Proposing Effective Ecotoxicity Test Species for Chemical Safety Assessment in East Asia: A Review.

East Asia leads the global chemical industry, but environmental chemical risk in these countries is an emerging concern. Despite this, only a few native species that are representative of East Asian environments are listed as test species in international guidelines compared with those native to Europe and America. This review suggests that Zacco platypus, Misgurnus anguillicaudatus, Hydrilla verticillata, Neocaridina denticulata spp., and Scenedesmus obliquus, all resident to East Asia, are promising test species for ecotoxicity tests. The utility of these five species in environmental risk assessment (ERA) varies depending on their individual traits and the state of ecotoxicity research, indicating a need for different applications of each species according to ERA objectives. Furthermore, the traits of these five species can complement each other when assessing chemical effects under diverse exposure scenarios, suggesting they can form a versatile battery for ERA. This review also analyzes recent trends in ecotoxicity studies and proposes emerging research issues, such as the application of alternative test methods, comparative studies using model species, the identification of specific markers for test species, and performance of toxicity tests under environmentally relevant conditions. The information provided on the utility of the five species and alternative issues in toxicity tests could assist in selecting test species suited to study objectives for more effective ERA.

Adverse effects of perfluoroalkyl acids on fish and other aquatic organisms: A review.

Perfluoroalkyl acids (PFAAs) have been widely used in many industrial and consumer products. They have been detected ubiquitously in ambient water along with other environmental matrices, and their adverse effects on aquatic organisms have been a subject of active investigation. Here, we intended to summarize and synthesize the existing body of knowledge on PFAA toxicity through an extensive literature review, and shed light on areas where further research is warranted. PFAA toxicity appears to be influenced by the sex and developmental stages of aquatic organisms, but not significantly by exposure route. PFAA-induced aquatic toxicity could be classified as metabolism disturbance, reproduction disruption, oxidative stress, developmental toxicity, thyroid disruption, etc. At the molecular level, these responses can be initiated by key events, such as nuclear receptor activation, reactive oxygen species induction, or interaction with a membrane, followed by a cascade of downstream responses. PFAA-induced toxicity involves diverse metabolic processes, and therefore elucidating crosstalk or interactions among diverse metabolic pathways is a challenging task. In the presence of other chemicals, PFAAs can function as agonists or antagonists, resulting in different directions of combined toxicity. Therefore, mixture toxicity with other groups of chemicals is another research opportunity. Experimental evidence supports the trans-generational toxicity of PFAAs, suggesting that their long-term consequences for aquatic ecosystems should become of concern. A recent global ban of several PFAAs resulted in an increasing dependence on PFAA alternatives. The lack of sufficient toxicological information on this emerging group of chemicals warrant caution and rigorous toxicological assessments.

n-Butyl acrylate-induced antioxidant system alteration through two generations in Oryzias latipes.

n-Butyl acrylate (nBA) is one of acrylate esters which has been applied to diverse industrial fields. For unveiling of xeno-estrogenic effects and oxidative stress induction by nBA under two-generational exposure regimen (17 weeks), the biomarkers relevant to an estrogenic effect and oxidative stress were analyzed. Acute toxicity value of nBA in Oryzias latipes was 7.2 mg/L (96 h-LC50). Over exposure time, the significant transcriptional change of cytochrome P450 19A (CYP19A) and vitellogenin 1/2 (VTG1/2) was not observed (one-way ANOVA, P < 0.05), meaning no estrogenic effect of nBA. Significant reduction of glutathione (GSH) content was observed in F0 male and female fish, while in F1 male, the content was increased (P < 0.05). Catalase (CAT) activity of male fish showed the significant decrease in both F0 and F1 fish, showing multi-generational suppressing effect of nBA on CAT activity. But in case of reactive oxygen species (ROS), expression level and glutathione S-transferase (GST) activity were not modulated in response to nBA. These findings suggest that nBA could affect an antioxidant system alteration through GSH depletion and inhibition of CAT activity which could be transferred to the next generation, whereas xeno-estrogenic effect would be questionable.

Metformin-induced endocrine disruption and oxidative stress of Oryzias latipes on two-generational condition.

