Use Of Sonication Research Articles

Ultrasound-Assisted Synthesis of Titania Nanoparticles, Characterization of Their Thin Films, and Activity in Photooxidation of β-Naphthol

Nanosized titania (TiO2) films and powders were prepared from titanium isopropoxide by ultrasonication then ultrasonic aerosol-assisted chemical vapor deposition (UAACVD). X-ray diffraction (XRD), used to study the crystal structure, phase, and crystallite size of TiO2 samples annealed at 500°C, revealed anatase was the main crystalline phase. Scanning electron microscopy and atomic force microscopy revealed the quasi-spherical morphology of the TiO2 nanoparticles; average size distribution was in the range 20–35 nm. Ultraviolet–visible spectroscopy was used to evaluate the photocatalytic activity of the anatase TiO2, on the basis of efficiency of degradation of β-naphthol. Pure TiO2 nanoparticles synthesized by use of sonication–UAACVD then calcination at 500°C enabled effective photodegradation under UV light. This method of synthesis of TiO2 is superior to the reflux–UAACVD method with titanium isopropoxide as precursor.

Microbial diagnosis of infection and colonization of cardiac implantable electronic devices by use of sonication

The clinical utility of sonication as an adjunctive diagnostic tool for the microbial diagnosis of cardiac implantable device-associated infections (CIDAIs) was investigated. The implants of 83 subjects were investigated, 15 with a CIDAI and 68 without a clinical infection. Clinical data were analyzed prospectively and sonication fluid cultures (83 patients, 100%) and traditional cultures (31 patients, 37.4%) were performed Generator pocket infection and device-related endocarditis were found in 13 (86.7%) and four (26.7%) subjects, respectively. The mean numbers of previous technical complications and infections were higher in the infected patients compared to the non-infected patients (8 vs. 1, p<0.001; 2 vs. 0, p<0.031, respectively). The sensitivity and specificity for detecting CIDAI was 73.3% (11/15) and 48.5% (33/68) for sonication fluid culture, and 26.7% (4/15) and 100% (16/16) for traditional culture (p<0.001), respectively. A higher number of organisms were identified by sonication fluid than by tissue culture (58 vs. 4 specimens; p<0.001). The most frequent organisms cultured were Gram-positive cocci (66.1%), mainly coagulase-negative staphylococci (35.5%). Thirty-five (51.5%) non-infected subjects were considered colonized due to the positive identification of organisms exclusively through sonication fluid culture. Sonication fluid culture from the removed cardiac implants has the potential to improve the microbiological diagnosis of CIDAIs.

Surface functionalization of nanodiamond with phenylphosphonate

HypothesisThis work explores arylation of oxidized nanodiamond (OND) and carboxylated nanodiamond (CND) using phenylphosphonate (PPA) to produce PPA-grafted nanodiamond (PPA@ND). The specific hypothesis is that PPA can be readily grafted onto the surfaces of OND and CND particles via the nanodiamonds’ reactive oxygen-rich surface functional groups. ExperimentsThis work explores the effect of varying reaction conditions (reactant ratio, heat, sonication) on PPA graft density and grafting mode. The products were characterized by a variety of techniques including: 31P MAS NMR and FTIR to confirm PPA grafting, XPS to estimate PPA grafted amounts, and TGA to assess thermal stability. The utility of PPA functionalization for improving ND dispersion in organic solvents was evaluated using dynamic light scattering. FindingsThe results confirm PPA grafting on both OND and CND, quantify the extent of PPA grafting, and suggest primarily bidentate grafting. For PPA grafting on OND, the grafted amount does not depend on PPA:ND ratio, application of heat, or use of sonication. The grafted amount of PPA on CND was significantly lower than that on OND. TGA data indicates that grafted PPA improves the thermal stability of both OND and CND. The starting OND disperses in water as aggregates with approximately 100nm particle size. However, PPA-grafted OND does not disperse at all in water: thus grafted PPA changes the ND surface from hydrophilic to hydrophobic. PPA@OND prepared without sonication does not completely disperse in either DMF or toluene. Only PPA@OND prepared with sonication disperses well in these solvents, suggesting that sonication promotes more complete exposure of the ND surface to PPA grafting.

