Enhancing cannabidiol bioaccessibility using ionic liquid as emulsifier to produce nanosystems: Characterization of structures, cytotoxicity assessment, and in vitro digestion.

The emulsifying potential of a biocompatible ionic liquid (IL) to produce lipid-based nanosystems developed to enhance the bioaccessibility of cannabidiol (CBD) was investigated. The IL (cholinium oleate) was evaluated at concentrations of 1 % and 2 % to produce nanoemulsions (NE-IL) and nanostructured lipid carriers (NLC-IL) loaded with CBD. The IL concentration of 1 % demonstrated to be sufficient to produce both NE-IL and NLC-IL with excellent stability properties, entrapment efficiency superior to 99 %, and CBD retention rate of 100 % during the storage period evaluated (i.e. 28 days at 25 °C). The in vitro digestion evaluation demonstrated that the NLC-IL provided a higher stability to the CBD, while the NE-IL improved the CBD bioaccessibility, which was mainly related to the composition of the lipid matrices used to obtain each nanosystem. Finally, it was observed that the CBD cytotoxicity was reduced when the compound was entrapped into both nanosystems.

Environmental impact assessment of dicationic ionic liquids with ammonium-phosphonium cations and amino acid anions.

Progress in the development of biodegradable or biobased ionic liquids (ILs) has led to the design of green compounds for several applications. Herein, four biocompatible dicationic ionic liquids (DILs) with ammonium-phosphonium cations and amino acid anions were synthesized and investigated their environmental impact. The structures of the DILs were confirmed by spectral analyses (1H, 13C and 31P NMR). Furthermore, physicochemical properties such as density, viscosity and refractive index were determined. Water content, bromide content and solubility were thereafter determined as the parameters needed for further studies. Subsequently, their antifeedant activity towards economically important pests of grain in storage warehouses: the granary weevil, the confused flour beetle, and the khapra beetle was examined, showing the dependence on structure. Moreover, selected DILs were investigated for toxicity towards white mustard, Daphnia magna, and Artemia franciscana to specify the environmental impact. These studies were complemented by understand the biodegradation of DILs by bacterial communities derived from soil at the agricultural land. The result was DILs with limited environmental footprints that have great potential for further application studies.

The Imidazolium Ionic Liquids Toxicity is Due to Their Effect on the Plasma Membrane.

Ionic liquids (ILs) are organic salts with a low melting point. This is due to the fact that their alkyl side chains, which are covalently connected to the ion, hinder the crystallization of ILs. The low melting point of ILs has led to their widespread use as relatively harmless solvents. However, ILs do have toxic properties, the mechanism of which is largely unknown, so identifying the cellular targets of ILs is of practical importance. In our work, we showed that imidazolium ILs are not able to penetrate model membranes without damaging them. We also found that inactivation of multidrug resistance (MDR) pumps in yeast cells does not increase their sensitivity to imidazolium ILs. The latter indicates that the target of toxicity of imidazolium ILs is not in the cytoplasm. Thus, it can be assumed that the disruption of the barrier properties of the plasma membrane is the main reason for the toxicity of low concentrations of imidazolium ILs. We also showed that supplementation with imidazolium ILs restores the growth of cells with kinetically blocked glycolysis. Apparently, a slight disruption of the plasma membrane caused by ILs can, in some cases, be beneficial for the cell.

Surface active dicationic ionic liquids as green oil spill dispersants.

The utilization of chemical dispersants as a way of mitigating of oil spills in marine eco-system has been extensively documented worldwide. Hence, in this research we have successfully synthesized two amphiphilic asymmetric Dicaionic Ionic Liquids (DILs). The efficacy of these synthesized DILs as dispersants was assessed using the baffled flask test (BFT). The results indicated a dispersant effectiveness ranging from 47.98 % to 79.76 % for the dispersion of heavy crude oil across various temperature ranges (10-30 °C). These dispersant-to-oil ratios (DOR) were maintained at 3: 100 (V%), showcasing promising dispersant capabilities for mitigating heavy crude oil spills. Additionally, acute toxicity tests conducted on Nile tilapia and Oreochromis niloticus have demonstrated the relatively low toxicity of the IL-dispersants, with Lethal Concentration 50 (LC50) values exceeding 100 ppm after 96 h. This suggests a practically slight toxic effect on the tested fish. In summary, the newly developed IL-dispersants are considered to be conducive to environmentally benign oil spill remediation.