Metformin has been treated for diabetes (type 2). Nowadays, this compound is frequently found in ambient water, influent/effluent of a wastewater treatment plant. To evaluate the metformin aquatic toxicity under a multi-generational exposure regimen, we exposed Oryzias latipes to metformin for two generations (133 d) and investigated its adverse effects. In the F0 generation, metformin significantly elevated gene expression for cytochrome P450 19a (CYP19a) and estrogen receptor α (ERα) in male fish; in female fish, the treatment decreased gene expression of vitellogenin (VTG2) and ERß1, suggesting endocrine disruption (one-way ANOVA, p < 0.05). Intersex occurrence of F0 female fish were found in a concentration-dependent manner, whereas no significant changes in fecundity and hatching rate were observed (p < 0.05). Metformin increased the reactive oxygen species (ROS) content, and decreased the glutathione (GSH) content in F0 male fish compared with those of the control (one-way ANOVA, p > 0.05). In F0 female fish, metformin increased catalase activity compared with that of the control (p > 0.05). The results demonstrated that metformin leads to oxidative stress and two-generation endocrine disruption in O. latipes. These results may be useful for better understanding metformin toxicity mechanism.

PFOA-induced metabolism disturbance and multi-generational reproductive toxicity in Oryzias latipes.

The aims of this study were to examine multi-generational reproductive toxicity and metabolism disturbances in Oryzias latipes exposed to 0.3, 3, and 30mg/L PFOA for 259-day. The highest concentration of PFOA suppressed fecundity over three generations from F0 to F2 and sac-fry survival rate in F2 generation, indicating that PFOA resulted in multi-generational reproductive toxicity (p<0.05). Histologically, in F1 and F2 generations, O. latipes exposed to 30mg/L PFOA revealed accelerated gonad development, and the atrophy and degeneration of thyroid follicular cell. Glucose content showed the highest increase in both genders in all metabolites. However, alanine, glutamine, threonine, and lactate content, which are converted into glucose showed decline tendency, suggesting that PFOA led to gluconeogenesis. Change of osmolyte content affecting osmosis such as a decrease of male myo-inositol (m-Ino), an increase of female trimethylamine N-oxide (TMAO) and an increase of male dimethylamine (DMA) suggest that PFOA might affect osmoregulation of O. latipes. Oxaloacetate of male fish and succinate of female fish showed significant alterations, indicating that PFOA may affect energy metabolism differently by sex. These findings will help elucidate the toxicity of PFOA in diverse biological responses including metabolism change.

Multi-generational xenoestrogenic effects of Perfluoroalkyl acids (PFAAs) mixture on Oryzias latipes using a flow-through exposure system.

To elucidate the multi-generational estrogenic potential of Perfluoroalkyl acids (PFAAs) mixture, vitellogenin (VTG) expression, growth indices, histological alteration, fecundity, hatching rate, larval survival rate, and sex ratio of Japanese medaka (Oryzias latipes) were investigated by exposing the fish to a mixture of perfluorooctane sulfonate (PFOS), perfluroroctanoic acid (PFOA), perfluorobutane sulfonate (PFBS), and perfluorononanoic acid (PFNA) for three generations (238 days). Mixture composition is in the ratio of 1:1:1:1. In addition, whole body burden for each PFAA was analyzed. According to the results, concentrated levels of the PFAAs in both F1 and F2 generation O. latipes were ordered PFOS > PFNA > PFOA > PFBS at both low concentration (0.5 µg/L) and high concentration (5 µg/L), whereas a significant difference in whole body burden based on sex or generation was not detected. Significant induction of VTG expression in F2 and the decline of the gonad somatic index (GSI) in F1 were observed following PFAAs mixture exposure (p < 0.05, one-way ANOVA). Furthermore, suppression level of reproduction rate relative to the control increased as generation was transferred to the next in response to PFAAs mixture or 17 ß-estradiol exposure, with the inhibition of hatchability observed in the F1 generation. The PFAA high concentration caused significant alteration of F1 generation sex ratio, suggesting the adverse effect of PFAA in population level (Chi-square test, P > 0.05). Overall, this study demonstrated that PFAA mixture could have the potential of multi-generational endocrine disruptors in O. latipes.