Usefulness of Sonication of Cardiovascular Implantable Electronic Devices to Enhance Microbial Detection

The cardiovascular implantable electronic device (CIED) infection rate is rising disproportionately to the rate of device implantation. Identification of microorganisms that cause CIED infections is not always achieved using present laboratory techniques. We conducted a prospective study to determine whether device vortexing-sonication followed by culture of the resulting sonicate fluid would enhance microbial detection compared with traditional swab or pocket tissue cultures. Forty-two subjects with noninfected and 35 with infected CIEDs were prospectively enrolled over 12 months. One swab each from the device pocket and device surface, pocket tissue, and the CIED were collected from each patient. Swabs and tissues were cultured using routine methods. The CIED was processed in Ringer's solution using vortexing-sonication and the resultant fluid semiquantitatively cultured. Tissue and swab growth was considered significant when colonies grew on ≥2 quadrants of the culture plate and device was considered significant when ≥20 colonies were isolated from 10 ml of sonicate fluid. In noninfected group, 5% of sonicate fluids yielded significant bacterial growth, compared with 5% of tissue cultures (p = 1.00) and 2% of both pocket and device swab cultures (p = 0.317 each). In infected group, significant bacterial growth was observed in 54% of sonicate fluids, significantly greater than the sensitivities of pocket swab (20%, p = 0.001), device swab (9%, p <0.001), or tissue (9%, p <0.001) culture. In conclusion, vortexing-sonication of CIEDs with semiquantitative culture of the resultant sonicate fluid results in a significant increase in the sensitivity of culture results, compared with swab or tissue cultures.

Characterization of Polyurethane Foam Added with Synthesized Acetic and Oleic-Modified TiO2 Nanocrystals

In this work, the effects due to the addition of nanoparticles in polyurethane foams on thermo-physical and mechanical properties have been evaluated. Two types of nanoparticles were used, acetic and oleic-modified titania nanocrystals TiO2. The nanoparticles were first dispersed in a polyol component via the use of sonication; then, the doped polyol was mixed with isocyanate. The different characterization techniques describe the state of the dispersion of fillers in foam. The effects of these additions in foam were evaluated according to UNI EN 826-UNI EN 12087- UNI EN 13165, in terms of thermo-physical and mechanical properties, i.e., diffusivity, conductivity, compressive strength and water uptake. The microstructure of the foam was analysed using scanning electron microscopy (SEM). The foam obtained with nanoadditives presented improved mechanical characteristics compared to neat foam, presumably due to the different shape of the nanoparticles. The addition of nanoparticles favoured the formation of nucleation centres; this effect was likely due to the size, shape and distribution of particles and due to their surface treatment.

Formulation, optimization and characterization of gemfibrozil nanocrystals prepared by wet milling technique

Today, nanotechnology has a variety of application areas. Pharmacy is one of the most important application fields of nanotechnology. Preparation of nanoparticular drug delivery systems such as nanocrystals could improve the solubility and bioavailability of poorly water soluble drugs. Gemfibrozil (GEM) is a low water soluble drug biopharmaceutical classification system II and used as a lipid regulating agent. In this study, a rapid and simple wet milling method was used for preparation of GEM nanosuspension (GEM NS). The use of sonication after wet milling process reduced the milling time significantly. Different concentrations of stabilizers (polyvinyl pyrrolidone K30 [PVP K30] and Tween 80) were tested for preparation of GEM NSs. The finest GEM NS was obtained by 0.5% w/v GEM, 1% w/v PVP K30 and 2% w/v Tween 80. The size and zeta potential of finest GEM NS were 238.2 ± 2.5 nm and - 19.6 ± 0.1 mV, respectively.The morphology of dried GEM NS was observed using atomic force microscopy. Differential scanning calorimetry of GEM and GEM NS confirmed that there was no interaction between GEM and stabilizers. Compared with GEM, the solubility of GEM NS increased significantly. Key words: Gmefibrozil, nanocrystal, poorly soluble drugs, wet milling

Improved Diagnosis of Infection Associated with Osteosynthesis by Use of Sonication of Fracture Fixation Implants

