Tributyltin-induced oxidative stress causes developmental damage in the cardiovascular system of zebrafish (Danio rerio).

Tributyltin (TBT) can be used as an antifouling agent with anticorrosive, antiseptic and antifungal properties and is widely used in wood preservation and ship painting. However, it has recently been found that TBT can be harmful to aquatic organisms. In this study, to gain insight into the effects of TBT with respect to the development of the cardiovascular system in zebrafish embryos, zebrafish embryos were exposed to different concentrations of TBT solutions (0.2 µg/L, 1 µg/L, and 2 µg/L) at 2 h post-fertilization (hpf) TBT exposure resulted in decreased hatchability and heart rate, deformed features such as pericardial edema, yolk sac edema, and spinal curvature in zebrafish embryos, and impaired heart development. Expression of cardiac development-related genes (vmhc, myh6, nkx2.5, tbx5a, gata4, tbx2b, nppa) is dysregulated. Transgenic zebrafish Tg (fli1: EGFP) were used to explore the effects of TBT exposure on vascular development. It was found that TBT exposure could lead to impaired development of intersegmental vessels (ISVs), common cardinal vein (CCV), subintestinal vessels (SIVs) and cerebrovascular. The expression of vascular endothelial growth factor (VEGF) signaling pathway-related genes (flt1, flt4, kdr, vegfa) was downregulated. Biochemical indices showed that ROS and MDA levels were significantly elevated and that SOD and CAT activities were significantly reduced. The expression of key genes for prostacyclin synthesis (pla2, ptgs2a, ptgs2b, ptgis, ptgs1) is abnormal. Therefore, it is possible that oxidative stress induced by TBT exposure leads to the blockage of arachidonic acid (AA) production in zebrafish embryos, which affects prostacyclin synthesis and consequently the normal development of the heart and blood vessels in zebrafish embryos.

Proteomic analysis of exosomes derived from fatty hepatocytes of grass carp.

Exosomes participate in intercellular communication by carrying proteins, messenger RNA, microRNAs, and non-coding RNA. Fatty liver is a common phenomenon in farmed fish, but there has been little study of fatty hepatocytes-derived exosomes. Here, we successfully isolated exosomes from hepatocytes of grass carp, named Exos (hepatocytes-derived exosomes) and OA-Exos (fatty hepatocytes-derived exosomes), from which 617 differentially expressed proteins were identified using liquid chromatography tandem mass spectrometry. Of these, 320 proteins were promoted and 297 proteins were restrained, which were gathered in biological processes and cellular components (cellular processes, cells, and intracellular structures). The results of kyoto encyclopedia of genes and genomes (KEGG) analysis revealed that the differential expression proteins were gathered in "carbohydrate transport and metabolism", "translation, ribosomal structure and biogenesis", "posttranslational modification, protein turnover, chaperones", and "intracellular trafficking, secretion, and vesicular transport". In addition, five differentially expressed exosomal proteins were further confirmed by parallel reaction monitoring, including 2-phospho-D-glycerate hydrolyase, cytochrome b5, fatty acid-binding protein domain-containing protein, metallothionein, and malate dehydrogenas, which were downregulated. These findings provided evidence that exosomes derived from fatty hepatocytes of grass carp may be biomarkers for the early diagnosis, treatment, and prevention of fatty liver in fishery development.

Regulation of lipid metabolism in grass carp primary hepatocytes by exosomes derived from fatty hepatocytes though GRP78.

Exosomes regulate lipid metabolism by carrying miRNAs, nucleic acids, and proteins, thereby influencing the function of receptor cells. Glucose-regulated protein 78 (GRP78) is also involved in the regulation of lipid metabolism. However, it remains unclear whether exosomes derived from fatty hepatocytes (OA-Exo) regulate lipid metabolism through the enrichment of GRP78. In this study, we observed the expression of GRP78 was significantly increased in fatty hepatocytes (incubating hepatocytes with oleic acid (OA) for 24 h) and OA-Exo (P < 0.05). In addition, OA-Exo (50 µg/mL) and GRP78 protein (1 µg/mL) significant increased the content of triacylglycerol (TG) and total cholesterol (TC), as well as up-regulated the expression of GRP78 and inositol-requiring enzyme-1alpha (IRE1α) protein (P < 0.05). We further used YUM70 (an inhibitor of GRP78) to inhibit endogenous GRP78, and compared with the YUM70 group, OA-Exo reversed the effect of YUM70 and increased the content of TG, TC, and the expression of GRP78 protein in hepatocytes (P < 0.05). Furthermore, the inhibition of the IRE1α pathway with 4µ8C resulted in a significant decrease in TG content compared to the control group (P < 0.05). However, when compared with the 4µ8C group, OA-Exo and GRP78 reversed the effect of 4µ8C and significantly increased TG content (P < 0.05). Taken together, these results indicated that OA-Exo activated IRE1α to promote lipid accumulation in hepatocytes through the enrichment of GRP78. This study provided a new perspective for further exploration of exosomal lipid metabolism in fish.

