In further applications the inventively used components are incorporated into soils, silts or water body sediments by positive mixers, rotary hoes or other mechanical mixers.
The precipitated phosphates may, provided that their pollutant content permits this, be reused as raw materials, for example as fertilizers, composting aids or in earthworks and agriculture.
The inventive use of the alkaline earth metal peroxides, in addition to increasing the enzyme activity of organisms present, also increases their microbiological activity in general, which leads to an accelerated and increased mineralization process, which can be observed via a decrease in the loss on ignition of the mineralized substrate.

Process for treating waters, soils, sediments and/or silts