Phenylbenzimidazole Research Articles

Electrochemical engineering of ZIF-7 electrode using ion beam technology for better supercapacitor performance

In the study, ZIF-7 material was exposed to 500 keV copper (Cu++) ions at dosages of 1 × 1014, 1 × 1015, and 5 × 1014 ions/cm2. The ZIF-7 unirradiated reveals a polycrystalline with an intense peak at 16.231° with a crystal plane of (012). The intensity of the peak in the XRD pattern broadens as the radiation of copper ions increases. In the ZIF-7 FTIR, the CC stretching vibration of the benzene ring is visible at 1469 cm−1, and the hydroxy phenyl benzimidazole (bIm) ligand's CH bending vibration is visible at 740 cm−1. Irradiated ZIF-7 reveals CH bending at 758, 759, and 754 cm−1 from the bIm ligand and CC stretching at 1456, 1460, and 1457 cm−1. The CV plots showed redox peaks, demonstrating faradaic processes. The ZIF-7's unirradiated estimated specific capacitances at scan rates of 50, 40, 30, 20, and 10 mV/s are 156, 195, 260, 390, and 781 F/g. The estimated specific capacitance of the irradiated ZIF-7 material with copper ions of 1 × 1014 ions/cm2 is 185, 308, 462, and 925 F/g. The estimated specific capacitance for ZIF-7 with copper ions of 1 × 1015 and 5 × 1014 ions/cm2 are 187, 234, 468, 937, and 1200 F/g, and 300, 375, 500, 750, and 1200 F/g respectively. The unirradiated (ZIF-7) shows bandgap energy of 3.53 eV which is suitable for applications in gas separation, catalysis, and optoelectronics. The irradiated sample with 1 × 1014 ions/cm2, 1 × 1015 ions/cm2 and 5 × 1014 ions/cm2 revealed bandgap energy of 2.52 to 2.87 eV which is suitable energy storage application.

Organic UV filters: Occurrence, risks and (anti-)progestogenic activities in samples from the Czech aquatic environment and their bioaccumulation in fish

The occurrence, environmental risks and contribution of organic UV filters to detected (anti-)progestogenic activities were examined in samples of wastewater treatment plant influents and effluents, various surface waters and fish from the Czech Republic. Of the 20 targeted UV filters, 15 were detected in the WWTP influent samples, 11 in the effluents, and 13 in the surface water samples. Benzophenone-3, benzophenone-4, and phenyl benzimidazole sulfonic acid (PBSA) were found in all water samples. Octocrylene, UV-327 and 4-methylbenzylidene camphor exceeded the risk quotient of 1 at some sites. In the anti-progestogenic CALUX assay, 10 out of the 20 targeted UV filters were active. Anti-progestogenic activities reaching up to 7.7 ng/L, 3.8 ng/L, and 4.5 ng/L mifepristone equivalents were detected in influents, effluents, and surface waters, respectively. UV filters were responsible for up to 37 % of anti-progestogenic activities in influents. Anti-progestogenic activities were also measured in fish tissues from the control pond and Podroužek (pond with the highest number of detected UV filters) and ranged from 2.2 to 9.5 and 1.9 to 8.6 ng/g dw mifepristone equivalents, respectively. However, only benzophenone was found in fish, but it does not display anti-progestogenic activity and thus could not explain the observed activities.

An Integrated Approach of Bioassays and Non-Target Screening for the Assessment of Endocrine-Disrupting Activities in Tap Water and Identification of Novel Endocrine-Disrupting Chemicals.