Previous studies have shown that sonication fluid cultures from removed orthopedic devices improved the microbiological diagnosis of orthopedic implant-associated infections; however, few of these investigations have applied sonication to the removed fracture fixation devices to evaluate its utility for the diagnosis of osteosynthesis-associated infection (OAI). We compared sonication fluid to conventional tissue cultures from 180 subjects with different sizes of plates and screws (n = 156), spinal implants (n = 26), and intramedullary nails (n = 3), of whom 125 and 55 subjects had OAI and noninfected osteosynthesis (NIO), respectively. The sensitivity for detecting OAI was 90.4% for sonication fluid culture and 56.8% for periprosthetic tissue cultures (P < 0.05), and the specificities were 90.9% and 96.4%, respectively. Sonication fluid culture detected more pathogens than peri-implant tissue culture (113 versus 71; P < 0.001), while polymicrobial infections were diagnosed by sonication fluid cultures and tissue cultures in 20.8% and 8% (P < 0.001), respectively. Microbiological diagnosis was achieved exclusively by sonication fluid cultures for 47 (90.4%) subjects, and among them, 18 (38.3%) had previously received antibiotics, whereas in five (9.6%) infected subjects, tissue culture was positive and the sonication fluid culture was negative. Among 39 (31.2%) OAI cases receiving antibiotics, the identification of the organisms occurred in 38.5% and 82.1% of the tissue and sonication fluid cultures, respectively (P < 0.049). We demonstrated that sonication fluid culture from removed osteosyntheses has the potential for improving the microbiological diagnosis of OAI.

Effects of sonication and ultraviolet-C treatment as a hurdle concept on quality attributes of Chokanan mango (Mangifera indica L.) juice.

The growing demand for fresh-like food products has encouraged the development of hurdle technology of non-thermal processing. In this study, freshly squeezed Chokanan mango juice was treated by paired combinations of sonication (for 15 and 30 min at 25 ℃, 40 kHz frequency) and UV-C treatment (for 15 and 30 min at 25 ℃). Selected physicochemical properties, antioxidant activities, microbial inactivation and other quality parameters of combined treated juice were compared to conventional thermal treatment (at 90 ℃ for 60 s). After thermal and combined treatment, no significant changes occurred in physicochemical properties. A significant increase in extractability of carotenoids (15%), polyphenols (37%), flavonoids (35%) and enhancement in antioxidant capacity was observed after combined treatment. Thermal and combined treatment exhibited significant reduction in microbial load. Results obtained support the use of sonication and UV-C in a hurdle technology to improve the quality of Chokanan mango juice along with safety standards.

Superior H2 production by hydrophilic ultrafine Ta2O5 engineered covalently on graphene

A H2O2-mediated hydrothermal method was developed for the fabrication of hydrophilic Ta2O5/graphene composite. The composite shows a superior H2 productivity, up to 30 mmol g−1 h−1 when used as a photocatalyst for water splitting, corresponding to an apparent quantum efficiency of 33.8% at 254 nm. This superior performance is due to the hydrophilic nature of the composite and more importantly due to the ultrafine Ta2O5 nanoparticles (about 4.0 ± 1.5 nm) which are covalently bonded with the conductive graphene. The hydrophilic property of the composite is attributed to the use of H2O2 in the hydrothermal process. The ultrafine size of the Ta2O5 particles which are covalently bonded with the graphene sheets is attributed to the use of sonication in the synthesis process. Furthermore, the hydrophilic Ta2O5/Gr composite is durable, which is beneficial to long term photocatalysis. The strategy reported here provides a new approach to designing photocatalysts with superior performance for H2 production.

An Easy, Convenient Cell and Tissue Extraction Protocol for Nuclear Magnetic Resonance Metabolomics

As a complement to the classic metabolomics biofluid studies, the visualisation of the metabolites contained in cells or tissues could be a very powerful tool to understand how the local metabolism and biochemical pathways could be affected by external or internal stimuli or pathologies. Therefore, extraction and/or lysis is necessary to obtain samples adapted for use with the current analytical tools (liquid NMR and MS). These extraction or lysis work-ups are often the most labour-intensive and rate-limiting steps in metabolomics, as they require accuracy and repeatability as well as robustness. Many of the procedures described in the literature appear to be very time-consuming and not easily amenable to automation. To find a fast, simplified procedure that allows release of the metabolites from cells and tissues in a way that is compatible with NMR analysis. We assessed the use of sonication to disrupt cell membranes or tissue structures. Both a vibrating probe and an automated bath sonicator were explored. The application of sonication as the disruption procedure led to reproducible NMR spectral data compatible with metabolomics studies. This method requires only a small biological tissue or cell sample, and a rapid, reduced work-up was applied before analysis. The spectral patterns obtained are comparable with previous, well-described extraction protocols. The rapidity and the simplicity of this approach could represent a suitable alternative to the other protocols. Additionally, this approach could be favourable for high- throughput applications in intracellular and intratissular metabolite measurements.

