Novel electrochemical sensor modified with molecularly imprinted polymers for determination of enrofloxacin in marine environment.

Molecularly imprinted polymers were synthesized by gel-sol method with multi-walled carbon nanotubes as support and enrofloxacin as a template and further modified on the surface of glassy carbon electrode to construct a molecularly imprinted electrochemical sensor. The performance of the imprinted electrochemical sensor was thoroughly investigated by using cyclic voltammetry and differential pulse voltammetry. The influence of imprinted polymers amount, electrolyte pH, and incubation time on the sensor performance was investigated for the detection of enrofloxacin. Under the optimal experimental conditions in a three-electrode system with the modified electrode as the working electrode the differential pulse voltammetry response current of the sensor had a good linear relationship at 0.2 V (vs. saturated calomel reference electrode) with the enrofloxacin concentration within 2.8 pM-28 µM and the limit of detection of the method was 0.9 pM. The competitive interference experiment showed that the imprinted electrochemical sensor could selectively recognize enrofloxacin. The method was applied to analyze spiked natural seawater, fish, and shrimp samples. The recovery was 96.4%-102%, and RSD was less than 4.3% (n = 3), indicating that the proposed imprinted electrochemical sensor was suitable for the determination of trace enrofloxacin in marine environment samples.

CdTe quantum dots coated with a molecularly imprinted polymer for fluorometric determination of norfloxacin in seawater.

A fluorescent nanoprobe consisting of CdTe quantum dots (QDs) and coated with molecularly imprinted layers was prepared and successfully applied to the determination of norfloxacin (NOR) in seawater and wastewater samples. The 3-mercaptopropionic acid capped QDs were prepared and then covered with a protective silica shell. A molecularly imprinted layer was finally synthesized around the silanized QDs using 3-aminopropyltriethoxysilane as functional monomer and norfloxacin as the template. Compared with the non-imprinted polymer, the fluorescence of the nanoprobe with imprinted polymer (measured at excitation/emission wavelengths of 300/596 nm) is strongly reduced in the presence of NOR, and the imprinting factor is 8.8. Under the optimal experimental conditions, the detection limit of the nanoprobe is 0.18 µM, and response is linear between 0.5 - 28 µM of NOR. The relative standard deviation of the detection of NOR is <7.2%. In order to evaluate the practicality of the probe, wastewater and seawater samples spiked with norfloxacin were analyzed by this method, and recoveries ranged from 96.2 - 106.0%. Graphical abstract Schematic presentation and fluorescence spectrum of fluorescent nanoprobe with selectivity for norfloxacin (NOR). CdTe quantum dots (QDs) are used as fluorescent carriers, 3-aminopropyltriethoxysilane (APTES) as template molecules, tetraethyl orthosilicate (TEOS) as crosslinking agent, and aqueous ammonia as initiator.

Determination of sulfadiazine in eggs using molecularly imprinted solid-phase extraction coupled with high-performance liquid chromatography.

The development of a simple and effective method for the isolation and purification of sulfadiazine residues in food of animal origin is of great significance since it is a great danger to human health. An off-line molecularly imprinted solid-phase extraction with high-performance liquid chromatography method was proposed for the selective pretreatment and determination of sulfadiazine in eggs, rapidly and effectively. The molecularly imprinted polymer was proved to have a homogeneous spherical structure and porous surface morphology with excellent adsorption capacity of 5258 µg/g for sulfadiazine. The newly established method showed a good linearity in the range of 0-200 µg/L, low limits of detection (0.06 µg/L), acceptable reproducibility (RSD, 2.60-5.03%, n = 3), and satisfactory relative recoveries (78.22-86.10%). It was demonstrated that the proposed molecularly imprinted solid-phase extraction with high-performance liquid chromatography method could be applied to determine sulfadiazine in eggs, which simplified the pretreatment procedure and improved the accuracy of the analysis process by reducing the loss of sulfadiazine in the fat-removing procedure compared with traditional methods. Molecularly imprinted solid-phase extraction with excellent selectivity and adsorption capacity is a simple, rapid, selective, and effective pretreatment method for the determination of sulfadiazine in egg samples.

Toxic effects of nSiO2 and mPS on diatoms Nitzschia closterium f. minutissima.