Adverse effects of MWCNTs on life parameters, antioxidant systems, and activation of MAPK signaling pathways in the copepod Paracyclopina nana.

Engineered multi-walled carbon nanotubes (MWCNTs) have received widespread applications in a broad variety of commercial products due to low production cost. Despite their significant commercial applications, CNTs are being discharged to aquatic ecosystem, leading a threat to aquatic life. Thus, we investigated the adverse effect of CNTs on the marine copepod Paracyclopina nana. Additional to the study on the uptake of CNTs and acute toxicity, adverse effects on life parameters (e.g. growth, fecundity, and size) were analyzed in response to various concentrations of CNTs. Also, as a measurement of cellular damage, oxidative stress-related markers were examined in a time-dependent manner. Moreover, activation of redox-sensitive mitogen-activated protein kinase (MAPK) signaling pathways along with the phosphorylation pattern of extracellular signal-regulated kinase (ERK), p38, and c-Jun-N-terminal kinases (JNK) were analyzed to obtain a better understanding of molecular mechanism of oxidative stress-induced toxicity in the copepod P. nana. As a result, significant inhibition on life parameters and evoked antioxidant systems were observed without ROS induction. In addition, CNTs activated MAPK signaling pathway via ERK, suggesting that phosphorylated ERK (p-ERK)-mediated adverse effects are the primary cause of in vitro and in vivo endpoints in response to CNTs exposure. Moreover, ROS-independent activation of MAPK signaling pathway was observed. These findings will provide a better understanding of the mode of action of CNTs on the copepod P. nana at cellular and molecular level and insight on possible ecotoxicological implications in the marine environment.

A microfluidic array for real-time live-cell imaging of human and rodent pancreatic islets.

In this study, we present a microfluidic array for high-resolution imaging of individual pancreatic islets. The device is based on hydrodynamic trapping principle and enables real-time analysis of islet cellular responses to insulin secretagogues. This device has significant advantages over our previously published perifusion chamber device including significantly increased analytical power and assay sensitivity, as well as improved spatiotemporal resolution. The islet array, with live-cell multiparametric imaging integration, provides a better tool to understand the physiological and pathophysiological changes of pancreatic islets through the analysis of single islet responses. This platform demonstrates the feasibility of array-based islet cellular analysis and opens up a new modality to conduct informative and quantitive evaluation of islets and cell-based screening for new diabetes treatments.

Multi-walled carbon nanotubes (MWCNTs) lead to growth retardation, antioxidant depletion, and activation of the ERK signaling pathway but decrease copper bioavailability in the monogonont rotifer (Brachionus koreanus).

To examine the toxic effects of multi-walled carbon nanotubes (MWCNTs) in the marine environment, we first exposed the monogonont rotifer (Brachionus koreanus) to MWCNTs in the presence of copper. The acute toxicity of copper decreased significantly with a decrease in copper bioavailability resulting from MWCNT exposure. Furthermore, we examined the effects of MWCNT exposure on reproductive capacity, population growth rate, growth patterns, antioxidant systems, and mitogen-activated protein kinase (MAPK) activation. Reproductive capacity, population growth rate, and body growth rate were significantly suppressed in B. koreanus in response to 1.3-4mg/L MWCNT exposure. Furthermore, MWCNTs induced the generation of reactive oxygen species (ROS) and decreased the antioxidant enzymatic activities of catalase (CAT) and glutathione reductase (GR). However, the enzymatic activity of glutathione S-transferase (GST) was up-regulated after a 24 h-exposure to 100mg/L MWCNTs. Exposure to 100mg/L MCWNTs induced extracellular signal-regulated kinase (ERK) activation in B. koreanus, suggesting that p-ERK may mediate the adverse effects of MWCNTs in B. koreanus via the MAPK signaling pathway. Our results provide insight into the mechanistic basis of the ecotoxicological effects of MWCNTs in the marine environment.

Effects of multi-walled carbon nanotube (MWCNT) on antioxidant depletion, the ERK signaling pathway, and copper bioavailability in the copepod (Tigriopus japonicus).

