Plastic Syringes Research Articles

Protein-polymer bioconjugates via a versatile oxygen tolerant photoinduced controlled radical polymerization approach

The immense application potential of amphiphilic protein-polymer conjugates remains largely unexplored, as established “grafting from” synthetic protocols involve time-consuming, harsh and disruptive deoxygenation methods, while “grafting to” approaches result in low yields. Here we report an oxygen tolerant, photoinduced CRP approach which readily affords quantitative yields of protein-polymer conjugates within 2 h, avoiding damage to the secondary structure of the protein and providing easily accessible means to produce biomacromolecular assemblies. Importantly, our methodology is compatible with multiple proteins (e.g. BSA, HSA, GOx, beta-galactosidase) and monomer classes including acrylates, methacrylates, styrenics and acrylamides. The polymerizations are conveniently conducted in plastic syringes and in the absence of any additives or external deoxygenation procedures using low-organic content media and ppm levels of copper. The robustness of the protocol is further exemplified by its implementation under UV, blue light or even sunlight irradiation as well as in buffer, nanopure, tap or even sea water.

Technique for “open sternal” chest closure in patients with assist devices and transplant recipients

Selection criteria in both cardiac transplantation and left ventricular assist device (LVAD) placement are expanding, leaving patients at risk for problems that require the sternum to be left open after surgery, such as coagulopathy or right ventricular dysfunction. Standard approaches for keeping the sternum open rely on plastic syringe struts to spread the sternum and a nonbiological barrier (ie, esmarch). A technique is described to achieve sternal separation while maintaining a biological barrier, which has been used selectively for delayed sternal closure (Video 1).

Interference of oleamide with analytical and bioassay results

During sample preparation and analysis, samples are coming in contact with different labware materials. By four unrelated analytical (phytochemical and pharmaceutical) case-studies and employing different analytical techniques, we demonstrated the potential misinterpretation of analytical results due to the use of contaminants-leaching labware during sample handling. Oleamide, a common polymer lubricant and a bioactive compound, was identified as a main analytical interference, leaching from different labware items into solvents, recognised as chemically compatible with the tested polymer material. Moreover, anti-inflammatory effect of oleamide at 100 μg mL−1 and considerable pro-inflammatory effect of the plastic syringe extractables (containing oleamide) at the same level were shown in a TLR4-based bioassay. Taking these results into account, together with the fact that oleamide can be a compound of natural origin, we would like to notify the professional public regarding the possible erroneous oleamide-related analytical and bioassay results due to the use of oleamide-leaching labware. Researchers are alerted to double check the real source of oleamide (labware or natural extract), which will prevent further reporting of false results. Analysis of procedural blanks with de-novo developed UHPLC-ESI-MS method is, among some other strategies, proposed for detection of oleamide interference and avoidance of misleading results of certain analyses.

Towards Eco-Flowable Concrete Production

Environmental concerns have increased due to the amount of unused/expired plastic medical waste generated in hospitals, laboratories, and other healthcare facilities, in addition to the fact that disposing of such wastes with extremely low degradation levels causes them to remain in the environment for extended periods of time. These issues have led researchers to develop more environmentally friendly alternatives for disposing of plastic medical waste in Australia. This study is an attempt to assess the impacts of using expired plastic syringes as fine aggregate on fresh and hardened characteristics of flowable concrete, which might provide a solution to environmental concerns. Six mixtures of flowable concrete with water-to-cement ratios of 0.38 were studied. It was found that using recycled aggregate in up to 20% can improve the workability and increase the V-funnel values of flowable concrete mixtures. However, using waste aggregates in more than 30% caused an inapt flowability. Adding waste aggregate at the 30%–50% replacement level led to a decrease in the L-box ratio. To verify the utility and the efficacy of this experiment, the connections between different rheological test measurements were also compared by implementing the Pearson correlation function. The mechanical properties of the mixes containing recycled aggregates were decreased at the age of seven days; however, at later ages, waste aggregates increased the strength at the 10%–30% replacement levels.

