Human coculture model of astrocytes and SH-SY5Y cells to test the neurotoxicity of chemicals.

In this study, we established a coculture model comprising human neuroblastoma SH-SY5Y cells and induced pluripotent stem cell-derived astrocytes, faithfully replicating the human brain environment for in vitro neurotoxicity assessment. We optimized the cell differentiation duration and cell ratios to obtain images conducive to neurite outgrowth evaluation. Subsequently, the neurotoxic effects in the coculture and monoculture of SH-SY5Y cells were confirmed using neurotoxic agents such as acrylamide (ACR) and hydrogen peroxide (H2O2). Disparities in the neurotoxic impacts of ACR and H2O2 within the coculture were mirrored in the expression of genes associated with early neuronal injury. Notably, the reduction in neurite outgrowth induced by neurotoxic agents revealed the coculture's lower sensitivity compared to monocultures. Furthermore, the coculture system exhibited distinct effects of test agents on nerve damage and manifested protective influences on nerve cells. The proposed methodology holds promise for large-scale chemical neurotoxicity screening through neurite change measurements. This in vitro coculture model, accounting for cell interactions, emerges as a valuable tool in toxicity testing, offering insights into the potential effects of chemicals within the human body.

ToxSTAR: drug-induced liver injury prediction tool for the web environment.

SUMMARY: Drug-induced liver injury (DILI) is a challenging endpoint in predictive toxicology because of the complex reactive metabolites that cause liver damage and the wide range of mechanisms involved in the development of the disease. ToxSTAR provides structural similarity-based DILI analysis and in-house DILI prediction models that predict four DILI subtypes (cholestasis, cirrhosis, hepatitis and steatosis) based on drug and drug metabolite molecules. AVAILABILITY AND IMPLEMENTATION: ToxSTAR is freely available at https://toxstar.kitox.re.kr/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Cardio- and neuro-toxic effects of four parabens on Daphnia magna.

Parabens can potentially disrupt the hormonal regulation of energy metabolism, leading to issues related to obesity, metabolic health, and the cardiovascular and nervous systems. However, the health effects of parabens have yielded conflicting research results. The impact of these substances on aquatic organisms, specifically their neuro- and cardio-toxic effects, has been insufficiently investigated. Hence, the primary goal of our research was to investigate and comprehensively assess the neuro- and cardio-toxic effects of four distinct parabens using the Daphnia magna model. After 48 h of exposure to various concentrations (0.1, 1, and 10 mg/L) of four parabens (methyl-, ethyl-, propyl-, and butyl-paraben), along with a solvent control, we conducted a series of physiological tests, behavioral observations, and gene transcription analyses, focusing on cardiomyopathy, serotonin, glutamate, dopamine, GABA, acetylcholine receptors, and ion flux. From a physiological perspective, the heart rate and thoracic limb activity of the exposed daphnids showed substantial time- and dose-dependent inhibitions. Notably, among the parabens tested, butylparaben exhibited the most potent inhibition, with significant alterations in cardiomyopathy-related gene transcription. In the context of neurotoxicity, all the parabens had a significant impact on gene expression, with methylparaben having the most pronounced effect. Additionally, significant changes were observed in parameters such as distance moved, the distance between individuals, and the extent of body contact among the daphnids. In summary, our findings indicate that each paraben has the capacity to induce neurobehavioral and cardiotoxic disorders in Daphnia magna. The effects of butylparaben on the cardiovascular and nervous systems were found to be the most pronounced. These discoveries showed the potential ecological implications of paraben exposure in aquatic ecosystems, particularly regarding the predator avoidance abilities of Daphnia magna.

Plausibility of Daphnia magna as an alternative experimental model to evaluate effects on eicosanoid synthesis.