To investigate the toxic mechanism of SiO2 nanoparticles (nSiO2) and polystyrene microplastics (mPS) on microalgae Nitzschia closterium f. minutissima, growth inhibition tests were carried out. The growth and biological responses of the algae exposed to nSiO2 (0.5, 1, 2, 5, 10, 30 mg L-1) and mPS (1, 5, 10, 30 and 75 mg L-1) were explored in f/2 media for 96 h. Both micro-/nano-particles (MNPs) inhibited the growth of N. closterium f. minutissima in a concentration- and time-dependent manner. The toxic effect of mPS on N. closterium f. minutissima is higher than that of nSiO2, because silicon is essential for diatoms to maintain cell wall integrity, and the addition of appropriate amounts of nSiO2 can be absorbed and used as a nutrient to promote diatom growth and protect the integrity of the siliceous shell to some extent. Both MNPs induce the production of excess oxidation and activate the cellular antioxidant defense system, leading to increased SOD and CAT activity as a means to resist oxidative damage to the cell, and eliminating excess ROS and maintaining normal cell morphology and metabolism. SEM is consistent with the results of MDA, showing that mPS with high concentrations attach to the surface of algal cells to produce heterogeneous aggregates and disrupt the cell wall and cell membrane, causing the cells to expand and rupture. This study contributes to the understanding of the size effect of MNPs on the growth of marine diatom.

Effects of polystyrene nanoplastics on growth and hemolysin production of microalgae Karlodinium veneficum.

There are few studies on the effects of nanoplastics on growth and hemolysin production of harmful algal bloom species at present. In this study, Karlodinium veneficum was exposed to different concentrations (0, 5, 25, 50, 75 mg/L) of polystyrene nanoplastics (PS-NPs, 100 nm) for 96 h. The effects of PS-NPs on growth of K. veneficum were investigated by measuring algal cell abundance, growth inhibition rate (IR), total protein (TP), malondialdehyde (MDA), glutathione reductase (GSH), superoxide dismutase (SOD), ATPase activity (Na+/K+ ATPase and Ca2+/Mg2+ ATPase). Scanning electron microscope and transmission electron microscope (SEM and TEM) images of microalgae with or without nanoplastics were also observed. The effects of PS-NPs on hemolysin production of K. veneficum were studied by measuring the changes of hemolytic toxin production of K. veneficum exposed to PS-NPs on 1, 3, 5 and 7 days. High concentrations (50 and 75 mg/L) of PS-NPs seriously affected the growth of K. veneficum and different degrees of damage to cell morphology and ultrastructure were found. Excessive free radicals and other oxidants were produced in the cells, which disrupted the intracellular redox balance state and caused oxidative damage to the cells, and the basic activities such as photosynthesis and energy metabolism were weakened. The athletic ability of K. veneficum was decreased, but the ability to produce hemolysin was enhanced. It was suggested that the presence of nanoplastics in seawater may strengthen the threat of harmful algal bloom species to aquatic ecosystems and human health.

Preparation of surface molecularly imprinted polymers with Fe3O4/ZIF-8 as carrier for detection of Dimethoate in cabbage.

In this study, molecularly imprinted polymers (MIPs) were prepared for the specific recognition of organophosphorus pesticides and a rapid, efficient and simple method was established for the detection of dimethoate (DIT) in food samples. Fe3O4 magnetic nanoparticles were synthesized by co-precipitation, and Fe3O4/ZIF-8 complexes were prepared by a modified in-situ polymerization method, and then magnetic molecularly imprinted polymers (MMIPs) were prepared and synthetic route was optimized by applying density functional theory (DFT). The morphological characterization showed that the MMIPs were coarse porous spheres with an average particle size of 50 nm. The synthesized materials are highly selective for the organophosphorus pesticide dimethoate with an adsorption capacity of 461.50 mg·g-1 and are effective resistance to matrix effects. A novel method for the determination of DIT in cabbage was developed using the prepared MMIPs in combination with HPLC. The practical results showed that the method can meet the requirements for the determination of DIT in cabbage with recoveries of 85.6-121.1 % and detection limits of 0.033 µg·kg-1.

Energy metabolism response induced by microplastic for marine dinoflagellate Karenia mikimotoi.