Subnanoliter precision piezo pipette for single-cell isolation and droplet printing

Although microliter-scale liquid handling with a handheld pipette is a routine task, pipetting nanoliter-scale volumes is challenging due to several technical difficulties including surface tension, adhesion and evaporation effects. We developed a fully automated piezoelectric micropipette with a precision of < 1 nanoliter, improving the efficiency of imaging-based single-cell isolation to above 90%. This improvement is crucial when sorting rare or precious cells, especially in medical applications. The compact piezoelectric micropipette can be integrated into various (bio)chemical workflows. It eliminates plastic tubes, valves, syringes, and pressure tanks. For high-quality phase-contrast illumination of the sample, e.g., cells or tiny droplets, we constructed rings of LEDs arranged concentrically to the micropipette. The same device can be readily used for single-cell printing and nanoliter-scale droplet printing of reagents using either fluorescent or transparent illumination on a microscope. We envision that this new technology will shortly become a standard tool for single-cell manipulations in medical diagnostics, e.g., circulating tumor cell isolation.

Holistic Extractables and Leachables Program: Evaluations of Prefilled Syringe Systems for Biotechnology Products.

Prefilled syringes (PFS) are a container and delivery device of choice for storing and administering therapeutic protein products to patients. Addressing concerns and regulatory expectations related to the risk to biologic drug product quality and patient safety from PFS requires implementation of an extractable and leachable program based on understanding of materials, risk assessment, review of existing literature, and testing supported by a sound scientific foundation. Extractables and leachables data generated as part of a thorough and holistic program are presented for five PFS systems, including glass and plastic syringes filled with 12 biologic drug products encompassing the implementation of traditional and single-use biotechnology manufacturing processes. The comprehensive extractables and leachables data presented demonstrate and substantiate a holistic extractable and leachable program designed to ensure product quality and patient safety.

Systematic Assessment of the Adsorption of 99mTc-Radiopharmaceuticals onto Plastic Syringes.

The phenomenon of adsorption of several 99mTc-radiopharmaceuticals onto disposable syringes is common knowledge and can reach a level of up to 50%, with the result being inadequate dosing. The resulting underdosing has a substantial influence on the quality of imaging, especially in pediatric patients. Therefore, we aimed to establish a standardized in vitro assessment to investigate the adsorption of several 99mTc-radiopharmaceuticals on various brands of syringes. Methods: The 99mTc-radiopharmaceuticals were prepared according to manufacturer instructions. For the assessment, the disposable syringes (n = 3) were filled to one third of capacity with the 99mTc preparation and incubated for 30 min at room temperature. The syringes were emptied into evacuated vials, and the radioactivity of the syringes was measured before and after they were emptied. Furthermore, the dilution effect of 99mTc preparations was studied. We used 2 different brands of syringes and systematically examined 99mTc-pertechnetate, 99mTc-butedronate, 99mTc-oxidronate, 99mTc-medronate, 99mTc-tetrofosmin, 99mTc-sestamibi, 99mTc(V)-dimercaptosuccinic acid, and 99mTc-succimer. Additionally, 99mTc-succimer was retested with 5 brands of syringes. Results: 99mTc-pertechnetate, 99mTc-phosphonates, and 99mTc(V)-dimercaptosuccinic acid showed no significant adsorption. The measured radioactive retention of 2%-5% was equivalent to the determined dead volume. Using 99mTc-tetrofosmin, we found a slight but significant adsorption of 4%-7%. The 99mTc-sestamibi preparation showed a nonsignificant retention of 3%-5%. However, when the 99mTc-sestamibi was diluted 1:10 with saline, the adsorption rate increased to 9%-13%. 99mTc-succimer displayed different adsorption levels depending on the brand of syringe and the preparation technique. The adsorption of 99mTc-succimer, prepared from kits according to the instructions, did not exceed 15%. The 1:10 saline dilution of a 99mTc-succimer kit preparation, as well as an in-house preparation, demonstrated a radioactive syringe adsorption rate of more than 30%. Conclusion: The results revealed the significance of syringe adsorption of radiopharmaceuticals in the prevention of underdosing. Therefore, a quality assurance assessment is recommended before the introduction of new brands of plastic syringes or routine application of diluted or in-house radiopharmaceuticals.

An 1H NMR study of the cytarabine degradation in clinical conditions to avoid drug waste, decrease therapy costs and improve patient compliance in acute leukemia.