Eicosanoids play important roles in inflammation, allergy, fever, and immune responses. In the eicosanoid pathway, cyclooxygenase (COX) catalyzes the conversion of arachidonic acid to prostaglandins and is a crucial target of nonsteroidal anti-inflammatory drugs (NSAIDs). Thus, toxicological studies on the eicosanoid pathway are important for drug discovery and the evaluation of adverse health outcomes due to environmental contaminants. However, experimental models are limited owing to concerns regarding ethical standards. Thus, new alternative models for evaluating toxic effects on the eicosanoid pathway must be developed. To this end, we adopted an invertebrate species, Daphnia magna, as an alternative model. D. magna was exposed to ibuprofen, a major NSAID, for 6 and 24 h. Transcription of eicosanoid-related genes (pla2, cox, pgd synthase, pgd2r2, ltb4dh, and lox) was analyzed by qPCR, eicosanoids (arachidonic acid, prostaglandin F2, dihydroxy prostaglandin F2, and 5-hydroxyeicosatetraenoate) were quantified by multiple reaction monitoring, and enzyme-linked immunosorbent assay was used to determine protein levels of arachidonic acid and prostaglandin E2 (PGE2). After 6 h of exposure, transcription of the pla2 and cox genes was downregulated. In addition, the whole-body level of arachidonic acid, an upstream of COX pathway, increased by over 1.5-fold. The levels of PGE2, a downstream of COX pathway, decreased after 24 h of exposure. According to our results, it is expected that the eicosanoid pathway might be conserved in D. magna, at least partially. This indicates the plausibility of D. magna as an alternative model for the screening of new drugs or chemical toxicity.

Cardiotoxicity and neurobehavioral effects induced by acrylamide in Daphnia magna.

Acrylamide has neurotoxic and/or cardiotoxic effects on humans however available information regarding the neuro- and cardiotoxicity currently is very limited for freshwater organism models. Using three distinct techniques, thus, we investigated the neuro- and cardiotoxic effects of acrylamide in the freshwater invertebrate model, Daphnia magna. We exposed D. magna to acrylamide at concentrations of 0.3, 2.7, and 11.1 mg/L for 48 h alongside a control group. We then conducted physiological (thoracic limb activity and heart rate) and behavioral tests (including distance moved, velocity, turn angle, moving duration, the distance between subjects, and body contact frequency), as well as gene transcription analyses (related to cardiomyopathy, the serotonergic synapse, neuroactive ligand-receptor interactions, the GABAergic synapse, and acetylcholine receptors). After acrylamide exposure, the thoracic limb activity and heart rates of D. magna showed time- and dose dependent inhibition. From low to high exposure concentrations, both heart rates and thoracic limb activity were decreased. Additionally, the distance between subjects and body contact frequencies was significantly reduced. At the gene transcription level, acrylamide significantly altered the transcription of five genes related to cardiomyopathy and eight genes related to the serotonergic synapse, neuroactive ligand-receptor interactions, and the GABAergic synapse. The signs of hindered neural and cardiac functions were shown in D. magna. This suggests that acrylamide exposure leads to cardiotoxicity and neurobehavior defects in D. magna. Because cardiotoxicity and neurobehavioral changes may cause an ecological imbalance via predation of D. magna, acrylamide may also be considered a threat to freshwater ecosystem.

Developmental neurotoxicity induced by glutaraldehyde in neuron/astrocyte co-cultured cells and zebrafish.

The genotoxicity, development toxicity, carcinogenicity, and acute or chronic toxic effects of glutaraldehyde (GA), particularly during occupational exposure through its use as a fixative, disinfectant, and preservative, are well-documented but its effects on neurotoxicity have not been investigated. We performed in vitro and in vivo studies to examine the developmental neurotoxicity (DNT) of GA. Neurite outgrowth was examined in an in vitro co-culture model consisting of SH-SY5Y human neuroblastoma cells and human astrocytes. Cell Counting Kit-8, lactate dehydrogenase assay, and high-content screening revealed that GA significantly inhibited neurite outgrowth at non-cytotoxic concentration. Further studies showed that GA upregulated the mRNA expression of the astrocyte markers GFAP and S100ß and downregulated the expression of the neurodevelopmental genes Nestin, ßIII-tubulin, GAP43, and MAP2. Furthermore, in vivo zebrafish embryo toxicity tests explored the effects of GA on neural morphogenesis. GA adversely affected the early development of zebrafish embryos, resulting in decreased survival, irregular hatching, and reduced heart rate in a time- and concentration-dependent manner. Furthermore, the width of the brain and spinal cord was reduced, and the myelination of Schwann cells and oligodendrocytes was decreased by GA in transgenic zebrafish lines. These data suggest that GAs have potential DNT in vitro and in vivo, highlighting the need for caution regarding the neurotoxicity of GA.