Determining the Potential Roles of Branched-Chain Amino Acids in the Regulation of Muscle Growth in Common Carp (Cyprinus carpio) Based on Transcriptome and MicroRNA Sequencing.

Branched-chain amino acids (BCAAs) can be critically involved in skeletal muscle growth and body energy homeostasis. Skeletal muscle growth is a complex process; some muscle-specific microRNAs (miRNAs) are involved in the regulation of muscle thickening and muscle mass. Additionally, the regulatory network between miRNA and messenger RNA (mRNA) in the modulation of the role of BCAAs on skeletal muscle growth in fish has not been studied. In this study, common carp was starved for 14 days, followed by a 14-day gavage therapy with BCAAs, to investigate some of the miRNAs and genes that contribute to the regulation of normal growth and maintenance of skeletal muscle in response to short-term BCAA starvation stress. Subsequently, the transcriptome and small RNAome sequencing of carp skeletal muscle were performed. A total of 43,414 known and 1,112 novel genes were identified, in addition to 142 known and 654 novel miRNAs targeting 22,008 and 33,824 targets, respectively. Based on their expression profiles, 2,146 differentially expressed genes (DEGs) and 84 differentially expressed miRNA (DEMs) were evaluated. Kyoto Encyclopedia of Genes and Genome pathways, including the proteasome, phagosome, autophagy in animals, proteasome activator complex, and ubiquitin-dependent protein catabolic process, were enriched for these DEGs and DEMs. Our findings revealed the role of atg5, map1lc3c, ctsl, cdc53, psma6, psme2, myl9, and mylk in skeletal muscle growth, protein synthesis, and catabolic metabolism. Furthermore, miR-135c, miR-192, miR-194, and miR-203a may play key roles in maintaining the normal activities of the organism by regulating genes related to muscle growth, protein synthesis, and catabolism. This study on transcriptome and miRNA reveals the potential molecular mechanisms underlying the regulation of muscle protein deposition and provides new insights into genetic engineering techniques to improve common carp muscle development.

Molecular identification of grass carp igfbp2 and the effect of glucose, insulin, and glucagon on igfbp2 mRNA expression.

The GH (growth hormone)/IGFs (insulin-like growth factors) system has an important function in the regulation of growth. In this system, IGFBPs play a crucial regulatory role in IGF functions. As a member of the IGFBP family, IGFBP2 can bind to IGF and regulate IGF functions to regulate development and growth. In addition, IGFBP2 shows key regulatory functions in cell proliferation and metabolism. In this study, the igfbp2 gene was cloned from grass carp (Ctenopharyngodon idellus) liver. The ORF of grass carp igfbp2 is 834 bp long and encodes 277 amino acids. The tissue distribution results showed that igfbp2 is expressed in multiple tissues in grass carp and has a high expression level in the liver. In the OGTT, igfbp2 expression was significantly decreased in the liver and brain after 6 h of treatment with glucose. In vitro, igfbp2 expression in grass carp's primary hepatocytes was significantly suppressed by insulin after treatment for 6 and 12 h. Moreover, igfbp2 expression was markedly increased in a dose-dependent manner with glucagon incubation in grass carp's primary hepatocytes. To the best of our knowledge, this is the first report about Igfbp2 in grass carp. These results will provide a basis for the in-depth study of grass carp Igfbp2.

Tissue distribution of olive flounder VDAC2 and its expression in fish cell lines.