Microplastic contaminations threaten the entire marine ecosystem and cause severe ecological stress. This study explored the energy metabolism change of Karenia mikimotoi under exposure to nanoplastics (NPs) and microplastics (MPs) (65 nm, 100 nm, and 1 µm polystyrene (PS), and 100 nm polymethyl methacrylate (PMMA)) at a concentration of 10 mg L-1. Membrane potential, esterase activity, polysaccharide content, and ATPase activity were detected to assess the energy metabolism of K. mikimotoi under MPs/NPs exposure. Transcriptome and metabolomic analyses were used to investigate the intrinsic mechanisms of energy metabolism changes. Smaller PS particles caused greater damage to the cell membrane potential, increased the polysaccharide content, and resulted in a heavier weakening of the ATPase enzymatic activity in K. mikimotoi cells, suggesting that smaller-sized PS had more influence on esterase activity and energy metabolism than the bigger-sized PS. The results evidenced that energy metabolism relates to the size and type of MPs/NPs, and nano-scale plastic particles could induce greater metabolic changes.

Highly Efficient Selective Extraction of Chlorpyrifos Residues from Apples by Magnetic Microporous Molecularly Imprinted Polymer Prepared by Reversible Addition-Fragmentation Chain Transfer Surface Polymerization.

Chlorpyrifos, as a moderate toxic organophosphorus pesticide, is prone to lingering in the environment and cannot be monitored easily. In this study, a magnetic, microporous, molecularly imprinted polymer was synthesized by using the reversible addition-fragmentation chain transfer polymerization method. The synthesized materials were properly characterized in terms of morphology, selectivity, and sorption capacity and used as sorbents for magnetic solid phase extraction for the selective determination of chlorpyrifos in apple samples. Results showed that the magnetic microporous molecularly imprinted materials were rough and porous spheres at an average size of 5 nm. The materials were highly selective toward chlorpyrifos with a superior sorption capacity of 167.99 mg·g-1 and were resistant to the interference of competitive pollutants. After optimization, the recoveries of chlorpyrifos reached 96.2-106.5%, and the detection limit was 0.028 µg·kg-1 by HPLC. Based on these analytical validation results, the developed method could be effective at determining chlorpyrifos in apples.

Toxic effects of pristine and aged polystyrene and their leachate on marine microalgae Skeletonema costatum.

The acute toxic effects of pristine and aged polystyrene (P-PS and A-PS) and their leaching solutions (L-PS) on microalgae Skeletonema costatum were investigated by measuring algal density and growth inhibition rate (IR), chlorophyll concentration and photosynthetic efficiency (Fv/Fm) over 96 h. Total protein (TP), superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) were measured to analyze the oxidative damage to microalgae by microplastics and their leachates. Hydrodynamic diameter of microplastics in seawater, FITR and SEM images were used to study the changes of polystyrene during aging. The interaction of algae cell with microplastics and the cellular ultrastructure changes of cells were analyzed combined with electron microscopy for a comprehensive and systematic understanding on the mechanisms of microplastic toxicity to microalgae. Both high concentration and small size of PS had significant inhibitory effect on the growth of microalgae, and the inhibitory effect was greater with increasing exposure time. The inhibition effect of aged microplastics was more obvious, which was speculated to be caused by the synergistic effect of aged PS itself and leaching solution. The negative effect of leaching solution on microalgae was due to the release of some additives during the aging process. The content of MDA reached the highest value of 54.41 nmol/mgprot in 1.0 µm 50 mg/L A-PS treatment group, and A-PS were found to be more prone to heterogeneous aggregation with algae cells by SEM.

Is hydrodynamic diameter the decisive factor? - Comparison of the toxic mechanism of nSiO2 and mPS on marine microalgae Heterosigma akashiwo.