Immunotoxicity of ionic liquid [C14mim]BF4 in rats.

Ionic liquid tetrafluoroborated-1-tetradecyl-3-methylimidazole salt ([C14mim]BF4) immunotoxicity was investigated in rats using three exposure groups (12.5, 25, and 50 mg kg-1), one recovery group (50 mg kg-1), and a control group without any treatment. The findings demonstrated that, at low doses, [C14mim]BF4 could raise WBC, NEU, and MID and lysozyme levels as well as spleen T-lymphocyte stimulation index in rats, however at high doses, the aforementioned indices were dramatically lowered. As the dose was raised, the proportion of RBC and PLT in the blood as well as CD4+ and CD8+ in the spleen increased, but the quantity of immunoglobulin IgG, IgA, and IgM in the serum as well as the number of NK cells in the spleen considerably dropped. Even though there were varying degrees of improvement 30 days after ceasing exposure, all these changes were unable to return to normal, and the number of NK cells was further decreased. The study demonstrates that [C14mim]BF4 can damage the specific immunity and non-specific immunity of rats, and cause immune dysfunction.

Biophysical Characterization and Cryopreservation of Mammalian Cells Using Ionic Liquids.

Ionic liquids (ILs) are a diverse class of solvents which can be selected for task-specific properties, making them attractive alternatives to traditional solvents. To tailor ILs for specific biological applications, it is necessary to understand the structure-property relationships of ILs and their interactions with cells. Here, a selection of carboxylate anion-based ILs were investigated as cryoprotectants, which are compounds added to cells before freezing to mitigate lethal freezing damage. The cytotoxicity, cell permeability, thermal behavior, and cryoprotective efficacy of the ILs were assessed with two model mammalian cell lines. We found that the biophysical interactions, including permeability of the ILs, were influenced by considering the IL pair together, rather than as single species acting independently. All of the ILs tested had high cytotoxicity, but ethylammonium acetate demonstrated good cryoprotective efficacy for both cell types tested. These results demonstrate that despite toxicity, ILs may be suitable for certain biological applications. It also demonstrates that more research is required to understand the contribution of ion pairs to structure-property relationships and that knowing the behavior of a single ionic species will not necessarily predict its behavior as part of an IL.

Ecotoxicity studies reveal that organic cations in dicamba-derived ionic liquids can pose a greater environmental risk than the herbicide itself.

The following research provides novel and relevant insights into potential environmental consequences of combination of various organic cations with commercial systemic herbicide (dicamba), in accordance with a 'herbicidal ionic liquids' (HILs) strategy. Toxicity assays of five dicamba-based HILs comprising different hydrophobic and hydrophilic cations, namely choline [CHOL][DIC], ethyl betainate [BETC2][DIC], decyl betainate [BETC10][DIC], hexadecyl betainate [BETC16][DIC] and didecyldimethylammonium [DDA][DIC]), have been tested towards bacteria (Pseudomonas putida, Escherichia coli, Bacillus subtilis), algae (Chlorella vulgaris), fresh and marine water crustaceans (Daphnia magna, Artemia franciscana). The structure of respective substituents in the cation emerged as a decisive determinant of toxicity in the case of tested species. In consequence, small ions of natural origin ([CHOL] and [BETC2]) demonstrated toxicity numerous orders of magnitude lower compared to fully synthetic [DDA]. These results emphasize the role of cations' hydrophobicity, as well as origin, in the observed acute toxic effect. Time-dependent toxicity assays also indicated that betaine-type cations comprising an ester bond can rapidly transform into less harmful substances, which can generally result in a reduction in toxicity by even several orders of magnitude. Nonetheless, these findings challenge the concept of ionic liquids with herbicidal activity and give apparent parallels to adjuvant-dependent toxicity issues recently noted in typical herbicidal formulations.

Effects of cationic head group structure on cytotoxicity and mitochondrial actions of amphiphilic ionic liquids.