The safety of drinking water is a significant environmental issue of great concern for human health since numerous contaminants are often detected in drinking water and its sources. Boiling is a common household method used to produce relatively high-quality drinking water in some countries and regions. In this study, with the aid of an integrated approach of in vitro bioassays and non-target analysis based on high-resolution mass spectrometry coupled with liquid chromatography, alterations in endocrine-disrupting activities in tap water samples without and with boiling were revealed, as well as the potential endocrine-disrupting chemicals (EDCs) contributing to these alterations were identified. The organic extracts of tap water had no significant (ant)agonistic activities against an estrogen receptor (ER), progesterone receptor (PR), glucocorticoid receptor (GR), and mineralocorticoid receptor (MR) at enrichment concentrations of ≤10 times, posing no immediate or acute health risk to humans. However, the presence of agonistic activities against PR and MR and antagonistic activities against ER, PR, GR, and MR in OEs of tap water at relatively higher enrichment concentrations still raise potential health concerns. Boiling effectively reduced antagonistic activities against these steroid hormone receptors (SHRs) but increased estrogenic and glucocorticoid activities in drinking water. Four novel potential EDCs, including one UV filter (phenylbenzimidazole sulfonic acid, PBSA) and three natural metabolites of organisms (beta-hydroxymyristic acid, 12-hydroxyoctadecanoic acid, and isorosmanol) were identified in drinking water samples, each of which showed (ant)agonistic activities against different SHRs. Given the widespread use of UV filters in sunscreens to prevent skin cancer, the health risks posed by PBSA as an identified novel EDC are of concern. Although boiling has been thought to reduce the health risk of drinking water contamination, our findings suggest that boiling may have a more complex effect on the endocrine-disrupting activities of drinking water and, therefore, a more comprehensive assessment is needed.

Novel 1-H Phenyl Benzimidazole Derivatives for IBD Therapy - An in-vitro and in-silico Approach to Evaluate its Effects on the IL-23 Mediated Inflammatory Pathway.

IBD is a chronic idiopathic gut condition characterised by recurring and remitting inflammation of the colonic mucosal epithelium. Benzimidazole is a prominent and attractive heterocyclic compound with diverse actions. Although seven locations in the benzimidazole nucleus can be changed with a number of chemical entities for biological activity, benzimidazole fused with a phenyl ring has caught our interest. To find and optimize novel 1-H phenyl benzimidazole compounds with favorable physicochemical features and drug-like characteristics for the treatment of IBD, in-silico studies and in-vitro approach were being used to identify and optimize these derivatives as potent inhibitors of IL-23 mediated inflammatory signaling pathway. All six compounds exhibit favorable drug-like properties with good intestinal absorption properties. Its high affinity for the target JAK and TYK, which is thought to be a key immunological signaling cascade in the pathophysiology of IBD, is revealed by docking studies. Because of their effects on decreasing iNOS-derived NO release and IL-23-mediated immune signaling by decreasing COX-2 and LOX activity, it's conceivable that the compounds CS3 and CS6 are better options for the treatment of IBD based on in-vitro cell line investigations.

Photosynthetic performance of symbiont-bearing foraminifera Heterostegina depressa affected by sunscreens

Foraminifera are abundant unicellular organisms that play an important role in marine element cycles. A large benthic foraminifer obligatory bearing photosymbionts is Heterostegina depressa. We studied potential impacts of sunscreens available on the market on the activity of photosymbionts on H. depressa by means of pulse-amplitude modulated (PAM) fluorescence microscopy. We included four different sunscreens, with two of them sold as “conventional” and two more stated as “eco-friendly”. Further, the impact of pure Ensulizole (phenylbenzimidazole sulfonic acid) was tested, which is a common agent of sunscreens. Foraminifera were incubated at varying concentrations (10, 50 and 200 mgL−1) of different sunscreens and the pure Ensulizole for 14 days. The photosynthetic performance was measured after 1,3, 7 and 14 days. Pure Ensulizole had a strong negative impact on the photobionts, which was reflected by a significant reduction of the areal fluorescence signal. “Eco-friendly” sunscreens affected the health of foraminifera more severely compared to “conventional” ones. We assume that metal nanoparticles like titanium dioxide or zinc oxide of “eco-friendly” sunscreens are causing this impact, because these substances were already classified as toxic for several microorganisms.

Dual Functions of Performance Improvement and Lead Leakage Mitigation of Perovskite Solar Cells Enabled by Phenylbenzimidazole Sulfonic Acid.

With the continuous improvement of performance of lead-based perovskite solar cells (PSCs), the potential harm of water-soluble lead ion (Pb2+ ) to environment and public health is emerging as a major obstacle to their commercialization. Herein, an amphoteric phenylbenzimidazole sulfonic acid (PBSA) that is almost insoluble in water is added to the perovskite precursor to simultaneously regulate crystallization growth, passivate defects, and mitigate lead leakage of high-performance PSCs. Through systematic research, it is found that PBSA can not only regulate the crystallization of perovskite grains to form the film, but also passivate the defects of annealed films mainly due to the strong interaction between the functional groups in PBSA and Pb2+ , which greatly improves the crystallinity and stability of perovskite films. Consequently, the highest power conversion efficiency of 23.27% is achieved in 0.09 cm2 devices and 15.31% is obtained for large-area modules with an aperture area of 19.32 cm2 , along with negligible hysteresis and improved stability. Moreover, the leakage of lead ions from unpackaged devices is effectively prevented owing to the strong coupling between PBSA molecules and water-soluble Pb2+ to form insoluble complexes in water, which is of great significance to promote the application of optoelectronic devices based on lead-based perovskite materials.