To investigate the toxic mechanism of SiO2 nanoparticles (nSiO2) and polystyrene microplastics (mPS) on microalgae Heterosigma akashiwo, growth inhibition tests were carried out. The growth and biological responses of the algae exposed to nSiO2 (0.5, 1, 1.5, 2, 5, 10 and 30 mg L-1) and mPS (1, 2, 5, 10, 30 and 75 mg L-1) were explored in f/2 media for 96 h. It was found that the hydrodynamic diameter of the particles seems to be one of the more important factors to influence the algae. nSiO2 and mPS with similar hydrodynamic diameters have the similar toxic mechanism on H. akashiwo, and the effects were dose- and time-dependent. High concentrations of micro-/nano-particles (MNPs) could inhibit the growth of algal cells, however, low concentrations of MNPs did not restrict or even promoted the growth of algae, known as "Hormesis" phenomenon. The 96 h-EC20 values of nSiO2 and mPS on H. akashiwo were 2.69 and 10.07 mg L-1, respectively, and chlorophyll fluorescence parameters indicated that the microalgal photosynthetic system were inhibited. The hydrophilic surface of nSiO2 increased the likelihood of nSiO2 binding to the hydrophilic functional group of microalgae, which may account for the slightly stronger toxic effect of nSiO2 than mPS. The algae continued to produce reactive oxygen species (ROS) under stress conditions. Total protein (TP) levels reduced, and superoxide dismutase (SOD) and catalase (CAT) levels increased to maintain ROS levels in the cells. The decrease in adenosine triphosphate (ATPase) indicated an impact on cellular energy metabolism. Cell membrane damage, cytoplasm and organelle efflux under stress were confirmed by scanning and transmission electron microscopy (SEM and TEM) images. This study contributes to the understanding of the size effect of MNPs on the growth of marine microalgae.

Microplastic-induced apoptosis and metabolism responses in marine Dinoflagellate, Karenia mikimotoi.

Microplastics (MPs) occur widely in marine environments, and disturb the balance of aquatic ecosystems. In this study, programmed cell apoptosis in marine dinoflagellate, Karenia mikimotoi exposed to 10 mg L-1 micro/nanoplastics (MPs/NPs; polystyrene and polymethyl methacrylate) for 72 h was assessed. Prior to the toxicity assay, MPs/NPs were dialyzed to remove possible additives. Cell viability, membrane integrity, cell apoptosis, and total DNA concentration were measured to assess programmed cell apoptosis in K. mikimotoi following exposure to MPs/NPs. A transcriptome analysis was used to explore the potential toxic mechanism of MPs to K. mikimotoi. Programmed cell apoptosis was related to the size of MPs/NPs, and NPs could more easily impair cell viability, and reduced cell membrane integrity and DNA concentration. NP particles caused continuous apoptosis of K. mikimotoi compared to MP particles. Size had the greatest effect on toxicity in K. mikimotoi. In conclusion, the results evidenced that both MPs and NPs have a negative impact on the marine dinoflagellate, K. mikimotoi. However, NPs were more harmful to K mikimotoi than MPs, highlighting the potential ecological problems associated with exposure to NPs.

The effects and mechanisms of polystyrene and polymethyl methacrylate with different sizes and concentrations on Gymnodinium aeruginosum.

In this study, Gymnodinium aeruginosum was exposed to polystyrene (PS) and polymethyl methacrylate (PMMA) of three particle sizes (0.1 µm, 1.0 µm and 100 µm) and two concentrations (10 mg/L and 75 mg/L) for 96 h. The density of algae cells, the endpoints that reactive oxygen species (ROS), total protein (TP), malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT), scanning and transmission electron microscopy (SEM and TEM) were used to explore the toxicity mechanism to the microalgae. At a concentration of 75 mg/L, the 96 h inhibition ratios (IR) with particle sizes of 0.1 µm, 1.0 µm and 100 µm on G. aeruginosum were 55.9%, 63.7% and 6.0% for PS, respectively, and 3.0%, 4.1% and -0.6% for PMMA, respectively. The most significant changes in ROS, TP, MDA, SOD and CAT were observed at 75 mg/L 1.0 µm of PS when treated for 96 h. When exposed to nanoplastics (NPs) and microplastics (MPs), the algae cells were damaged, and the antioxidant system was activated. Extracellular polymeric substance (EPS) could help to detoxify the algae. In general, PS was more toxic than PMMA. The toxicity of small MNPs (0.1 µm and 1.0 µm) was related to the concentrations, while large MNPs (100 µm) did not.

Size-dependent oxidative stress effect of nano/micro-scaled polystyrene on Karenia mikimotoi.