Advanced graphene oxide-based paper sensor for colorimetric detection of miRNA.

MicroRNAs (miRNAs), found in blood and body fluids, have emerged as potential non-invasive biomarkers for disease and injury. miRNAs are quantitatively evaluated using typical RNA analysis methods such as the quantitative reverse transcription polymerase chain reaction, microarrays, and Northern blot, all of which require complex procedures and expensive reagents. To utilize miRNAs as practical biomarkers, it will be helpful to develop simple and user-friendly sensors. In this study, a paper-based miRNA sensor was developed by combining two methods: (1) target-recycled DNAzyme (Dz) amplification and (2) graphene oxide-assisted Dz blotting on paper. The Dz spots on paper caused a miRNA-dependent color change in presence of colorimetric reagents and facilitated the quantification of absolute amount of the target miRNA, irrespective of the volume, with high reproducibility. This approach is technologically straightforward and enables quantification of as low as 7.75 fmol miRNA using a portable smartphone.

Developmental and Neurotoxicity of Acrylamide to Zebrafish.

Acrylamide is a commonly used industrial chemical that is known to be neurotoxic to mammals. However, its developmental toxicity is rarely assessed in mammalian models because of the cost and complexity involved. We used zebrafish to assess the neurotoxicity, developmental and behavioral toxicity of acrylamide. At 6 h post fertilization, zebrafish embryos were exposed to four concentrations of acrylamide (10, 30, 100, or 300 mg/L) in a medium for 114 h. Acrylamide caused developmental toxicity characterized by yolk retention, scoliosis, swim bladder deficiency, and curvature of the body. Acrylamide also impaired locomotor activity, which was measured as swimming speed and distance traveled. In addition, treatment with 100 mg/L acrylamide shortened the width of the brain and spinal cord, indicating neuronal toxicity. In summary, acrylamide induces developmental toxicity and neurotoxicity in zebrafish. This can be used to study acrylamide neurotoxicity in a rapid and cost-efficient manner.

A fluorescence/colorimetric dual-mode sensing strategy for miRNA based on graphene oxide.

MicroRNAs (miRNAs) are small non-coding RNAs, which are involved in RNA silencing and post-transcriptional regulation of gene expression. Numerous studies have determined the expression of certain miRNAs in specific tissues and cell types, and their aberrant expression is associated with a variety of serious diseases such as cancers, immune-related diseases, and many infectious diseases. This suggests that miRNAs may be attractive and promising non-invasive biomarkers of diseases. In this study, we established a graphene oxide (GO)-based fluorescence/colorimetric dual sensing platform for miRNA by using a newly designed probe. The probe was designed to form a hairpin-like configuration with a fluorescent dye-labeled long tail, possessing a guanine (G)-rich DNAzyme domain in the loop region and target binding domain over the stem region and tail. By introducing this new hairpin-like probe in a conventional GO-based fluorescence platform, we observed both the miRNA-responsive color change by direct observation and sensitive fluorescence increase even below the nanomolar levels in a single solution without an additional separation step.

Novel (1E,3E,5E)-1,6-bis(Substituted phenyl)hexa-1,3,5-triene Analogs Inhibit Melanogenesis in B16F10 Cells and Zebrafish.

The present study aimed to evaluate the anti-melanogenic activity of 1,6-diphenyl-1,3,5-hexatriene and its derivatives in B16F10 murine melanoma cells and zebrafish embryos. Twenty five (1E,3E,5E)-1,6-bis(substituted phenyl)hexa-1,3,5-triene analogs were synthesized and their non-cytotoxic effects were predictively analyzed using three-dimensional quantitative structure-activity relationship approach. Inhibitory activities of these synthetic compounds against melanin synthesis were determined by evaluating melanin content and melanogenic regulatory enzyme expression in B16F10 cells. The anti-melanogenic activity was verified by observing body pigmentation in zebrafishes treated with these compounds. Compound #2, #4, and #6 effectively decreased melanogenesis induced by α-melanocyte-stimulating hormone. In particular, compound #2 remarkably lowered the mRNA and protein expression levels of microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), and TYRP2 in B16F10 cells and substantially reduced skin pigmentation in the developed larvae of zebrafish. These findings suggest that compound #2 may be used as an anti-melanogenic agent for cosmetic purpose.