Sonication as a Diagnostic Approach Used to Investigate the Infectious Etiology of Prosthetic Hip Joint Loosening

The purpose of the study was to evaluate the usefulness of sonication for the diagnosis of prosthetic joint infections (PJIs) by its comparison with periprosthetic tissues (PTs) and synovial fluid (SV-F) cultures. The study groups included 54 patients undergoing exchange of total hip prostheses for so called "aseptic" loosening occurring without clinical manifestations of an accompanying PJI and 22 patients who developed a sinus tract communicating with the prosthesis which was indicative of an ongoing infectious process. Significant positive culture results were obtained among 10 (18.5%) patients with "aseptic" implant failure and in 18 (81.8%) patients who developed a sinus tract. Sonicate-fluid (S-F) yielded bacterial growth in all culture-positive patients with "aseptic" loosening vs. 15 patients with presumed PJIs. There was a concordance in terms of bacterial species isolated from S-F and conventional cultures from individual patients. Coagulase-negative staphylococci were isolated most frequently. Sensitivity of sonication (75%) exceeded that estimated for PTs (69%) and SV-F (45%) cultures. We conclude that identification of causative agents of PJIs which is critical to further therapeutic decisions is aided by the combination of sonication and conventional culture.

Single stage electrochemical exfoliation method for the production of few-layer graphene via intercalation of tetraalkylammonium cations

We present a non-oxidative production route to few layer graphene via the electrochemical intercalation of tetraalkylammonium cations into pristine graphite. Two forms of graphite have been studied as the source material with each yielding a slightly different result. Highly orientated pyrolytic graphite (HOPG) offers greater advantages in terms of the exfoliate size but the source electrode set up introduces difficulties to the procedure and requires the use of sonication. Using a graphite rod electrode, few layer graphene flakes (2nm thickness) are formed directly although the flake diameters from this source are typically small (ca. 100–200nm). Significantly, for a solvent based route, the graphite rod does not require ultrasonication or any secondary physical processing of the resulting dispersion. Flakes have been characterized using Raman spectroscopy, atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS).

Effects of thermal treatment and sonication on quality attributes of Chokanan mango (Mangifera indica L.) juice

Ultrasonic treatment is an emerging food processing technology that has growing interest among health-conscious consumers. Freshly squeezed Chokanan mango juice was thermally treated (at 90°C for 30 and 60s) and sonicated (for 15, 30 and 60min at 25°C, 40kHz frequency, 130W) to compare the effect on microbial inactivation, physicochemical properties, antioxidant activities and other quality parameters. After sonication and thermal treatment, no significant changes occurred in pH, total soluble solids and titratable acidity. Sonication for 15 and 30min showed significant improvement in selected quality parameters except color and ascorbic acid content, when compared to freshly squeezed juice (control). A significant increase in extractability of carotenoids (4–9%) and polyphenols (30–35%) was observed for juice subjected to ultrasonic treatment for 15 and 30min, when compared to the control. In addition, enhancement of radical scavenging activity and reducing power was observed in all sonicated juice samples regardless of treatment time. Thermal and ultrasonic treatment exhibited significant reduction in microbial count of the juice. The results obtained support the use of sonication to improve the quality of Chokanan mango juice along with safety standard as an alternative to thermal treatment.

Diagnosis and management of prosthetic joint infection

Prosthetic joint infection remains a devastating complication of arthroplasty associated with significant patient morbidity. The demand for arthroplasty is rapidly growing with a corresponding increase in the number of infections involving the prosthesis. The diagnosis and treatment of prosthetic joint infections presents a significant challenge to orthopaedic and infectious diseases clinicians. The underlying pathogenesis of prosthetic joint infections is due to the ability of the microorganisms to form a biofilm. The biofilm provides protection against host immune responses and antimicrobial therapy. In addition, it impedes standard laboratory diagnostic techniques. This review will examine new investigations to improve the diagnostic yield and rapidity of diagnosis of infections, including the use of sonication to disrupt the biofilm, new molecular tests to improve the detection of infecting microorganisms and new imaging techniques such as (18)F-fluoro-deoxyglucose PET. The successful treatment of prosthetic joint infections is dependent on eliminating the biofilm dwelling microorganisms whilst maintaining patient mobility and quality of life. This review will examine current understanding of management approaches for these infections, with a particular focus on antimicrobial therapy with activity against the biofilm, such as rifampicin and fluoroquinolones.