Aramid-film pH sensitive fluorescence enhancement based on benzimidazole intermolecular hydrogen bonds

The aramid-film containing benzimidazole units was synthesized, and its pH sensitive fluorescence enhancement effect was investigated. After surface-treated by acidic ethanol solution (pH 1), the aramid film exhibits noticeable emissive enhancement as high as 33 times than the blank sample. The fluorescence lifetimes of aramid films increase significantly from 0.9 × 10−10 s to 8.2 × 10−10 s. The driving force is ascribed to the decrease of nonradiative transition probability, which comes from the strengthening molecular rigidity of phenyl benzimidazole chromophore within the polymer chains, induced by hydrogen bonds between N-H in benzimidazole group and C=O in amide group. The hydrogen-bond effect is thoroughly proved through examining the repeating molecular units of the aramid polymer chains by the methods of XRD, UV–vis, photoluminescence, molecular simulation, SEM and etc. This pH sensitive fluorescence change should have great potential in the sensor-device design of NH3, heavy metal ions or other systems due to the subsequent fluorescence quench effect, because of the excellent mechanical and processability of aramid polymers.

HPLC Method Validation for the Quantitative Fast Determination of 8 Organic Uv-Filters in Products with a Level of Sun Protection Factor Emulsion-Type

A fastHigh Performance Liquid Chromatography (HPLC) method was developed and validated for the simultaneous quantitative determination of phenylbenzimidazole sulfonic acid (PBMSA), benzophenone-3 (BZ3), diethylamino hydroxybenzoyl hexyl benzoate (DHHB), octocrylene (OC), butyl methoxydibenzoylmethane (BMBM), ethylhexyl methoxycinnamate (EMC), ethylhexyl salicylate (ES), and homosalate (HS) which are eightorganic UV-filters of the most commonly used in cosmetics and sunscreens. A solution pH 2.0 of phosphate buffer, methanol, and acetonitrile in proportions 15:3:82 respectively as mobile phase was used in isocratic conditions, combined with a Purospher Star ® Performance RP-18e column and eight minutes of analysis time. The proposed method was validated following the recommendations of International Council on Harmonization (ICH) and the Food and Drug Administration (FDA) guidelines. All parameters examined were found to be well within the stated guidelines. From the robustness test, it was possible to identify that pH of the buffer and the vial material are the critical methodological parameters for the analysis of butyl methoxydibenzoylmethane (BMBM), whereas for phenylbenzimidazole sulfonic acid (PBMSA) they were, the vial material, volume ofNaOH solution and sonication time and temperature.

High Anthropogenic Organic Matter Inputs during a Festival Increase River Heterotrophy and Refractory Carbon Load.

Streams and rivers metabolize dissolved organic matter (DOM). Although most DOM compounds originate from natural sources, recreational use of rivers increasingly introduces chemically distinct anthropogenic DOM. So far, the ecological impact of this DOM source is not well understood. Here, we show that a large music festival held adjacent to the Traisen River in Austria increased the river’s dissolved organic carbon (DOC) concentration from 1.6 to 2.1 mg L–1 and stream ecosystem respiration from −3.2 to −4.5 mg L–1. The DOC increase was not detected by sensors continuously logging absorbance spectra, thereby challenging their applicability for monitoring. However, the fluorescence intensity doubled during the festival. Using parallel factor analysis, we were able to assign the increase in fluorescence intensity to the chemically stable UV-B filter phenylbenzimidazole sulfonic acid, indicating organic compounds in sunscreen and other personal care products as sources of elevated DOC. This observation was confirmed by liquid chromatography coupled with mass spectrometry. The elevated respiration is probably fueled by anthropogenic DOM contained in beer and/or urine. We conclude that intense recreational use of running waters transiently increases the anthropogenic DOM load into stream ecosystems and alters the fluvial metabolism. We further propose that chemically distinct, manmade DOM extends the natural range of DOM decomposition rates in fluvial ecosystems.