Voltage-dependent anion channel (VDAC) is located in the mitochondrial outer membrane, which plays a crucial role in regulating cell life and death. In this study, the tissue distribution of olive flounder Paralichthys olivaceus VDAC2 (PoVDAC2) was detected by quantitative real-time PCR and Western blot analysis. The qRT-PCR results showed that the expression level of PoVDAC2 was abundant in heart, muscle and gill tissues. Western blot analysis revealed a protein of 32 kDa detected in all six tissues. Furthermore, a recombinant eukaryotic expression plasmid pEGFP-N3-PoVDAC2 was successfully constructed and transiently expressed the fusion protein in fish cell lines. Subcellular localization indicated that PoVDAC2-GFP was distributed in a punctate mitochondria-like pattern throughout the cytoplasm in flounder embryonic cells (FEC). The distribution of native VDAC2 in untransfected fish cells was also investigated by immunofluorescence microscopy. The punctate VDAC2 fluorescence signals of both FEC and EPC cells were identically observed in the cytoplasm but not in the nucleus. These results laid a foundation for investigating the functional relevance of VDAC response to pathogens in flounder.

Multiple components are integrated to determine leaf complexity in Lotus japonicus.

Transcription factors and phytohormones have been reported to play crucial roles to regulate leaf complexity among plant species. Using the compound-leafed species Lotus japonicus, a model legume plant with five visible leaflets, we characterized four independent mutants with reduced leaf complexity, proliferating floral meristem (pfm), proliferating floral organ-2 (pfo-2), fused leaflets1 (ful1) and umbrella leaflets (uml), which were further identified as loss-of-function mutants of Arabidopsis orthologs LEAFY (LFY), UNUSUAL FLORAL ORGANS (UFO), CUP-SHAPED COTYLEDON 2 (CUC2) and PIN-FORMED 1 (PIN1), respectively. Comparing the leaf development of wild-type and mutants by a scanning electron microscopy approach, leaflet initiation and/or dissection were found to be affected in these mutants. Expression and phenotype analysis indicated that PFM/LjLFY and PFO/LjUFO determined the basipetal leaflet initiation manner in L. japonicus. Genetic analysis of ful1 and uml mutants and their double mutants revealed that the CUC2-like gene and auxin pathway also participated in leaflet dissection in L. japonicus, and their functions might influence cytokinin biogenesis directly or indirectly. Our results here suggest that multiple genes were interplayed and played conserved functions in controlling leaf complexity during compound leaf development in L. japonicus.

Genome-wide comparative identification and analysis of membrane-FADS-like superfamily genes in freshwater economic fishes.

Membrane fatty acid desaturase (FADS)-like superfamily proteins (FADSs) are essential for the synthesis of unsaturated fatty acids (UFAs). Recently, studies on FADS in fishes have mostly focused on marine species, and a comprehensive analysis of the FADS superfamily, including the FADS, stearoyl-CoA desaturase (SCD), and sphingolipid delta 4-desaturase (DEGS) families, in freshwater economic fishes is urgently required. To this end, we conducted a thorough analysis of the number, gene/protein structure, chromosomal location, gene linkage map, phylogeny, and expression of the FADS superfamily. We identified 156 FADSs genes in the genome of 27 representative species. Notably, FADS1 and SCD5 were lost in most freshwater fish and other teleosts. All FADSs proteins contain 4 transmembrane helices and 2-3 amphipathic α-helices. FADSs in the same family are often linked on the same chromosome; moreover, FADS and SCD or DEGS are frequently collocated on the same chromosome. In addition, FADS, SCD, and DEGS family proteins share similar evolutionary patterns. Interestingly, FADS6, as a member of the FADS family, exhibits a similar gene structure and chromosome location to that of SCD family members, which may be the transitional form of FADS and SCD. This study shed light on the type, structure, and phylogenetic relationship of FADSs in freshwater fishes, offering a new perspective into the functional mechanism analysis of FADSs.

Assessment of the therapeutic potential of probiotics against carbon quantum dots-induced neurotoxicity in common carp (Cyprinus carpio).