Towards a reliable and high sensitivity O2-independent glucose sensor based on Ir oxidenanoparticles

The primary goal of this work is the development of a rapidly responding, sensitive, and biocompatible Ir oxide (IrOx)-based glucose sensor that regenerates solely via IrOx-mediation in both O2-free and aerobic environments. An important discovery is that, for films composed of IrOx nanoparticles, Nafion® and glucose oxidase (GOx), a Michaelis–Menten constant (K′m) of 20–30mM is obtained in the case of dual-regeneration (O2 and IrOx), while K′m values are much smaller (3–5mM) when re-oxidation of GOx occurs only through IrOx-mediation. These smaller K′m values indicate that the regeneration of GOx via direct electron transfer to the IrOx nanoparticles is more rapid than to O2. Small K′m values, which are obtained more commonly when Nafion® is not present in the films, are also important for the accurate measurement of low glucose concentrations under hypoglycemic conditions. In this work, the sensing film was also optimized for miniaturization. Depending on the IrOx and GOx surface loadings and the use of sonication before film deposition, the imax values ranged from 5to 225μAcm−2, showing very good sensitivity down to 0.4mM glucose.

An improved plant leaf protein extraction method for high resolution two-dimensional polyacrylamide gel electrophoresis and comparative proteomics

We report here a simple and universally applicable protocol for extracting high quality proteins from plant leaf tissues. The protocol provides improved resolution and reproducibility of two-dimensional polyacrylamide gel electrophoresis (2-DE) and reduces the time required to analyze samples. Partitioning rubisco by polyethylene glycol (PEG) fractionation provides clearer detection of low-abundance proteins. Co-extraction of interfering substances increases the sample conductivity, which results in poor electrophoretic separation. Re-extraction of PEG-fractionated samples with phenol effectively eliminated interfering substances, which results in optimal conductivity during separation in the first dimension of the isoelectric focusing. Smooth focusing reduces analysis time and provides superior resolution in 2-DE gels. Incubating the samples at −80° C instead of −20° C reduced protein precipitation time to 2–3 h. Removal of nonprotein contaminants and the use of sonication increased protein solubility without additional reagents. These changes enabled loading and separation of maximum amounts of proteins, which permitted improved protein identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). An immunological approach revealed that little or no ribulose-1, 5-bisphosphte bisphosphate carboxylase oxygenase was present in the PEG supernatant. In addition, low-abundance proteins, such as myelocytomatosis transcription factor (MYC) and alpha subunit of heterotrimeric guanine nucleotide-binding protein complex (Gα), were detected only in the modified PEG supernatant and not in the total protein. These results suggest that our protocol produced high quality proteins and made many low-abundant proteins available for proteomic analysis. The successful application of this protocol for analyzing the leaf proteomes of soybean, Miscanthus sinensis, barley, Chinese cabbage, peanut and tea (Camellia sinensis) suggests that it could be used for comparative proteomic analysis of a wide range of plant leaves.

Effect of ultrasound alone or ultrasound coupled with CO2 on the chemical composition, cheese-making properties and sensory traits of raw milk

This study aimed to evaluate the effects of different intensities and durations of ultrasound on the composition, oxidation, cheese-making properties and sensory traits of raw milk. Moreover, the effects of the addition of CO2 and HCl prior to the sonication were tested to determine whether these chemicals could reduce the production of off-flavours. Treating milk with ultrasound led to certain changes in milk composition. We observed significant increases in free fatty acid levels, oxidation, and decreases in somatic cell count and pH; improved coagulation traits were also observed. The results of sensory evaluations revealed a significant increase in a burnt off-flavour with increasing intensity and duration of the ultrasound treatment. The addition of CO2 appeared to significantly reduce the disruptive effect of ultrasound, the formation of oxidation products and the detection of the burnt off-flavour, whereas the sour flavour was increased. Among the new technologies that are used to improve milk sanitisation and shelf life, ultrasound treatment has been demonstrated to inactivate many bacterial species and to be a viable sanitisation procedure. In this study the milk coagulation properties of sonicated milk were dramatically improved following the ultrasound (US) treatments, indicating a possible use for US in the cheese-making industry. Moreover, despite the evident production of off-flavours caused by US in milk, the use of CO2 to reduce pyrolytic processes and thus the formation of oxidation products, appeared to be promising with respect to the increased use of sonication on food and other organic matrices.