Developmental and reproductive effects of tamoxifen on Daphnia magna.

Although medicines are less toxic than other toxicants, increased production and usage of pharmaceuticals have led to many concerns regarding their toxic effects on human and non-target organisms. Additionally, reproductive toxicity after long-term exposure is difficult to anticipate. Tamoxifen (TAM), a selective estrogen receptor modulator, has been widely used as an anticancer drug for mammalian breast and endometrial cancers. With increased TAM usage, it has frequently been reported that TAM is a potential endocrine disruptor capable of interfering with reproduction in non-target organisms. However, the mode of action of TAM in the endocrine system is unknown. In this study, we performed a 21-day chronic toxicity test using the crustacean Daphnia magna and investigated the transcriptional modulation of major genes related to the endocrine system, molting, development, and reproduction (i.e., Dm-vtg2, vmo1, cyp314, usp, and ecrb) after TAM exposure for 3, 6, 12, and 24 h. Our results showed a concentration-dependent decrease in the total number of offspring per individual, except for the concentration 25 µg/L; additionally, the expression of oogenesis-related genes was induced early but was later inhibited by TAM exposure. Additionally, molting-related genes were also downregulated in a time-dependent manner. Our findings suggested that TAM regulates reproduction by interfering with the molecular mechanisms involved in oogenesis and molting. This study supports the hypothesis that D. magna are a useful model to rapidly evaluate the reproductive effects of pharmaceuticals.

Time-Dependent Toxicity Responses in Daphnia magna Exposed to CuO and ZnO Nanoparticles.

Aggregation and dissolution of CuO and ZnO nanoparticles (NPs) increased with increasing exposure time (24, 48, and 72 h). Acute toxicity of CuO NPs to Daphnia magna also increased significantly with increasing exposure time (p < 0.05), whereas exposure time did not significantly affect acute toxicity of ZnO NPs. The dissolved Cu concentration of CuO NPs was much lower than the median effective concentration (EC50) value (44 µg L-1 at 72 h), implying that the increase in acute toxicity was caused by particles rather than by dissolved ions. However, the dissolved Zn concentration of ZnO NPs was higher than the EC50 value (600 µg L-1 at 72 h), suggesting this acute toxicity may be caused by dissolved ions. Moreover, CuO NPs induced greater lipid peroxidation than Cu ions did at an exposure time of 72 h, whereas converse results were observed for ZnO NPs.

Comparative Analysis of Transcriptional Profile Changes in Larval Zebrafish Exposed to Zinc Oxide Nanoparticles and Zinc Sulfate.

Many studies of the toxic effects of zinc oxide nanoparticles (ZnO NPs) in aquatic organisms have been performed because of increasing ZnO NP use. However, the toxicological pathways are not understood. In this study, ZnO NPs were found to be more toxic than ZnSO4 to zebrafish larvae, but ZnO NP toxicity did not involve transcript alterations. Biological processes affected by ZnO NPs and ZnSO4 were investigated by performing ingenuity pathway analysis on differently expressed genes in larvae exposed to sub-lethal ZnO NP and ZnSO4 concentrations. We identified upregulated and downregulated differently expressed genes in fish exposed to ZnO NPs and ZnSO4, and found that ZnO NPs slightly induced cell differentiation and pathways associated with the immune system and activated several key genes involved in cancer cell signaling. The results may be key to predicting and elucidating the mechanisms involved in ZnO NP and ZnSO4 toxicity in zebrafish larvae.

In situ impact assessment of wastewater effluents by integrating multi-level biomarker responses in the pale chub (Zacco platypus).