Multi-walled carbon nanotubes (MWCNTs) are nanoparticles widely applicable in various industrial fields. However, despite the usefulness of MWCNTs in industry, their oxidative stress-induced toxicity, combined toxicity with metal, and mitogen-activated protein kinase (MAPK) activation have not been widely investigated in marine organisms. We used the intertidal copepod Tigriopus japonicus as a test organism to demonstrate the adverse effects induced by MWCNTs in aquatic test organisms. The dispersion of the MWCNTs in seawater was maintained over 48 h without aggregation. MWCNTs caused a decrease in acute copper toxicity compared to the copper-only group in response to 20 and 100 mg/L MWCNTs, but not in response to 4 mg/L MWCNT, indicating that MWCNT may suppress acute copper toxicity. Reactive oxygen species (ROS) and enzymatic activities of glutathione S-transferase (GST) and catalase were significantly down-regulated in response to 100 mg/L MWCNT exposure. Glutathione (GSH) and glutathione reductase (GR) activity did not change significantly, indicating that MWCNTs may cause failure of the antioxidant system in T. japonicus. However, MWCNT induced extracellular signal-regulated kinase (ERK) activation without p38 and c-jun NH2-terminal kinase (JNK) activation, suggesting that ERK activation plays a key role in cell signaling pathways downstream of CNT exposure. This suggests that this pathway can be used as a biomarker for CNT exposure in T. japonicus. This study provides a better understanding of the cellular-damage response to MWCNTs.

Significance of adverse outcome pathways in biomarker-based environmental risk assessment in aquatic organisms.

In environmental risk assessments (ERA), biomarkers have been widely used as an early warning signal of environmental contamination. However, biomarker responses have limitation due to its low relevance to adverse outcomes (e.g., fluctuations in community structure, decreases in population size, and other similar ecobiologically relevant indicators of community structure and function). To mitigate these limitations, the concept of adverse outcome pathways (AOPs) was developed. An AOP is an analytical, sequentially progressive pathway that links a molecular initiating event (MIE) to an adverse outcome. Recently, AOPs have been recognized as a potential informational tool by which the implications of molecular biomarkers in ERA can be better understood. To demonstrate the utility of AOPs in biomarker-based ERA, here we discuss a series of three different biological repercussions caused by exposure to benzo(a)pyrene (BaP), silver nanoparticles (AgNPs), and selenium (Se). Using mainly aquatic invertebrates and selected vertebrates as model species, we focus on the development of the AOP concept. Aquatic organisms are suitable bioindicator species whose entire lifespans can be observed over a short period; moreover, these species can be studied on the molecular and population levels. Also, interspecific differences between aquatic organisms are important to consider in an AOP framework, since these differences are an integral part of the natural environment. The development of an environmental pollutant-mediated AOP may enable a better understanding of the effects of environmental pollutants in different scenarios in the diverse community of an ecosystem.

Multiwall Carbon Nanotube-Induced Apoptosis and Antioxidant Gene Expression in the Gills, Liver, and Intestine of Oryzias latipes.

Multiwall carbon nanotubes (MWCNTs) have many attractive properties with potential applications in various fields. Despite their usefulness, however, the associated waste can be hazardous to the environment. To examine adverse effects in aquatic environments, Oryzias latipes were exposed to MWCNTs dispersed in water for 14 days and apoptosis and antioxidant gene expression were observed. This work showed that in gills exposed to 100 mg/L MWCNTs for 4 days, there was significant p53, caspase-3 (Cas3), caspase-8 (Cas8), and caspase-9 (Cas9) gene expression relative to the controls, while catalase (CAT) and glutathione-S-transferase (GST) expression were reduced. At 14 days, CAT, GST, and metallothionein (MT) were induced significantly in the gills and Cas3, Cas8, and Cas9 were induced in the liver. No significant gene induction was seen in intestine. Intracellular reactive oxygen species (ROS) were increased significantly only at 14 days. Histologically, no apoptosis was observed with exposure to 100 mg/L MWCNTs for 21 days. The gills were more sensitive to MWCNT toxicity than the other organs. Males had higher apoptosis gene induction than females. These results demonstrated that MWCNTs could cause apoptosis in a manner influenced by tissue and gender in aqueous environments.

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.