Carbon quantum dots (CQDs) have received increasing attention in recent years for their potential toxicity. However, little is known about their neurobehavioral toxicity. This study aimed to investigate the potential mechanisms by which probiotics reduce CQDs neurotoxicity from a brain-gut axis perspective by exposing carp to CQDs and/or probiotics for five weeks. The results showed that CQDs accumulation in the brain reduces the expression of blood-brain-barrier (BBB) related genes in carp, leading to brain damage. In addition, CQDs impaired motor behavior and inhibited acetylcholinesterase activity. These abnormalities were alleviated by probiotic supplementation. Microbiomic analysis showed that probiotics improved the imbalance of intestinal flora caused by CQDs and increased the abundance of Firmicutes. Serum metabolomic analysis showed that probiotic supplementation restored the abnormal metabolic levels associated with neurological, inflammatory, and apoptotic cell death caused by CQDs. Overall, probiotic supplementation improved the CQDs-induced changes in brain damage, gut microbiology, and systemic metabolism. These results suggests that CQDs may cause neurotoxicity via the brain-gut microbial axis.

Long-term exposure to polyethylene microplastics and glyphosate interferes with the behavior, intestinal microbial homeostasis, and metabolites of the common carp (Cyprinus carpio L.).

Polyethylene microplastics (PE-MPs) and glyphosate (GLY) occur widely and have toxic characteristics, resulting in increased research interest. In this study, common carp were used to assess the individual and combined toxicity of PE-MPs (0, 1.5, or 4.5 mg/L) and GLY (0, 5, or 15 mg/L) on the brain-gut axis. After 60 days of exposure, the developmental toxicity, blood-brain barrier (BBB), locomotor behavior, intestinal barrier (physical barrier, chemical barrier, microbial barrier), and intestinal content metabolism of common carp were evaluated. Results showed that 15 mg/L of GLY exposure significantly reduced the mRNA expression of tight-junction genes (occludin, claudin-2, and ZO-1) in the brain, and acetylcholinesterase (AChE) activity was clearly inhibited by high concentrations of GLY. However, different concentrations of PE-MPs had no significant effect on the activity of AChE. Furthermore, the free-swimming behavior of common carp was distinctly inhibited by treatment with a combination of 15 mg/L GLY and 4.5 mg/L PE-MPs. Histological studies indicated that PE-MPs alone and in combination with GLY could disrupt the physical and chemical intestinal barriers of common carp. Additionally, the abundance and diversity of gut microbiota in common carp were significantly changed when exposed to a combination of PE-MPs and GLY. Metabolomics further revealed that PE-MPs combined with GLY triggered metabolic changes and that differential metabolites were related to amino acid and lipid metabolism. These findings illustrate that exposure to PE-MPs or GLY alone is toxic to fish and results in physiological changes to the brain-gut axis. This work offers a robust analysis to understand the mechanisms underlying GLY and MP-induced aquatic toxicity.

Bisphenol AF induces multiple behavioral and biochemical changes in zebrafish (Danio rerio) at different life stages.

As common environmental endocrine-disrupting chemicals (EDCs), bisphenol AF (BPAF) raises potential concerns for aquatic organisms due to its widespread presence and continued release in the aquatic environment. This research aimed to use zebrafish embryos and adult fish to explore the effects of environmentally relevant concentrations (5 µg/L), 50 µg/L and 500 µg/L of BPAF on zebrafish embryonic development, behavioral alterations, and the potential mechanisms driving these effects. The results showed that 500 µg/L of BPAF severely affected the growth and development of embryos. In behavioral experiments, all concentrations of BPAF significantly inhibited the locomotor activity of larvae, 50 and 500 µg/L BPAF significantly altered the anxiety-like and aggressive behavior of adult zebrafish. Furthermore, environmentally relevant concentrations and higher concentrations of BPAF induced varying degrees of oxidative stress in both embryonic and adult fish. The most significant histopathological changes and decreased acetylcholinesterase (AChE) activity were observed in the brain at 50 and 500 µg/L of BPAF. We hypothesized that oxidative stress is an important cause of behavioral disturbances in larvae and adult fish. To our best knowledge, the present experiment is a pioneer in studying the effects of BPAF on a variety of complex behaviors (swimming performance, anxiety-like, social behavior, aggression) in zebrafish, which emphasizes the potential health risk of higher concentrations of BPAF in terms of induced neurotoxicity.

Toxic effects of glyphosate on the intestine, liver, brain of carp and on epithelioma papulosum cyprinid cells: Evidence from in vivo and in vitro research.