The effects of polystyrene (PS) of different sizes of diameter (65 nm, 100 nm and 1 µm) with different treat concentrations (control, 1 mg L-1 and 10 mg L-1) on growth and oxidative stress for K. mikimotoi were assessed across PS short-term exposures (3 d) and long-term exposures (13 d). The endpoints of physiological parameters such as cell density, antioxidant enzyme activity of SOD and CAT, the content of MDA and ROS level were monitored. The results showed that the CAT activity, SOD activity, MDA content and the relative ROS level reached to 377 U mgprot-1, 164 U mgprot-1, 157 nmol mgprot-1 and 10.8% when treated with 10 mg L-1 PS of 65 nm diameter; the CAT activity, SOD activity and MDA content in single K. mikimotoi reached to 0.46 U mgprot-1, 0.36 U mgprot-1 and 0.16 nmol mgprot-1 under 10 mg L-1 PS of 65 nm diameter on the third day. The relative ROS level in single K. mikimotoi was 71% under 10 mg L-1 PS of 100 nm diameter on the 13th day. The works found that the size of nano/micro-PS was a key factor that cannot be ignored. Smaller size had more serious negative effects on the growth, oxidative stress and cell microstructure. The potential cytotoxicity mechanisms were that monodisperse nanoscaled PS crossed the biological barriers and the agglomerate nanoparticles caused physical blockage, while microscaled PS may not have such an equally strong negative effects. Visualized SEM images also proved that exposing to nano/micro-PS of varies diameters led to apparent size-dependent effects. The arms race of systematic oxidative defensive and offensive between K. mikimotoi and nano/micro-PS would have considerable value in deliberating the relationship between nano/microplastics and marine phytoplankton.

Growth inhibition of the microalgae Skeletonema costatum under copper nanoparticles with microplastic exposure.

In order to investigate the combined toxicities of copper nanoparticles (nano-Cu) with microplastic on microalgae Skeletonema costatum, growth inhibition tests were carried out. The toxic effects of copper nanoparticles and microplastic on the microalgae under singleness and coexistence conditions were investigated. Both copper nanoparticles and microplastic inhibited the growth of S. costatum. The growth inhibition ratio (IR) increased with the increasing of particle concentrations and incubation time. The toxicity of copper nanoparticles was reduced with the addition of microplastic. The concentrations of Cu2+ in the medium with or without addition of microplastic were determined. It was found that adsorption of Cu2+ on microplastic and aggregation between copper nanoparticles and microplastic are the main reasons for attenuation of toxicity of nano-Cu with adding microplastic. The adhesion and aggregate interactions between microalgae and nanomaterial were also approved by the observations through scanning electron microscopy.

The interactions between micro polyvinyl chloride (mPVC) and marine dinoflagellate Karenia mikimotoi: The inhibition of growth, chlorophyll and photosynthetic efficiency.

Microplastics pose a great threat to entire marine ecosystems, but little is known about their impacts on phytoplankton, especially for the harmful dinoflagellates. In this study, effects of micro polyvinyl chloride (mPVC) on the growth, chlorophyll content and photosynthetic efficiency of the dinoflagellate Karenia mikimotoi at different periods (0, 24, 48, 72 and 96 h) were assessed using gradient concentrations (0, 5, 25, 50 and 100 mg L-1) of mPVC with a size of 1 µm. PVC microplastics had dose-dependent adverse effects on K. mikimotoi growth, chlorophyll content and photosynthetic efficiency. The density of algal cell decreased with increasing mPVC concentrations and the highest inhibitory rate (IR) was 45.8% at 24 h under 100 mg L-1 of mPVC. The total chlorophyll content and chlorophyll content in a single algal cell decreased at 96 h and the ФPSⅡ and Fv/Fm decreased 25.3% and 17.1%, respectively. The SEM images provided an intuitive visual method to observe the behaviors and interactions between microplastics and microalgae. It was found from the SEM images that microalgae was wrapped by microplastic beads. The physical blockage and aggregation were also responsible for the cytotoxicity of K. mikimotoi. Our study clarified that PVC microplastics can reduce algal growth, chlorophyll content and photosynthetic efficiency, and it is beneficial to evaluate the possible impact of plastics on aquatic ecosystems.

Application of molecular imprinting polymer anchored on CdTe quantum dots for the detection of sulfadiazine in seawater.