Cytarabine, the 4-amino-1-(β-D-arabinofuranosyl)-2(1H)-pyrimidinone, (ARA-C) is an antimetabolite cytidine analogue used worldwide as key drug in the management of leukaemia. As specified in the manufacturers’ instructions, once the components—sterile water and cytarabine powder—are unpackaged and mixed, the solution begins to degrade after 6 hours at room temperature and 12 hours at 4°C. To evaluate how to avoid wasting the drug in short-term, low-dose treatment regimens, the reconstituted samples, stored at 25°C and 4°C, were analyzed every day of the test week by reversed-phase HPLC and high-field NMR spectroscopy. All the samples remained unchanged for the entire week, which corresponds to the time required to administer the entire commercial drug package during low-dose therapeutic regimens. The drug solution was stored in a glass container at 4°C in an ordinary freezer and drawn with sterile plastic syringes; during this period, no bacterial or fungal contamination was observed. Our findings show that an cytarabine solution prepared and stored in the original vials retains its efficacy and safety and can, therefore, be divided into small doses to be administered over more days, thus avoiding unnecessary expensive and harmful waste of the drug preparation. Moreover, patients who require daily administration of the drug could undergo the infusion at home without need to go to hospital. The stability of the aliquots would help decrease hospitalization costs.

A new method for pharmaceutical compounding and storage of anti-VEGF biologics for intravitreal use in silicone oil-free prefilled plastic syringes

Intravitreal injections of antibody-based biologics targeting vascular endothelial growth factor (VEGF) are highly effective and have markedly decreased the risk of visual impairment associated with prevalent retinal diseases, such as neovascular age-related macular degeneration and diabetes macular oedema. The diseases are chronic in their nature, and most patients need long-term therapy to suppress disease activity. We previously reported a compounding method for repackaging and storage of aflibercept (Eylea), a commonly used anti-VEGF biologic, in silicone oil-coated plastic syringes without compromising drug stability or activity. In addition to improving safety and time spent per patient, compounding of anti-VEGF biologics enables single-dose vials to be split into multiple syringes, thereby considerably reducing waste and drug expenses. However, symptomatic silicone oil droplets may deposit in the eye’s vitreous body after repetitive injections. To fully avoid this complication, we here report on a novel pharmaceutical compounding method using silicone oil-free syringes and a 33 G × 9 mm Low Dead Space Needle hub injection needle. We evaluate the method for three anti-VEGF biologics commonly used in ophthalmology: aflibercept, ranibizumab (Lucentis) and bevacizumab (Avastin). Our results show that compounding and storage for one week does not compromise the functional activity of the biologics and allows for safe and cost-effective compounding of anti-VEGF biologics for intravitreal injections in prefilled silicone oil-free syringes.

Unpredictable Nanoparticle Retention in Commonly Used Plastic Syringes Introduces Dosage Uncertainties That May Compromise the Accuracy of Nanomedicine and Nanotoxicology Studies

In recent animal experiments with suspensions of radiolabeled TiO2 nanoparticles large and highly variable radioactivity fractions were retained in disposable plastic syringes. After unloading between 10% and up to 70% of the loaded dose were still present in the syringes. As a consequence the effectively delivered nanoparticle dose to the animals was frequently much smaller than the nominal dose of the nanoparticles loaded into the syringe. The high variability of this nanoparticle retention challenges the application of a precise, predefined dose and creates a major error source when normalizing organ and tissue contents to the dose loaded into the syringe, which is usually set as the applied dose. A control study was performed employing six commonly used syringe types with seven types of radiolabeled oxide and metallic nanoparticles. For this purpose the syringes were loaded with a given volume of nanoparticle suspension, the radioactivity was measured, the syringe was unloaded and the activity measurement was repeated with the empty syringe. The highest retention values were found when using TiO2 nanoparticle suspensions with Tuberkulin type syringes. In the worst case between 6.6% and 79.1% of the nanoparticles were retained in the syringe. When using the same nanoparticle suspension with an insulin-type syringe the retention was reduced to 1.4% to 20.6%. For amorphous silica nanoparticles the maximum observed retention was 8% and for Au nanoparticles it was 5.1%. Further data gathered from in vivo animal imaging studies show that nanoparticle retention in syringes also affects experiments with nanoparticles such as exosomes, polymersomes, and protein-based nanoparticles investigated for possible applications in nanomedicine. Since the retention is highly variable the effectively applied dose cannot be determined by applying a simple syringe retention factor. The present work shall alert to the problem and illustrate its possible magnitude and unpredictable variability. As mitigation strategy adequate checks with different syringe types are proposed in order to find out whether a given combination of syringe type and nanoparticle suspension is affected by nanoparticle retention and, if necessary, to select a different syringe type that minimizes retention.