Nanovesicle-based formulations for photoprotection: a safety and efficacy approach

Vesicular nanosystems are versatile and they are able to encapsulate actives with different solubilities, such as lipophilic and hydrophilic compounds. The most well-known vesicular nanosystems are liposomes and niosomes, the last one is formed by non-ionic surfactants. In the present work, we developed photoprotective niosomes containing sunscreens (octyl methoxycinnamate, diethylamino hydroxybenzoyl hexyl benzoate and phenylbenzimidazole sulfonic acid), non-ionic surfactants, cholesterol and stearylamine (positive-charged lipid). Studies based on dynamic light scattering techniques, entrapment efficiency and morphology by transmission electron microscopy were performed to characterize the niosomes. In addition, rheology, pH, in vitro sun protection factor (SPF) efficacy and toxicity and in vivo and in vitro safety were determined for the niosome formulations F-N1 and F-N2. The mean sizes of N1 and N2 were 168 ± 5 nm and 192 ± 8 nm, respectively, and their morphologies were spherical, unilamellar and with an entrapment efficiency of more than 45% for each sunscreen. Both formulations, F-N1 and F-N2 presented characteristics of pseudoplastic non-Newtonian fluids, showing declining viscosity with increasing shear rate applied. SPF values were considered satisfactory, 34 ± 8 for formulation F-N1 and 34 ± 5 for F-N2. The formulations did not present toxicity when tested in macrophages and the pH was compatible with skin, which minimizes allergies. The in vitro safety assay showed lipophilic sunscreens greater affinity for the epidermis, since this layer contains natural lipids. In vivo safety assay suggests that the increased skin retention of N2 is directly correlated with the positive charge of stearylamine. Stable photoprotective niosomes were obtained and were shown to be promising nanostructures to be used against solar radiation.

Per capita loads of organic UV filters in Australian wastewater influent

Per capita loads of six UV filters were estimated in wastewater influent samples from 36 wastewater treatment plants in Australia collected over a weekend period during the 2016 Australian Census. Of the analysed samples, 99% contained at least one of the target compounds. Phenyl benzimidazole sulfonic acid (PBSA) was the most prevalent (99%), followed by benzophenone 4 (BP4) (97%), benzophenone 3 (BP3) (87%), benzophenone 1 (BP1) (84%), 4-methylbenzylidene camphor (4-MBC) (22%) and isoamyl 4-methoxycinnamate (IMC) (1.5%). The highest concentrations were 3780 and 5070 ng L−1 for PBSA and BP4, respectively. Total per capita UV filter loads in influent across all plants were calculated using population data from the Australian Census and ranged from 0.21 to 3.4 mg day−1 person−1. Notably, a relationship was found between latitude and total per capita daily mass load of UV filters with an increase in mass load from southern to northern catchments. Compared to international studies, mass loads were generally similar with higher loads of BP4 found in Australia. This study provides insight into the occurrence of UV filters in influent wastewater from across Australia and provides the first comprehensive nationwide baseline of UV filter loads.

Design of efficient non-doped blue emitters: toward the improvement of charge transport.

Charge transport and electronic transition properties of a series of newly designed anthracene-based non-doped blue emitters were investigated by density functional theory calculations. For a highly efficient non-doped device, Cz3PhAn-based emitters were designed to suppress the hole and electron reorganization energies required for structural relaxation with respect to the changes of charged states. As a result, the hole hopping rates of triphenylamine (TPA) and phenylbenzimidazole (PBI) substituted Cz3PhAn derivatives (1, 4, and 5–7) were tremendously enhanced as compared to that of Cz3PhAn due to the suppression of the reorganization energy of holes, λh. Moreover, 1 and 4 emitters showed almost identical hopping rates of holes and electrons, which can possibly lead to a perfect charge balance and high efficiency. The photo-physical properties showed that the emission energy of all 1–10 emitters is in 439–473 nm range. It is expected that our rational design strategy can help develop non-doped blue fluorescent emitters for high efficiency.