Ionic liquids (ILs) are a class of low melting point salts with physicochemical properties that make them suitable for a range of industrial applications. Accumulating evidence suggests that certain ILs are cytotoxic and potential environmental pollutants, thus understanding the structural features that promote IL cytotoxicity is important. Amphiphilic ionic liquids (AmILs), a class of ILs with lipophilic N-alkyl chains, containing aromatic head groups are generally more cytotoxic than their aliphatic counterparts, however the impact of other head group properties are less clear. This study therefore sought to provide new structure activity relationship (SAR) insights regarding the role of the cationic head group on AmIL cytotoxicity. A series of AmILs bearing a range of structurally diverse aromatic cations varying in size, charge, and lipophilicity was synthesised and screened against human MDA-MB-231 breast cancer cells. It was found that larger and more lipophilic head groups increased cytotoxicity, although the magnitude of the changes were modest. The mitochondrial effects of representative ILs were assessed. The AmILs induced mitochondrial dysfunction in MDA-MB-231 cells at cytotoxic concentrations, suggesting that they target mitochondria. The new SAR information from this study may assist in the design of AmILs with controlled cytotoxicity.

Advances in Cryopreservatives: Exploring Safer Alternatives.

Cryopreservation of cells, tissues, and organs is widely used in the biomedical and research world. There are different cryopreservatives that are used for this process; however, many of them, such as DMSO, are used despite the problems they present, mainly due to the toxicity it presents to certain types of samples. The aim of this Review is to highlight the different types of substances used in the cryopreservation process. It has been shown that some of these substances are well-known, as in the case of the families of alcohols, sugars, sulfoxides, etc. However, in recent years, other compounds have appeared, such as ionic liquids, deep eutectic solvents, or certain polymers, which open the door to new cryopreservation methods and are also less toxic to frozen samples.

Intergenerational toxic effects of 1-methyl-3-octylimidazolium chloride and 1-dodecylpyridinium chloride on the water flea, Moina macrocopa.

Ionic liquids (ILs) are generally considered eco-friendly alternatives to conventional industrial solvents, but they are hard to degrade and easily accumulate in the environment. Therefore, their long-term toxicities are especially vital to estimate their potential risk. However, the chronic toxicities of ILs over generations lacked intensive investigation. In the present work, acute toxicity and chronic toxicity of 1-methyl-3-octylimidazolium chloride ([Omim]Cl) and 1-dodecylpyridinium chloride ([DPy]Cl) were studied on Moina macrocopa with the first exposed generation (F0) and two successive recovery generation (F1 to F2). The acute results showed that both [Omim]Cl and [DPy]Cl exhibited high toxicity to M. macrocopa. The chronic results indicated that the exposure of [Omim]Cl and [DPy]Cl could inhibit the survivorship, body length, and reproduction of M. macrocopa and exhibited a significant dose-related decrease. Furthermore, these two types of ILs presented intergenerational toxicity in the water flea. And the toxic effects of [Omim]Cl disappeared in the recovery tests of F2 generation, while the [DPy]Cl toxic effects continued. Our research suggested a potential risk for the aquatic ecosystem induced by ILs. And the damage done by these chemicals to the aquatic environment is worthy of attention.

In vitro screening of imidazolium and pyrrolidinium based ionic liquids toxicity on subcellular fractions of the Mediterranean mussel Mytilus galloprovincialis.

Ionic liquids (ILs) have been considered eco-friendly alternatives to conventional organic solvents. However, several studies have reported that ILs exert toxicity towards aquatic invertebrates. Applying in vitro methodology, the aim of the present study was to evaluate the potential effect of three ILs on the biochemical performance of exposed Mytilus galloprovincialis digestive gland and gills cellular fractions. Carboxylesterase might be involved in the derived toxicity mechanism of ILs as activity levels increased significantly in digestive gland exposed fractions. This group of ILs did not seem to induce genotoxicity, except in gills cellular fractions exposed to 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide. In the literature, in vitro methodology has been suggested as an important complement to animal testing and in silico studies. The present research underlines its efficacy as a quick pre-screening before in vivo testing, particularly with heterogenic groups of substances with high variability in composition, such as ILs and deep eutectic solvents.

Aggregation, toxicity, and biodegradability study of an ionic liquid-based formulation for effective oil spill remediation.