The texture and preference of “mentsuyu (Japanese noodle soup base) caviar” prepared from sodium alginate and calcium lactate

Molecular cooking involves preparing cuisines in a home and/or restaurant kitchen. Spherification is one of the processes of molecular cooking in which sodium alginate and calcium salts are used to produce spherical gels called molecular caviars. In the present study, we used a liquid and seasoned Japanese noodle soup base (mentsuyu) and applied spherification to create mentsuyu caviar (MC). Therefore, the effects of different time course after preparation, gelation times, salt concentrations, and on rupture force and sensory scores in MC were studied. Mentsuyu containing 1% (w/w) sodium alginate was added to 5% (w/w) calcium lactate solution in a drop-by-drop manner using a plastic syringe; the solution immediately gelled to form MC. Rupture forces of MCs exhibited a time-dependent increase after preparation. MCs rupture force after 2.5 min of exposure to calcium lactate was higher than that after 1.5 min of exposure. Moreover, rupture force was higher for MCs made with low-salt (NaCl) mentsuyu than those made with normal salt mentsuyu. In sensory evaluation, MC after 30 min of preparation was stronger than that immediately after preparation. Overall, MC harden over the time after preparation, or with long exposure to calcium lactate, or with low NaCl concentration in foods. The scores for visual appearance and the impact of visual appearance on preference resulting from a combination of MC and Japanese thin wheat noodles (somen) were higher than scores for these properties when combining liquid mentsuyu and somen. Thus, the visual appearance of MC was novel and high-impact compared with liquid mentsuyu. These results suggest that MC is useful for developing novel and high-impact Japanese cuisine (Washoku).

Stability of Insulin Detemir Injection in Different Primary Packaging Systems at Room Temperature

Purpose: To assess the stability of insulin detemir at controlled room temperature (RT) at 25°C in different packaging systems over 7 days. Methods: The degradation characteristics of insulin detemir were determined based on the assay results in different packaging systems (pinhole glass vial, closed glass vial, glass syringe, and plastic syringe) at RT using a reverse-phase high-performance liquid chromatography (HPLC) assay method for insulin injection. Each packaging system was compared to insulin detemir stored in the original packaged closed glass vial at 2°C to 8°C. Results: Insulin detemir stored in a closed glass vial and a glass syringe showed minor degradation at the end of day 7 (98.96% ± 1.49% and 99.78% ± 0.10%, respectively). Insulin detemir stored in plastic syringe decreased to 94.90% ± 2.50% by day 3 and to 93.52% ± 0.29% by day 7. Storage in pin-hole glass vial showed an increase in the assay (152.13% ± 0.12%) by day 7. Conclusion: Stability studies in different packaging systems demonstrated that insulin detemir remained stable for at least 7 days in a closed glass vial or glass syringe, but for only 3 days in a plastic syringe at RT. This study will allow pharmacists in the hospital setting to deliver patient-specific insulin doses into an insulin syringe with confidence in the stability.

Physicochemical stability of extemporaneously prepared clonidine solutions for use in neonatal abstinence syndrome.

Extemporaneously prepared clonidine admixture is increasingly used for the management of neonatal abstinence syndrome. However, its stability beyond 15minutes at room temperature is currently unknown. Therefore, healthcare professionals must prepare clonidine admixtures multiple times a day while the treatment is indicated, resulting in subsequent limitations and problems. The aim of this study was to investigate the physicochemical stability of clonidine in commonly used pharmaceutical diluents at clinically relevant concentrations and temperatures. Glass bottles (n=18) and plastic syringes (n=18) containing 0.5 and 5µg/mL of clonidine in either 5% glucose, 10% glucose or 0.9% normal saline were prepared and stored at 4°C for 7days and at 35°C for 24hours, respectively. Aliquots were withdrawn at predefined time points and analysed for the concentration of clonidine, changes in pH and colour, and particle content. No evidence of particle formation, or colour or pH change was observed throughout the study period. Clonidine retained more than 98% of its initial concentration when stored in the tested diluents at 4°C for 7days and at 35°C for 24hours. Our findings will allow healthcare professionals to prepare weekly dose of clonidine in glass bottles for storage in a refrigerator. The daily required dose of clonidine can be drawn aseptically from the glass bottle each day and stored in a plastic syringe at room temperature. Clonidine present in a plastic syringe can be administered via the nasogastric route 4-6 times a day. This practice would not only save nursing time and avoid delays in the timely administration of clonidine, but also reduce the risk of potential medication errors as well as preparation-associated costs.