The integration of biomarker responses ranging from the molecular to the individual level is of great interest for measuring the toxic effects of hazardous chemicals or effluent mixtures on aquatic organisms. This study evaluated the effects of wastewater treatment plant (WWTP) effluents on the freshwater pale chub Zacco platypus by using multi-level biomarker responses at molecular [mRNA expression of catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), and metallothionein (MT)], biochemical (enzyme activities of CAT, SOD, GST, and concentration of MT), and physiological [condition factor (CF) and liver somatic index (LSI)] levels. The mRNA expression levels of GST and MT in Z. platypus from a site downstream of a WWTP significantly increased by 2.2- and 4.5-fold (p<0.05) when compared with those from an upstream site. However, the enzyme activities of CAT, SOD, and GST in fish from the downstream site significantly decreased by 43%, 98%, and 13%, respectively (p<0.05), except for an increase in MT concentration (41%). In addition, a significant increase in LSI (46%) was observed in Z. platypus from the downstream site (p<0.05). Concentrations of Cu, Zn, Cd, and Pb in the liver of Z. platypus were higher (530%, 353%, 800%, and 2,200%, respectively) in fish from a downstream site than in fish from an upstream location, and several multi-level biomarker responses were significantly correlated with the accumulated metals in Z. platypus (p<0.05). Integrated biomarker responses at molecular, biochemical, and physiological levels (multi-level IBR) were much higher (about 4-fold) at the downstream site than at the upstream site. This study suggests that the multi-level IBR approach is very useful for quantifying in situ adverse effects of WWTP effluents.

Use of the Integrated Biomarker Response to Measure the Effect of Short-term Exposure to Dibenz[a,h]anthracene in Common carp (Cyprinus carpio).

Dibenz[a,h]anthracene (DbA) is a polycyclic aromatic hydrocarbon that is released into the environment through incomplete combustion of gasoline, cigarettes, and coal tar. The effects of short-term (10 days) exposure of common carp (Cyprinus carpio) to DbA (0-50 µg L(-1)) were evaluated using the following four biomarkers: DNA damage, 7-ethoxyresorufin-O-deethylase (EROD) activity, acetylcholinesterase (AChE) activity, and vitellogenin (VTG) levels. An integrated biomarker response (IBR) was calculated for exposure to DbA, and the results were compared with those in our previous study of two other PAHs, benzo[k]fluoranthene (BkF) and benzo[a]pyrene (BaP). DbA exposure resulted in a significant (p < 0.05) increase in DNA damage, EROD activity, and VTG levels relative to the control. By contrast, DbA did not affect AChE activity. The IBR increased as the concentration of DbA increased. Based on the IBR values, the order of toxicity for the PAHs was BkF > BaP > DbA. Our results suggest that the IBR can be used as a quantitative tool for evaluating the responses of multiple biomarkers to PAH exposure.

Cloning metallothionein gene in Zacco platypus and its potential as an exposure biomarker against cadmium.

Zacco platypus, pale chub, is an indigenous freshwater fish of East Asia including Korea and has many useful characteristics as indicator species for water pollution. While utility of Z. platypus as an experimental species has been recognized, genetic-level information is very limited and warrants extensive research. Metallothionein (MT) is widely used and well-known biomarker for heavy metal exposure in many experimental species. In the present study, we cloned MT in Z. platypus and evaluated its utility as a biomarker for metal exposure. For this purpose, we sequenced complete complementary DNA (cDNA) of MT in Z. platypus and carried out phylogenetic analysis with its sequences. The transcription-level responses of MT gene following the exposure to CdCl2 were also assessed to validate the utility of this gene as an exposure biomarker. Analysis of cDNA sequence of MT gene demonstrated high conformity with those of other fish. MT messenger RNA (mRNA) expression and enzymatic MT content significantly increased following CdCl2 exposure in a concentration-dependent manner. The level of CdCl2 that resulted in significant MT changes in Z. platypus was within the range that was reported from other fish. The MT gene of Z. platypus sequenced in the present study can be used as a useful biomarker for heavy metal exposure in the aquatic environment of Korea and other countries where this freshwater fish species represents the ecosystem.

Estrogenic potency of bisphenol S, polyethersulfone and their metabolites generated by the rat liver S9 fractions on a MVLN cell using a luciferase reporter gene assay.