Glyphosate (GLY) is the most widely used organophosphorus herbicide in agriculture. The present study aimed to analyze the comprehensive toxicological effects of GLY on juvenile common carp and an epithelioma papulosum cyprinid (EPC) cell line. In the in vivo experiments, exposure to GLY (5 and 15 mg/L) for 30 days induced liver inflammation and oxidative damage in common carp and changed the physical barrier of the intestine. Histopathological analysis of the intestine, liver, brain, and changes in oxidative stress biomarkers provided evidence of damage and immune system responses to GLY. Moreover, an inhibitory effect of 15 mg/L GLY on acetylcholinesterase (AChE) activity was found in the brain, which may be an important reason for the significant decrease in both swimming distance and average acceleration of common carp. Cell experiments showed that 0.65 and 3.25 mg/L GLY inhibited the viability of EPCs. Furthermore, oxidative DNA damage, mitochondrial dysfunction, and reactive oxygen species (ROS) production were observed in EPC cells following GLY exposure. Taken together, this study not only highlights the negative effects of GLY on common carp but also enriches the knowledge of the cytotoxicity mechanism to further clarify the comprehensive toxicity of GLY in common carp.

The effects of fipronil exposure on oxidative stress, non-specific immunity, autophagy, and apoptosis in the common carp.

The increase in the area treated with the insecticide fipronil has caused concern for aquatic organisms such as fish. Here, we assessed the effect of fipronil on carp indexes of non-specific immunity, oxidative stress, autophagy, and apoptosis following exposure to 0.074 mg/L and 0.185 mg/L of fipronil in the aqueous environment for 1 day, 3 days, 5 days, and 7 days. It was found that glutathione (GSH), malonaldehyde (MDA), and superoxide dismutase (SOD) in gills were significantly reduced (P < 0.05). The increase in exposure time increases the impact on GSH, SOD, and MDA parameters in the liver and intestine. Liver acid phosphatase (ACP), alkaline phosphatase (AKP), and lysozyme (LZM) activity levels increased significantly in the treatment group on the first day after exposure, except for the 0.074 mg/L group of ACP (P < 0.05). The mRNA expression levels of autophagy-related genes ATG12, ATG5, ATG16L, LC3-II, and BECN1 were generally elevated in the liver and intestine during the initial exposure period (P < 0.05), while mTOR was significantly reduced on the first and third days after treatment (P < 0.05). From the results of Western blotting (WB), we can see that the amount of LC3-II was significantly higher than that of LC3-I at 1, 3, and 5 days of exposure (P < 0.05). Furthermore, the apoptosis-related gene Bcl-2 reached its peak in the liver, intestine, and gill on the first day, and caspase3 was significantly downregulated throughout the exposure period (P < 0.05). The results showed that fipronil was potentially harmful to carp and should be used moderately to reduce the damage to aquatic ecosystems. This study complements the mechanism theory of fipronil on fish toxicology and has a certain value for human health risk assessment.

The effects of Aeromonas hydrophila infection on oxidative stress, nonspecific immunity, autophagy, and apoptosis in the common carp.

Although the toxicity of Aeromonas hydrophila infection to common carp has been characterized, the mechanisms underlying this toxicity have not been fully explored. The present study assessed the effects of A. hydrophila infection on oxidative stress, nonspecific immunity, autophagy, and apoptosis in the common carp (Cyprinus carpio). We measured the effects of 7.55 × 105 CFU/mL and 4.87 × 107 CFU/mL A. hydrophila on carp after 1, 3, 5, and 7 d of infection. GSH and SOD activity levels in the serum, liver, intestine, and gills generally increased during the early stage of infection, but significantly decreased (P < 0.05) on the seventh day. In addition, MDA levels were significantly increased throughout the infection period. The activity levels of ACP, AKP, and LZM in the liver and intestine increased on the first day after infection, then decreased on the fifth and seventh days. The mRNA expressions levels of the autophagy-associated genes atg12, atg5, LC3-II, and BECN1 in the liver, kidney, and brain substantially increased on the third day after infection (P < 0.05), while mTOR was significantly downregulated on the first and third days (P < 0.05). Western blot analysis indicated that the ratio of LC3B-ǁ/LC3B-ǀ significantly increased (P < 0.05) on days 3 and 5 post infection. Furthermore, the apoptosis-related gene Bcl-2 was significantly (P < 0.05) upregulated in the liver, kidney, and brain of the treatment group on the first and third days, while caspase3 was significantly downregulated (P < 0.05). In conclusion, our results demonstrate that A. hydrophila infection causes oxidative stress, stimulates nonspecific immune reactions, and results in autophagy in the common carp, possibly initiating apoptosis in the late stage of infection. The results of this study provide new insights into the mechanism of A. hydrophila infection in carp.