Chemical dispersants are extensively used for marine oil spill remediation. However, the increased toxicity and low biodegradability of these dispersants restrict their employment in the marine environment. Hence, in this work, we have developed an eco-friendly formulation composed of an ionic liquid,1-butyl-3-methylimidazolium lauroyl sarcosinate [BMIM][Lausar] and sorbitan monooleate (Span) 80. Micellar and interfacial parameters, dispersion effectiveness, as well as the toxicity and biodegradability of the developed formulation were investigated. Micellar properties confirmed a high degree of synergism among the surfactant molecules and the formation of stable micelle. The dispersion effectiveness, at dispersant-to-oil ratio (DOR) of 1:25 (v/v), against three crude oils (Arab, Ratawi, and Doba) was assessed. We achieved a dispersion effectiveness of 68.49%, 74.05%, and 83.43% for Ratawi, Doba, and Arab crude oil, respectively, using a 70:30 (w/w) ratio of Span 80 to [BMIM][Lausar]. Furthermore, the results obtained from optical microscopy and particle size analysis (PSA) indicated that the oil droplet size decreased with higher DOR. Additionally, acute toxicity experiments were conducted on zebrafish (Danio rerio) using the developed formulation, confirming its non-toxic behavior, with LC50 values of 800 mg/L after 96 h. The formulation also exhibited high biodegradability, with only 25.01% of the original quantity remaining after 28 days. Hence, these results suggest that the new formulation has the potential to be a highly effective and environmentally friendly dispersant for oil spill remediation.

Unveiling the molecular interactions between alkyl imidazolium ionic liquids and human serum albumin: Implications for toxicological significance.

Alkyl imidazolium-based ionic liquids (ILs) are promising for diverse industrial applications; however, their growing prevalence has raised concerns regarding human exposure and potential health implications. A critical aspect to be clarified to address the adverse health effects associated with ILs exposure is their binding mode to human serum albumin (HSA). In this study, we delved into the binding interactions between three alkyl imidazolium ILs (1-hexyl-3-methyl-imidazolium (C6[MIM]), 1-ethyl-3-methyl-imidazolium chloride (C8[MIM]) and 1-decyl-3-methyl-imidazolium (C10[MIM]) and human serum albumins (HSAs) using a comprehensive approach encompassing molecular docking and multi-spectroscopy (UV-visible, Fluorescence, Circular Dichroism, FTIR). Furthermore, for the first time, we developed an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) approach time to quantify plasma protein binding rates. Our results revealed that the ILs primarily bind to the hydrophobic cavity of HSA through hydrogen bonding and van der Waals forces, forming stable complexes via static quenching. This affected HSA's secondary structure, reducing α-helical content, particularly around specific residues. Equilibrium dialysis and ultrafiltration coupled with UPLC-MS/MS analysis showed modest plasma protein binding rates (17.84%-31.85%) for the three ILs, with no significant influence from alkyl chain effects or concentration relationship. Lower plasma protein binding rates can affect bioavailability and distribution of ILs, potentially influencing their toxicity. These findings provide critical insights into the potential toxicological implications at the molecular level, thereby contributing to continuous efforts to evaluate the risk profiles and ensure the safe utilization of these compounds.

Modelling enzyme inhibition toxicity of ionic liquid from molecular structure via convolutional neural network model.

Deep learning (DL) methods further promote the development of quantitative structure-activity/property relationship (QSAR/QSPR) models by dealing with complex relationships between data. An acetylcholinesterase inhibitory toxicity model of ionic liquids (ILs) was established using a convolution neural network (CNN) combined with support vector machine (SVM), random forest (RF) and multilayer perceptron (MLP). A CNN model was proposed for feature self-learning and extraction of ILs. By comparing with the model results through feature engineering (FE), the model regression results based on the CNN model for feature extraction have been substantially improved. The results showed that all six models (FE-SVM, FE-RF, FE-MLP, CNN-SVM, CNN-RF, and CNN-MLP) had good prediction accuracy, but the results based on the CNN model were better. The hyperparameters of six models were optimized by grid search and the 10-fold cross validation. Compared with the existing models in the literature, the model performance has been further improved. The model could be used as an intelligent tool to guide the design or screening of low-toxicity ILs.

Insights into the quantitative structure-activity relationship for ionic liquids: a bibliometric mapping analysis.