BACKGROUND: Bisphenol A (BPA) is an applied chemical that is used in many industrial fields and is a potential endocrine disruption chemical (EDC) that is found in the environment. Bisphenol S (BPS) and polyethersulfone (PES) have been suggested as putative BPA alternatives. In this study, the estrogenic potency induced by the binding of 17-beta-estradiol (E2), BPA, BPS, PES and their metabolites formed by the rat liver S9 fraction to the human estrogen receptor (ER) was estimated. METHODS: We used an in vitro bioassay based on the luciferase reporter assay in MVLN cells to evaluate the estrogenic activity of 17-beta-estradiol (E2), BPA, BPS, PES (E2: 0.001 to 0.3 nM; BPA, BPS and PES: 0.0001 to 5 microM) and their metabolites (E2: 0.05 microM; BPA, BPS and PES: 0.1 mM) according to incubation times (0, 20 and 40 min). After chemical treatment to MVLN cells for 72 hrs, and the cell viability and luciferase intensity induced were estimated, from which the estrogenic activity of the chemicals tested was evaluated. RESULTS: BPA and BPS induced estrogenic activity whereas PES did not show any estrogenic activity in the concentrations tested. In an in vitro assay of metabolites, BPA metabolites displayed comparable estrogenic activity with BPA and metabolites of both BPS and PES showed increasing estrogenic activity. CONCLUSIONS: The results suggest that the metabolites of BPS and PES have estrogenic potential and the need for the assessment of both chemicals and their metabolites in other EDC evaluation studies. The estrogenic potency of PES and its metabolites is the first report in our best knowledge.

Uptake, tissue distribution, and depuration of total silver in common carp (Cyprinus carpio) after aqueous exposure to silver nanoparticles.

The increased use and disposal of silver nanoparticles (AgNPs) has led to their release from wastewater treatment plants into surface waters and concern over potential for negative effects in aquatic organisms. Investigations of the toxicity of AgNPs in fish have considered various species, exposure routes, and test end points; however, the toxicokinetics of total silver has not been studied in fish exposed to aqueous AgNPs. In this study, we investigated the toxicokinetics of total silver in common carp (Cayprinus carpio) exposed to AgNPs [0.62 ± 0.12 (mean ± standard deviation) mg L(-1)] for 7 days followed by a 2 week depuration period. During exposure and depuration, fish were sampled, tissues were excised (gills, brain, skeletal muscle, gastrointestinal tract, liver, and blood) and digested in acid, and total silver concentrations were analyzed by inductively coupled plasma-optical emission spectrometry. Total silver in tissues increased during the 7 day exposure, and mean concentrations were 5.61 mg/kg of liver, 3.32 mg/kg of gills, 2.93 mg/kg of gastrointestinal tract, 0.48 mg/kg of skeletal muscle, 0.14 mg/kg of brain, and 0.02 mg/kg of blood. Transmission electron microscopy energy-dispersive spectroscopy confirmed the presence of silver in the tissues. After depuration for 14 days, total silver returned to control levels in all tissues except liver (4.22 mg/kg), gastrointestinal tract (1.26 mg/kg), and gills (0.77 mg/kg).

Integration of multi-level biomarker responses to cadmium and benzo[k]fluoranthene in the pale chub (Zacco platypus).

The Cd exposure for 14 days significantly increased both the molecular (DNA single-strand breaks) and biochemical (metallothionein concentrations) biomarkers in the freshwater pale chub, Zacco platypus, whereas changes in the histological and physiological biomarker responses were negligible. The BkF exposure for 14 days led to significant increases in the mRNA expression of catalase and superoxide dismutase, 7-ethoxyresorufin-O-deethylase enzymatic activity and DNA single-strand breakage at the molecular and biochemical levels. In addition, exposure to 50µg/L of BkF induced histological alteration in the liver, with significant changes to the liver somatic index and condition factor at the physiological level. The integration of multi-level biomarker responses at the molecular, biochemical and physiological levels was highly correlated with the concentrations of Cd and BkF.

Toxicity of citrate-coated silver nanoparticles differs according to method of suspension preparation.

To evaluate substance toxicity, it is critical to maintain specific concentrations of test substances throughout the exposure period. During the last decade, the need to improve methods for nanoparticle (NP) suspension preparations has gained attention because many published results on NPs toxicity have been inconsistent. Here, we compared the toxicity of citrate-coated silver nanoparticles (AgNPs) suspended by two different methods (fractionated vs. colloidal) in freshwater organisms (daphnia and medaka). Analytical methods (ICP-OES, DLS and UV absorbance) were employed to characterize behavior of AgNPs in suspension. Results showed that fractionated (stirred and settled) solution was less toxic to daphnia (13.8 µg/L) than colloidal solution (6.1 µg/L), suggesting that method of preparation was a critical factor that affected toxicity. However, differences in toxicity caused by suspension methods were not observed in medaka. Results indicate that the method used to prepare suspensions of NPs can affect toxicity, and that differences can exist among test organisms.