Personal care products in surface, ground and wastewater of a complex aquifer system, a potential planning tool for contemporary urban settings

The use and discharge of personal care products (PCPs) result in their presence in the aquatic environment. This study investigates the occurrence and fate of some PCPs in wastewater, surface and groundwater in an urbanized area in the North of Italy. We investigated four UV filters: phenylbenzimidazole sulfonic acid (PBSA), benzophenone-3 (BP3), benzophenone-4 (BP4) and 4 methyl-benzilidine-camphor (4-MBC), and two antibacterial agents: triclosan (TCS) and triclocarban (TCC). BP3, BP4 and PBSA were detected in all WWTPs and concentrations ranged 27–822 ng/L (BP4 > PBSA > BP3). TCS was the only disinfectant detected in wastewater and ranged from <0.2 to 1690 ng/L. Removal efficiencies in WWTPs were good for BP3 and TCS (80–100%), but were quite low for PBSA and BP4 (0–40%). Consequently, PBSA and BP4 were the most abundant substances in surface water, detected up to 560.4 ng/L. TCS was also found in surface water (<0.2–161.0 ng/L). Only PBSA and TCS were found in untreated groundwater, and levels were higher in wells close to rivers, suggesting the contribution of surface water to this contamination, but not from the catchment and the sewer networks. These PCPs were confirmed to be ubiquitous in all the aquifers sampled, being reliable descriptors of human presence. The use of these data as direct indicators of pollutant's loads for the aquifers deriving from human presence could provide early warnings on chemicals that are continuously introduced into surface waters, identifying dynamic urban trends and suggesting paths for the planning in urban regions and for appropriate investment and rehabilitation strategies of infrastructure.

Sensitive Synchronous Spectrofluorimetric Study of Certain Sunscreens Using Fluorescence Enhancers in Cosmeceutical Formulations.

Synchronous spectrofluorimetric methods could be successfully adopted for simultaneous determination of Octinoxate (OMC), Avobenzone (AVO), Octyltriazone (OT), and Phenyl benzimidazole sulfonic acid (PBSA) in moisturizing sunscreen lotion, utilizing β-CD as fluorescence enhancer, and determination of Avobenzone (AVO), Homosalate, Tinosorb M and Phenyl benzimidazole sulfonic acid (PBSA) in presence of Octocrylene (OCR) in whitening sunscreen cream, using micellar medium of Sodium Dodecyl Sulfate (SDS) to enhance fluorescence intensity. For first product, zero order synchronous spectrofluorimetric method was used for determination of OMC and AVO, and derivative synchronous spectrofluorimetric technique was utilized for OT and PBSA in quaternary mixture. Linear calibration curves were obtained in a concentration range of 0.5-8μg mL- 1 for OMC and AVO, and in range of 0.05-3μg mL- 1 for OT and 0.001-5μg mL- 1 for PBSA, by measuring the fluorescence at 370, 405, 333.2 and 340.6nm, respectively. For second product, first derivative synchronous fluorescence method was used for each UV-filter. A linear calibration curves were obtained in a concentration range of 0.5-8μg mL- 1 for AVO, in range of 0.1-8μg mL- 1 for Homosalate, 2-10μg mL- 1 for Tinosorb M and 0.001-5μg mL- 1 for PBSA, by measuring the fluorescence at 409.8, 373, 307.2 and 316.8nm, respectively. The detection limits are well below the maximum admissible concentration. The proposed methods were validated according to ICH guidelines and successfully applied to determine sunscreens in pure form and in Cosmeceutical formulations. All the results obtained were compared with those of published methods, where no significant difference was observed.

Risk assessment of phenylbenzimidazole sulfonic acid in cosmetic products

Risk assessment of phenylbenzimidazole sulfonic acid in cosmetic products

Novel biocomposite of carboxymethyl chitosan and pineapple peel carboxymethylcellulose as sunscreen carrier