Environmental protection and sustainability is the development goal that countries all over the world are pursuing. Ionic liquids (ILs), as a new type of green material, have a great application prospect. And the quantitative structure-activity relationship (QSAR) is significant for the research of ILs. To better understand the role played by QSAR in the research of ILs, 4139 literatures published in the WOS database from 2002 to 2022 were used for bibliometric analysis, and different types of knowledge maps were mapped to obtain the current status and trends of IL research applied QSAR. The distribution pattern of the literature output chronology, country, institution, author cooperation, and major source journals can be obtained through the research of the distribution of literature. Through core literature, dual-map overlays, and evolutionary path analysis, the research knowledge base was obtained mainly including ionic liquid toxicological properties research, environmental protection and sustainability, ionic liquid design, and mild steel corrosion inhibition; through the co-occurrence and evolution of keywords, the current research hotspots are basic properties of ILs, corrosion inhibition of mild steel, the effect of toxicity on the environment, QSAR modeling methods, solvent application of ILs, and drug design.

Hydroxyapatite and ionic liquid coupled with hybrid membranes for toxic pollutant removal and remediation.

Access to clean water is the mandatory requirement for every living being to sustain life. So, membrane-based integrated approach of adsorption and separation technology has recently been preferred by scientists over other conventional techniques, for wastewater treatment. Current research focused on the synthesis of novel imidazolium (A1) based IL, which was further functionalized with hydroxyapatite (HAp; extracted from Labeo rohita scales), to create possible solutions towards environmental remediation. Cellulose acetate (CA) was used for the fabrication of three different ionic liquid membranes. All the synthesized products were characterized by FTIR, XRD and TGA. Two dyes of different nature, i.e., congo red (anionic) and crystal violet (cationic) were selected because of their highly toxic and carcinogenic effects, for batch adsorption experiments. Antibacterial activity of the synthesized membranes was also evaluated against S. aureus. Results of the study revealed that CA-HA1 1:2 acted as the best adsorbent towards the removal of crystal violet, exhibiting removal efficiency of 98% with the contact time of 24 h while in case of congo red adsorption, CA-HA1 (1:2) proved as prime adsorbent with the removal efficiency of 96% for the same preceding contact time. Considering the antibacterial character of the synthesized membranes, CA-A1 (1:1) emerged as very efficient antibacterial agent with the inhibition zone of 50 mm after 48 h. The overall behavior of monolayer and multilayer adsorption was witnessed for both dyes while kinetic studies favored the pseudo-second order reaction for all adsorbents.

Chronic and intergenerational toxic effects of 1-decyl-3-methylimidazolium hexafluorophosphate on the water flea, Moina macrocopa.

With the increasing use and production of "green solvents" ionic liquids (ILs) and their known stability in the environment, the potential adverse effects of ILs have become a focus of research. In the present study, acute, chronic, and intergenerational toxic effects of an imidazolium-based ionic liquid, 1-decyl-3-methylimidazolium hexafluorophosphate ([Demim]PF6), on Moina macrocopa were investigated following the parental exposure. The results showed that [Demim]PF6 exhibited high toxicity to M. macrocopa, and the long-term exposure significantly inhibited the survivorship, development, and reproduction of the water flea. Furthermore, it is also observed that [Demim]PF6 induced toxic effects in the following generation of M. macrocopa, resulting in the complete cessation of reproduction in the first offspring generation, and the growth of the organisms was also significantly affected. These findings provided a novel insight into the intergenerational toxicity induced by ILs to crustaceans and suggested that these compounds pose potential risks to the aquatic ecosystem.

Removal of mercury by silica-supported ionic liquids: Efficiency and ecotoxicological assessment.

Anthropogenic impacts have affected the coastal environment and contributed to its contamination. Mercury (Hg) is widespread in nature and has been shown to be toxic in even the smallest amounts, negatively affecting not only the marine ecosystem but also the entire trophic chain due to its biomagnification. Mercury ranks third on the Agency for Toxic Substances and Diseases Registry (ATSDR) priority list and it is therefore imperative to develop more effective methods than those currently available to avoid the persistence of this contaminant in aquatic ecosystems. The present study aimed to evaluate the effectiveness of six different silica-supported ionic liquids (SIL) in removing Hg from contaminated saline water, under realistic conditions ([Hg] = 50 µg/L), and to ecotoxicologically evaluate the safety of the SIL-remedied water, using as test model the marine macroalga Ulva lactuca. The results revealed that SIL [Si][C3C1im][SCN] (250 mg/L) was the most effective in removing Hg from solution, with a efficiency up to 99 % in just 6 h, that enable to obtain < 1 µg/L Hg (European guideline in drinking water). U. lactuca exposed to either the SIL and/or the remedied water showed no significant changes in relative growth rate and chlorophyll a and b levels, compared to the control condition. Biomarker analysis (LPO, GSH, GSSG, SOD, GPx, CAT and GRed) also showed no significant changes in the biochemical performance of U. lactuca. Therefore, it could be assumed that water treatment with SIL or its presence in an aqueous environment does not pose toxicity levels that could inhibit the metabolism or cause cell damage to U. lactuca.