Determination of activity meter settings for the PET nuclides 44Sc and 89Zr

Radionuclide activity meters depend on “calibration setting numbers” that are specific for a certain combination of radionuclide, volume and geometry. This work describes their determination for two PET emerging nuclides (44Sc and 89Zr), three commercial activity meters (CAPINTEC CRC15 BETA, ATOMLAB BIODEX 500 and VEENSTRA VDC 405) and two geometries: 1 mL plastic syringes and 5 mL glass vials. For 44Sc, only values for the dial setting for 5 mL ampoules were reported previously and its use for the assayed geometries would lead to significant differences. For 89Zr more data were available in the literature, and results are provided for new geometries. All results rely on the absolute standardisation in activity of solutions of both radionuclides.

Development of a sterile morphine hydrogel for the local treatment of painful skin ulcers

ObjectivesIt is often difficult to relieve the pain from skin ulcers. In several patients, topical morphine, applied as a hydrogel, has been described as useful. There is no such product...

Automated matrix-matching calibration using standard dilution analysis with two internal standards and a simple three-port mixing chamber

Simple data processing and unattended calibration are achieved in automated standard dilution analysis (aSDA) using two internal standards and an inline lab-made mixing chamber furnished from a common plastic syringe. Only two calibration solutions are required per sample, which minimizes reagent consumption and waste generation. Solution 1 contains 50% sample and 50% of a standard containing the analytes and internal standard 1 (IS1). Solution 2 has 50% sample and 50% of a blank containing internal standard 2 (IS2). The concentration of analyte in the sample is calculated from (i) the slope and intercept of an analyte vs. IS1 plot, (ii) the concentration of analyte in the standard added to Solution 1, and (iii) the intercept of a second plot with IS1vs. IS2. The aSDA method was used to determine Cd, Co, Cr, Cu, Pb and Zn in tap and creek water, beer, cola soft drink, mouthwash, cough syrup and cachaça by ICP OES. Addition/recovery experiments involving these same samples and other challenging matrices (i.e. 40% v/v HNO3, and 1% m/v Na, Ca or C) were performed to evaluate the method's accuracy. The results were compared with values obtained with external standard calibration (EC), internal standardization (IS) and standard additions (SA). Considering all samples and analytes evaluated, aSDA provided the best accuracy, with an average absolute error (ε‾=|analytepercentrecovery−100|) of 4% (EC, IS and SA had 13%, 9% and 7% errors, respectively). The aSDA strategy is a simple and inexpensive alternative to traditional methods. It has great potential for broad implementation with existing ICP OES instrumentation, as it requires little modification to systems already in place in routine laboratories.

A study to assess the knowledge regarding biomedical waste management among nursing officers in selected areas of PCMC Hospital, Pune

The biomedical waste has become a serious health hazard in many hospitals. The waste has been disposed off illegally in to the garbage and into the severs in most of the parts of the world including India. In many places, the waste is disposed carelessly and indiscriminate disposal of this waste by healthcare. So many hospitals waste spread serious disease such as hospitals and AIDS (HIV) among though who handle it and also among the general public the waste disposal is at generator site and nurses are highly responsible for biomedical waste management they play a leading role in managing and supervising hospital waste.Problem statement A study to assess the knowledge regarding biomedical waste management among Nursing officers in selected areas of PCMC Hospital, Pune.Methodology A Non Probability Purposive Sampling Technique was used for this studyMaterials and methods Research Approach: In This Study Used evaluative approachResearch Design was used Non Experimental descriptive design: The setting for this study was the selected hospitals in PCMC, Pune. The population of the present study conducted in selected hospitals at PCMC, Pune. Sample for this study consists of Nursing officers working in selected areas of PCMC Hospital, Pune. The General Systems Theory was used for the study which is developed by Ludwig Von Bertalanffy. The setting for this study was the selected hospitals in PCMC, Pune.Results The study concluded that Half (50%) of the Nursing officers had average knowledge (Score 7-13), 26.7% of them had poor knowledge (Score 0-6) and 23.3% of them had good knowledge (score 14-20) regarding biomedical waste management.60.0% of the Nursing officers knew the personal protective measures. 53.3% of them were aware of the colour of the container in which are needles and blades disposed. 46.7% of them knew Plastic syringe will be discarded in what colour bin. 46.7% of them knew Cotton dressing soiled with blood goes in which colour code.50% of them knew General and domestic waste is put in what colour bag. 60% of them knew the waste disposal methods. 43.3% of them knew what the disease caused during hospitalization are called. 30% of them knew Hospital infection control concerns personnel. The investigator concluded that in the wake of increased demand on quality care and accreditation also in the best concern of the clients there is a need to improve the knowledge level of Nursing officers regarding biomedical waste management. Staff training activity like continuing nursing education, workshops and seminars on the concerned topic should be undertaken. Also periodic assessment through competency matrix and evaluation is required.