Use of sonication and vacuum infiltration for Agrobacterium – mediated transformation of an Indian lentil (Lens culinaris Medik.) cultivar

The regeneration and transformation protocol for lentil employing both sonication and vacuum infiltration of whole seed explants is described. This protocol overcomes the non-competence of whole seed explants to Agrobacterium mediated transformation though the regeneration potential of this explant is high as compare to other agro-competent explants. The decoated seeds of four genotypes were cultured on regeneration media that consisted of Murashige and Skoog (MS) salts and Gamborg (B5) vitamins (MSB5V) supplemented with different concentrations of 6-benzylaminopurine (2.5–20μM) and thidiazuron (0.1–10μM) separately. Best regeneration response with an average of 9 shoots per explant was observed in 95% of the cultures on MSB5V medium fortified with 5.0μM thidiazuron. The protocol was found independent to the effect of genotype. Among the effects of physical treatments, it was found that sonication at 20KHz for 60s followed by vacuum infiltration at −660mm of Hg for 5min resulted in highest transient GUS expression (68%) with no significant effect on regeneration. Rooting was observed in 45% of shoots on rooting medium containing half strength MS salts and full strength MS vitamins supplemented with 2.5μM IBA. Regenerated plantlets were established in soil with 50% survival rate and grown to maturity. GUS activity in leaves and roots of primary transformants recovered on kanamycin containing selection media confirmed the expression of transgene. These plants were tested and found positive for nptII gene by PCR and Southern analysis with an overall transformation frequency of 0.66%. PCR analysis of T0 progeny revealed the inheritance of transgene.

Scalable Method for the Reductive Dissolution, Purification, and Separation of Single-Walled Carbon Nanotubes

As synthesized, bulk single-walled carbon nanotube (SWNT) samples are typically highly agglomerated and heterogeneous. However, their most promising applications require the isolation of individualized, purified nanotubes, often with specific optoelectronic characteristics. A wide range of dispersion and separation techniques have been developed, but the use of sonication or ultracentrifugation imposes severe limits on scalability and may introduce damage. Here, we demonstrate a new, intrinsically scalable method for SWNT dispersion and separation, using reductive treatment in sodium metal-ammonia solutions, optionally followed by selective dissolution in a polar aprotic organic solvent. In situ small-angle neutron scattering demonstrates the presence of dissolved, unbundled SWNTs in solution, at concentrations reaching at least 2 mg/mL; the ability to isolate individual nanotubes is confirmed by atomic force microscopy. Spectroscopy data suggest that the soluble fraction contains predominately large metallic nanotubes; a potential new mechanism for nanotube separation is proposed. In addition, the G/D ratios observed during the dissolution sequence, as a function of metal:carbon ratio, demonstrate a new purification method for removing carbonaceous impurities from pristine SWNTs, which avoids traditional, damaging, competitive oxidation reactions.

Ecotoxicity test methods for engineered nanomaterials: Practical experiences and recommendations from the bench

Ecotoxicology research is using many methods for engineered nanomaterials (ENMs), but the collective experience from researchers has not been documented. This paper reports the practical issues for working with ENMs and suggests nano-specific modifications to protocols. The review considers generic practical issues, as well as specific issues for aquatic tests, marine grazers, soil organisms, and bioaccumulation studies. Current procedures for cleaning glassware are adequate, but electrodes are problematic. The maintenance of exposure concentration is challenging, but can be achieved with some ENMs. The need to characterize the media during experiments is identified, but rapid analytical methods are not available to do this. The use of sonication and natural/synthetic dispersants are discussed. Nano-specific biological endpoints may be developed for a tiered monitoring scheme to diagnose ENM exposure or effect. A case study of the algal growth test highlights many small deviations in current regulatory test protocols that are allowed (shaking, lighting, mixing methods), but these should be standardized for ENMs. Invertebrate (Daphnia) tests should account for mechanical toxicity of ENMs. Fish tests should consider semistatic exposure to minimize wastewater and animal husbandry. The inclusion of a benthic test is recommended for the base set of ecotoxicity tests with ENMs. The sensitivity of soil tests needs to be increased for ENMs and shortened for logistics reasons; improvements include using Caenorhabditis elegans, aquatic media, and metabolism endpoints in the plant growth tests. The existing bioaccumulation tests are conceptually flawed and require considerable modification, or a new test, to work for ENMs. Overall, most methodologies need some amendments, and recommendations are made to assist researchers.