Potential for cardiac toxicity with methylimidazolium ionic liquids.

Methylimidazolium ionic liquids (MILs) are solvent chemicals used in industry. Recent work suggests that MILs are beginning to contaminate the environment and lead to exposure in the general population. In this study, the potential for MILs to cause cardiac toxicity has been examined. The effects of 5 chloride MIL salts possessing increasing alkyl chain lengths (2 C, EMI; 4 C, BMI; 6 C; HMI, 8 C, M8OI; 10 C, DMI) on rat neonatal cardiomyocyte beat rate, beat amplitude and cell survival were initially examined. Increasing alkyl chain length resulted in increasing adverse effects, with effects seen at 10-5 M at all endpoints with M8OI and DMI, the lowest concentration tested. A limited sub-acute toxicity study in rats identified potential cardiotoxic effects with longer chain MILs (HMI, M8OI and DMI) based on clinical chemistry. A 5 month oral/drinking water study with these MILs confirmed cardiotoxicity based on histopathology and clinical chemistry endpoints. Since previous studies in mice did not identify the heart as a target organ, the likely cause of the species difference was investigated. qRT-PCR and Western blotting identified a marked higher expression of p-glycoprotein-3 (also known as ABCB4 or MDR2) and the breast cancer related protein transporter BCRP (also known as ABCG2) in mouse, compared to rat heart. Addition of the BCRP inhibitor Ko143 - but not the p-glycoproteins inhibitor cyclosporin A - increased mouse cardiomyocyte HL-1 cell sensitivity to longer chain MILs to a limited extent. MILs therefore have a potential for cardiotoxicity in rats. Mice may be less sensitive to cardiotoxicity from MILs due in part, to increased excretion via higher levels of cardiac BCRP expression and/or function. MILs alone, therefore may represent a hazard in man in the future, particularly if use levels increase. The impact that MILs exposure has on sensitivity to cardiotoxic drugs, heart disease and other chronic diseases is unknown.

Intergenerational toxic effects of parental exposure to [Cn mim]NO3 (n = 2,4,6) on nervous and skeletal development in zebrafish offspring.

Ionic liquids (ILs) are thought to have negative effects on human health. Researchers have explored the effects of ILs on zebrafish development during the early stages, but the intergenerational toxicity of ILs on zebrafish development has rarely been reported. Herein, parental zebrafish were exposed to different concentrations (0, 12.5, 25, and 50 mg/L) of [Cn mim]NO3 (n = 2, 4, 6) for 1 week. Subsequently, the F1 offspring were cultured in clean water for 96 h. [Cn mim]NO3 (n = 2, 4, 6) exposure inhibited spermatogenesis and oogenesis in F0 adults, even causing obvious lacunae in the testis and atretic follicle oocytes in ovary. After parental exposure to [Cn mim]NO3 (n = 2, 4, 6), the body length and locomotor behavior were measured in F1 larvae at 96 hours post-fertilization (hpf). The results showed that the higher the concentration of [Cn mim]NO3 (n = 2, 4, 6), the shorter the body length and swimming distance, and the longer the immobility time. Besides, a longer alkyl chain length of [Cn mim]NO3 had a more negative effect on body length and locomotor behavior. RNA-seq analysis revealed several downregulated differentially expressed genes (DEGs)-grin1b, prss1, gria3a, and gria4a-enriched in neurodevelopment-related pathways, particularly the pathway for neuroactive ligand-receptor interaction. Moreover, several upregulated DEGs, namely col1a1a, col1a1b, and acta2, were mainly associated with skeletal development. Expression of DEGs was tested by RT-qPCR, and the outcomes were consistent with those obtained from RNA-Seq. We provide evidence showing the effects of parental exposure to ILs on the regulation of nervous and skeletal development in F1 offspring, demonstrating intergenerational effects.