Igfbp3 in grass carp (Ctenopharyngodon idellus): Molecular identification and mRNA expression under glucose, insulin and glucagon.

IGFBPs play a pivotal role in regulating the physiological function of IGFs (insulin-like growth factors). As an important member of IGFBPs, IGFBP3 is involved in the regulation of physiological functions of IGFs. To investigate the functional role of Igfbp3 in a herbivorous fish species, grass carp igfbp3 (GenBank accession no. MN251843) was isolated from the liver by molecular cloning. The ORF of grass carp igfbp3 was 882 bp, which encoded 293 amino acids, and the first 22 amino acids comprised the signal peptide. The predicted molecular weight of grass carp Igfbp3 is 31.95 kDa, and the theoretical isoelectric point is 8.32. Grass carp Igfbp3 displayed a high identity with its counterparts of common carp and zebrafish. And phylogenetic tree analysis showed that the grass carp Igfbp3 was clustered into the Igfbp3 subgroups of common carp and zebrafish. Tissue distribution study showed that igfbp3 was ubiquitously expressed in all tissues examined in the present study. High expression level of igfbp3 was detected in the heart, brain and fat of grass carp. The OGTT demonstrated that igfbp3 mRNA expression was significantly elevated in the liver of grass carp in response to glucose treatment. In vitro study showed that insulin could markedly stimulated igfbp3 mRNA expression in primary grass carp hepatocytes. Moreover, igfbp3 mRNA levels were also regulated by glucagon in grass carp primary hepatocytes. These results may provide the theoretical foundation for investigating the role of Igfbp3 in fish metabolism.

Protective effect of lycopene on fluoride-induced ameloblasts apoptosis and dental fluorosis through oxidative stress-mediated Caspase pathways.

Fluoride is an environmental toxicant and induces dental fluorosis and oxidative stress. Lycopene (LYC) is an effective antioxidant that is reported to attenuate fluoride toxicity. To determine the effects of LYC on sodium fluoride (NaF) -induced teeth and ameloblasts toxicity, rats were treated with NaF (10 mg/kg) and/or LYC (10 mg/kg) by orally administration for 5 weeks; ameloblasts were treated with NaF (5 mM) and/or LYC (2 µM) for 6 h. We found that the concentrations of fluoride, malondialdehyde (MDA) and reactive oxygen species (ROS), gene expressions and activities of Caspase-9 and Caspase-3, and the gene expressions of Bax were significantly decreased, while the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX), the gene expression of Bcl-2 were significantly increased in the LYC + NaF-treated rats group; concentrations of MDA and ROS, gene expressions and activities of Caspase-9 and Caspase-3, and the gene expression of Bax, and ameloblasts apoptosis rate were significantly decreased, while the activities of SOD and GPX, the gene expression of Bcl-2 were significantly increased in the LYC + NaF-treated ameloblasts group. These results suggest that LYC significantly combated NaF-induced ameloblasts apoptosis and dental fluorosis by attenuation oxidative stress and down-regulation Caspase pathway.

Photoelectrocatalytic degradation of triclosan on TiO2 nanotube arrays and toxicity change.

Triclosan, one of the most widely used disinfectants, has been found to be toxic to animals and human beings. In this paper, triclosan was degraded on TiO2 nanotube arrays, using a photoelectrocatalytic (PEC) process under UV illumination, with Na2SO4 as the supporting electrolyte. The effect of bias potential was investigated and the results showed that 0V was the most appropriate potential for the degradation of triclosan. In 30min, 78.7% of triclosan had degraded during the PEC process. Intermediate analysis showed that 2,7-dichlorodibenzodioxin (DCDD) had formed during the degradation. The toxicity change during the PEC process was investigated using a luminescent bacteria test, with the results demonstrating that the toxicity of the reaction liquid decreased at the beginning and subsequently increased to a stable level. The indications were that some intermediates such as 2,7-dichlorodibenzodioxin was more toxic and stable than triclosan in the solution.