This study aims to prepare of biocomposite of carboxymethyl chitosan (CM-chitosan) and carboxymethylcellulose (CMC) from Ananas comosus (pineapple) peel for use as broad spectrum sunscreen carrier. Biocomposite was produced by using ferulic acid (FA), a plant extract, as crosslinker with the optimal ratio of CMC: CM-chitosan: FA at 1:2:4%w. FT-IR technique demonstrated that crosslinking may occur at amine group of CM-chitosan and carboxyl group of FA and hydrogen bonding between hydroxyl group of CMC and carboxyl group of FA. Biocomposite is pale yellow powder and present fibre bundle-like surface in the SEM image. DSC, TGA and XRD results indicated that new compound was formed. The particle size of biocomposite is 626nm determined by using Zetasizer. Hydrophilic TiO2 and phenylbenzimidazole sulphonic acid (PBSA) were used as sunscreen agent at ratio of TiO2: PBSA at 2:1%w. The biocomposite sunscreen possesses the SPF value of 2.47 with boost star rating of 3 at 2% compound. The results obtained indicate that the biocomposite was successfully prepared from CM-chitosan and pineapple peel CMC and the system can be used as matrix delivery system for hydrophilic sunscreens.

Dual Encapsulation of Electron Transporting Materials To Simplify High-Efficiency Blue Thermally Activated Delayed Fluorescence Devices

The charge flux balance and interfacial optimization are two core concerns when simplifying blue thermally activated delayed fluorescence (TADF) diodes, which reflects the more stringent demand on carrier transporting materials (CTM) as the embodiment of the contradiction between charge transportation and quenching suppression with the opposite requirement on intermolecular interactions. Herein, phenylbenzimidazole (PBI) was used as the core substituted with two diphenylphosphine oxide (DPPO) groups to form six dual-encapsulated charge–exciton separation (CES)-type electron transporting materials (ETM) with the collective name of xyPBIDPO. Through tuning the substitution positions of DPPO group, its two functions of resonance and steric effects were integrated and optimized to enhance charged moiety encapsulation without cost of reducing electroactivity. As the result, among xyPBIDPO, mmPBIDPO successfully realizes the balance of favorable electrical performance and interfacial interaction suppressions in...

Synthesis and characterization of green-emitting phosphorescent Ir(III) complexes based on phenyl benzimidazole ligand

Several new Ir(III) complexes with 2-(4-bromophenyl)-1H-benzo[d]imidazole or 2-(4-bromophenyl)- 1-methyl-benzo[d]imidazole ligands as cylcometalated ligand and acetylacetonate or picolinate as the ancillary ligand were synthesized and their structures and photophysical properties were characterized. HOMO and LUMO energy levels and the molecular structures of Ir(III) complexes were scrutinized by DFT calculations. The complexes exhibited green luminescence at the maximum emission peaks at ca 495–522 nm. The methyl group substituent and replacing of acetylacetonate with picolinate complex can enhance the complex thermal stability. HOMO energy levels of the complexes vary from –4.99 to –5.44 eV, the LUMO energy levels are between –1.52 and –1.97 eV.

Synthesis, Analgesic and Anti-Inflammatory Activity of Some 2-[(4-Amino acetyl) amino] Substituted Phenyl Benzimidazole Derivatives.

Synthesis, Analgesic and Anti-Inflammatory Activity of Some 2-[(4-Amino acetyl) amino] Substituted Phenyl Benzimidazole Derivatives.

Presence of UV filters in surface water and the effects of phenylbenzimidazole sulfonic acid on rainbow trout (Oncorhynchus mykiss) following a chronic toxicity test

UV filters belong to a group of compounds that are used by humans and are present in municipal waste-waters, effluents from sewage treatment plants and surface waters. Current information regarding UV filters and their effects on fish is limited. In this study, the occurrence of three commonly used UV filters – 2-phenylbenzimidazole-5-sulfonic acid (PBSA), 2-hydroxy-4-methoxybenzophenone (benzophenone-3, BP-3) and 5-benzoyl-4-hydroxy-2-methoxy-benzenesulfonic acid (benzophenone-4, BP-4) – in South Bohemia (Czech Republic) surface waters is presented. PBSA concentrations (up to 13μgL−1) were significantly greater than BP-3 or BP-4 concentrations (up to 620 and 390ngL−1, respectively). On the basis of these results, PBSA was selected for use in a toxicity test utilizing the common model organism rainbow trout (Oncorhynchus mykiss). Fish were exposed to three concentrations of PBSA (1, 10 and 1000µgL−1) for 21 and 42 days. The PBSA concentrations in the fish plasma, liver and kidneys were elevated after 21 and 42 days of exposure. PBSA increased activity of certain P450 cytochromes. Exposure to PBSA also changed various biochemical parameters and enzyme activities in the fish plasma. However, no pathological changes were obvious in the liver or gonads.