Bevacizumab for Intravitreal Injection: Impact of Sub-Visible Particles on the Shelf-Life of Repackaged Bevacizumab.

Purpose: Bevacizumab (Avastin) is a humanized monoclonal antibody approved by the European Medicines Agency for the intravenous treatment of cancer. However, it is often used as an intravitreal injection for the treatment of macular degeneration or edema. For this purpose, bevacizumab is repackaged from glass vials into plastic syringes. The formation of particles during this compounding process as well as during storage is a source of concern. The aim of this study was to test the sub-visible particulate contamination in bevacizumab material, both in the glass vial and after repackaging into polycarbonate BD Luer-Lok™ syringes. Methods: Syringes with repackaged bevacizumab from 3 different compounding hospital pharmacies were tested for sub-visible particles at different time points during storage at 2-8°C. Results: The batches of bevacizumab starting product complied with the European Pharmacopoeia (Ph. Eur.) for small-volume parenterals. Repackaging into syringes led to an immediate increase in small particles. The number of particles ≥25 μm increased 1.3-fold, and the number of particles ≥10 μm increased 5-fold, respectively. Storage of up to 37 days did not lead to an additional increase in particle counts. All batches complied with the national criteria for particles in intravitreal solutions. Conclusions: Particle count increased due to the repackaging process, but no substantial increase was observed during storage. Formation of sub-visible particles does not impact the shelf-life of bevacizumab repackaged into BD Luer-Lok syringes.

Setup for Semimicro Pressure Filtration

Semimicro pressure filtration is carried out by pressurizing a suspension through pushing the piston of a gas-filled plastic syringe whose needle perforates the closure of a cylindrical filter funnel. Basic features as well as pros and cons of vacuum and pressure filtration are pointed out and compared to support rational choice between the two procedures.

Colourful Chemistry – from Hybrid Flow Batteries to a Powerful Redox Flow Battery with Impressive Colour Changes for a Phenomenological Approach

The energy transition towards a larger share of renewables requires energy storage devices with redox flow batteries playing a central role for stationary large-scale storage. The most promising and developed one is the vanadium redox flow battery. However, vanadium compounds are toxic, harmful to the environment and hardly suitable for use in schools – in addition, the worldwide occurrence of vanadium minerals is limited. In Freiburg, the aim of a research project was to make the topic of storage systems accessible to students both at university and in chemistry classes, because they will be the generation that will or must help shape this development. Therefore, hybrid flow batteries with harmless electrolytes, a common metal electrode and a simplified construction needed to be built for school application. Utensils from medical technology such as plastic syringes or extension lines are well suited for building cost-effective hybrid flow batteries for chemistry lessons. With such materials, electrolytes can be circulated with a pump that generates an efficient electrolyte flow. Freiburg’s design makes it possible to charge and discharge electrolytes in just a few minutes and the electrochemical reactions get visible through colour changes. In the field of redox flow batteries where the energy is stored in two electrolytes, a high-performance cell based on iron and cerium salts is also presented. With this battery, the anolyte and catholyte can be oxidised or reduced in a short time and electrolytes which change their colours during the electrochemical reaction are used as well. Thus, the operating principle of flow batteries can impressively be demonstrated by changing the properties of the electrolytes at the phenomenological level.