Benzo(a)pyrene-induced cytochrome p4501A expression of four freshwater fishes (Oryzias latipes, Danio rerio, Cyprinus carpio, and Zacco platypus).

Oryzias latipes, Danio rerio, Cyprinus carpio, and Zacco platypus are useful indicator species for CYP1A biomarker studies; however, comparative studies have not been performed. To compare susceptibility, dose- and time-dependent CYP1A induction at the mRNA and protein levels in response to benzo(a)pyrene (BaP) exposure was analyzed. At the mRNA level, a statistically significant difference was found among the four species; however, such was not observed at the protein level. C. carpio showed the highest CYP1A induction level and the steepest slope in the dose-response curve. To assess susceptibility, the difference in CYP1A mRNA induction among species must be considered, and C. carpio was the most sensitive species of the four evaluated in terms of CYP1A expression.

Inhibitory effects of biocides on transcription and protein activity of acetylcholinesterase in the intertidal copepod Tigriopus japonicus.

Acetlycholinesterase (AChE) is a serine esterase that plays an important role in the hydrolytic degradation of acetylcholine. We investigated the modulatory potential of T. japonicus-AChE (TJ-AChE) for biocide response by cloning, sequencing, and characterizing the full-length genomic DNA of the TJ-AChE1 and TJ-AChE2 genes. The deduced TJ-AChE proteins were highly conserved across species and were distinctively separated into two subtypes, AChE1 and AChE2. Each TJ-AChE protein was closely phylogenetically clustered with invertebrate AChE1 and AChE2 proteins. Transcriptional level of TJ-AChE1 was higher than TJ-AChE2 in all developmental stages. TJ-AChE1 mRNA decreased in response to five biocides (alachlor, chlorpyrifos, dimethoate, endosulfan, lindane,) but not in the molinate-exposed group. TJ-AChE2 decreased significantly only in response to chlorpyrifos and lindane. TJ-AChE enzymatic activity was significantly inhibited when exposed to alachlor, chlorpyrifos, endosulfan, or lindane for 24 h. This study elucidates potential endogenous mechanisms of biocide-induced neurotoxicity in T. japonicas.

Modulated expression and enzymatic activity of the monogonont rotifer Brachionus koreanus Cu/Zn- and Mn-superoxide dismutase (SOD) in response to environmental biocides.

Superoxide dismutases (SODs) are important antioxidant enzymes whose expression levels are often used as biomarkers for oxidative stress. To investigate the biomarker potential of the monogonont rotifer Brachionus koreanus SOD genes, the full-length Cu/Zn-SOD (Bk-Cu/Zn-SOD) and Mn-SOD (Bk-Mn-SOD) genes were cloned from genomic DNA and characterized. All amino acid residues involved in the formation of tertiary structure and metal binding in Bk-Cu/Zn-SOD and Bk-Mn-SOD were highly conserved across species. Phylogenetic analysis revealed that Bk-Mn-SOD, in particular, was closely clustered with mitochondrial Mn-SOD. Transcript analysis after exposure to six different biocides (alachlor, chlorpyrifos, dimethoate, endosulfan, lindane, and molinate) revealed that the transcriptional level of Bk-Cu/Zn-SOD was significantly increased in a dose-dependent manner. In contrast, the level of Bk-Mn-SOD transcript was significantly increased compared with control cells in response to chlorpyrifos, endosulfan, and molinate at their no observed effect concentrations (NOECs). However, exposure to alachlor, chlorpyrifos, and molinate significantly reduced the enzymatic activity of total SOD protein, while a decreased pattern was observed in all biocide treatments. Taken together, these results indicate that exposure to waterborne environmental biocides induces the transcription of Bk-Cu/Zn-SOD and Bk-Mn-SOD, but inhibits the enzymatic activity of Bk-SODs. These results contribute to our understanding of the modes of action of oxidative stress-mediating biocides on rotifer.

Cytochrome P450 system expression and DNA adduct formation in the liver of Zacco platypus following waterborne benzo(a)pyrene exposure: implications for biomarker determination.