A molecularly imprinted polymer (MIP) anchored on the surface of CdTe quantum dots (QDs) was fabricated and used as a fluorescent probe for sulfadiazine (SDZ) detection in seawater. CdTe QDs was used as photoluminescent material, SDZ as the template, 3-aminopropyltriethoxysilane (APTES) as the functional monomer and tetraethyl orthosilicate (TEOS) as the cross-linking agent. Characterizations of MIP-QDs were analyzed by Fourier transform infrared (FT-IR), Transmission electron microscopy (TEM) and Scanning electron microscope (SEM). The conditions were optimized for the detection of MIP-QDs to SDZ. The mechanism of fluorescence quenching was studied by UV-Vis absorption spectroscopy and fluorescence spectroscopy. Under optimal conditions, the fluorescence intensity of MIP-QDs decreased linearly between 4- and 20 µM SDZ with a good correlation coefficient of 0.995. The limit of detection is 0.67 µM and the recovery is between 91.8 and 109.4% with RSD lower than 3.9%. These results indicated that MIP-QDs for SDZ detection in seawater was developed successfully.

Separation and detection of trace atrazine from seawater using dummy-template molecularly imprinted solid-phase extraction followed by high-performance liquid chromatography.

A novel sample pretreatment strategy for separation and detection of atrazine from seawater was established with molecular imprinting solid-phase extraction (MISPE). Cyromazine was used as dummy-template based on computational analysis to synthesize dummy-template molecularly imprinted polymers (DMIPs) as sorbent of MISPE for atrazine analysis. The DMIPs were irregular loose porous layered structure characterized by scanning electron microscopy and showed higher binding capacity than non-imprinted polymers through adsorption experiments. An offline MISPE procedure using DMIPs as sorbent coupled with high-performance liquid chromatograph was developed for separation and purification of atrazine from seawater samples. The recoveries of atrazine in the spiked seawater samples ranged from 86.7% to 98.6%, and the relative standard deviation was less than 4.07% (n = 3) under optimal conditions indicating that the proposed method was suitable for the detection of trace residual atrazine in seawater. In addition, no atrazine was detected in three seawater samples from Jiaozhou bay, China.

Multipoint recognition of domoic acid from seawater by dummy template molecularly imprinted solid-phase extraction coupled with high-performance liquid chromatography.

Due to the high cost of domoic acid (DA), different carboxylic acid compounds including indole-3-acetic acid (IAA), pyrrole-2-formic acid (PFA), pyridine-2,3-dicarboxylic anhydride (PDA), trimesitinic acid (TA) and citric acid (CA) were investigated as dummy templates for the molecularly imprinted solid-phase extraction (MISPE) for selective isolation and pre-concentration of an amnesic shellfish poison (ASP), DA. The highest binding amount of the polymers towards DA was obtained when CA was used as dummy template owing to its high hydrophilicity. In addition, the "four-point" recognition site constructed by three COOH groups and a OH group in CA was also speculated to be the reason for the high binding amount of CA-MIPs and the rebinding of DA can be depend on the three COOH groups and a NH group with conformational change in the recognition process. Finally, the CA-MISPE column was chosen for DA isolation and pre-concentration and effective result was obtained with recoveries higher than 90% and relative standard deviation (RSD) less than 5% (n=3). This new polymer can be effectively applied to the monitoring and predicting the existence of trace DA.

Toxic effects of microplastic on marine microalgae Skeletonema costatum: Interactions between microplastic and algae.

To investigate toxic effects of microplastic on marine microalgae Skeletonema costatum, both algal growth inhibition test and non-contact shading test were carried out, and algal photosynthesis parameters were also determined. The SEM images were used to observe interactions between microplastic and algae. It was found that microplastic (mPVC, average diameter 1 µm) had obvious inhibition on growth of microalgae and the maximum growth inhibition ratio (IR) reached up to 39.7% after 96 h exposure. However, plastic debris (bPVC, average diameter 1 mm) had no effects on growth of microalgae. High concentration (50 mg/L) mPVC also had negative effects on algal photosynthesis since both chlorophyll content and photosynthetic efficiency (ΦPSⅡ) decreased under mPVC treatments. Shading effect was not one reason for toxicity of microplastic on algae in this study. Compared with non-contact shading effect, interactions between microplastic and microalage such as adsorption and aggregation were more reasonable explanations for toxic effects of microplastic on marine microalgae. The SEM images provided a more direct and reasonable method to observe the behaviors of microplastic.