Benzo(a)pyrene (BaP) is a polycyclic aromatic hydrocarbon that causes mutations and tumor formation. Zacco platypus is a sentinel species that is suitable for monitoring aquatic environments. We studied cytochrome P450 system (CYP system) expression and DNA adduct formation in the liver of Z. platypus following waterborne exposure to BaP. The results showed both dose and time dependency. The significant induction levels of CYP system mRNA and protein reached maximums at 2 days and 14 days, respectively, and hepatosomatic index was maximally induced at 4 days during 14 days BaP exposure. DNA adduct formation was significantly induced compared to corresponding controls (t-test, p < 0.01) after 4 days of exposure in 100 µg/L BaP. These results indicate that the only use of mRNA expression level of CYP system as a biomarker make us underestimate prolonged toxicity (4-14 days) of BaP and the only use of protein expression level of CYP system make us underestimate acute toxicity (1-2 days) of BaP. Therefore, we suggests that a combinational use of the mRNA expression level and protein expression level of CYP system, hepatosomatic index is a useful biomarker in risk assessment of waterborne BaP exposure. In addition, DNA adduct formation was a useful biomarker in risk assessment of waterborne BaP exposure at 4 days. CYP1A was a more sensitive biomarker than CYP reductase for BaP exposure when considering both the mRNA and protein level. Furthermore, our results show that Z. platypus is a useful species for assessing the risk of waterborne BaP exposure.

Cyanide-bridged W(V)Mn(III) bimetallic chains composed of a blocked W hexacyanide precursor: geometry-related magnetic couplings and magnetostructural correlation.

Five one-dimensional bimetallic W(V)Mn(III) complexes 1-5, consisting of [W(CN)6(bpy)](-) anions and [Mn(Schiff base)](+) cations, were prepared. The central coordination geometry around each W atom is determined as a distorted dodecahedron (DD) for 1 and 2, and a distorted square antiprism (SAPR) for 3-5. Magnetic analyses demonstrate that compounds 1, 4, and 5 exhibit antiferromagnetic interactions between magnetic centers, which are different from the ferromagnetic couplings in 2 and 3. For the distorted DD geometry, the Mn-N(ax) (ax = axial) bond length increases when moving from 1 to 2, with the Mn-N(ax)-C(ax) angle remaining constant. The elongation of the bond length is responsible for the reduction in orbital overlap and consequent ferromagnetic coupling in 2. In comparison, for 3-5 with the distorted SAPR geometry, given that the Mn-N(ax) bond lengths are similar across all the samples, the increase in the Mn-N(ax)-C(ax) angles accounts for the enhanced magnetic strength. Notably, a correlation between structure and magnetic exchange coupling is established for the first time in W(V)Mn(III) bimetallic systems based on the [W(CN)6(bpy)](-) precursor.

Syntheses, structures, and magnetic properties of cyano- and phenoxide-bridged Fe(III)-Mn(III) tetrameric assemblies formed by blocked fac-Fe tricyanide with aliphatic rings.

Three tetranuclear clusters [(Tp(Me,mt3))Fe(CN)3Mn(L)]2 (1, L = 5-Clacphmen; 2, L = 5-Clsalen; 3, L = 5-MeOsalen; Tp(Me,mt3) = hydrotris(3-methyl-4,5-propylene-5-methylpyrazol-1-yl)borate) were prepared by assembling the fac-Fe tricyanide with the corresponding Mn Schiff bases. The assembled molecules are linked by cyanide and phenoxide bridges. Weak π-π contacts between molecules are evident in these clusters. Compounds 1-3 exhibit slow relaxation of the magnetization. The phenoxide linkers mediate ferromagnetic coupling between the Mn centers, which is ascribed to the long axial Mn-O* length. The overall magnetic exchange coupling nature of the Fe-C≡N-Mn route is accounted for by the important structural parameters of bridging pathways such as Mn-N(cyano) length, Mn-N(cyano)-C(cyano) angle, and C(eq)-Fe···Mn-N(eq) (eq = equatorial) torsion angle.

Designed cyanide- and phenoxide-bridged Fe(III)Mn(III) single-molecule magnet constructed by highly blocked paramagnetic precursors.

A tetranuclear Fe(III)(2)Mn(III)(2) compound was prepared using highly blocked precursors. The well-isolated molecular entity associated with appropriate magnetic anisotropy allows for single-molecule magnet behavior.