Anopheles mosquitoes are the primary vectors of human malaria, which is a serious threat to public health worldwide. To reduce the spread of malaria and identify the malaria infection rates in mosquitoes, accurate species identification is needed. Malaria re-emerged in 1993 in the Republic of Korea (ROK), with numbers peaking in 2004 before decreasing to current levels. Eight Anopheles species  are distributed throughout Korea. Members of the Anopheles Hyrcanus group currently cannot be identified morphologically. The other species of Anopheles can be identified morphologically, except when specimens are damaged in traps. The purpose of this study was to develop a rapid and accurate method for simultaneous molecular identification of the eight Anopheles species present in the ROK.Genus Anopheles spp. used in this study were collected near/in the demilitarized zone in ROK, where most malaria cases are reported. DNA from 165 of the Anopheles specimens was used to develop a multiplex PCR assay. The internal transcribed spacer 2 (ITS2) region of each species was sequenced and analysed for molecular identification.Anopheles specimens was identified to species using a multiplex diagnostic system. These included: 20 An. sinensis, 21 An. koreicus, 17 An. lindesayi, 25 An. kleini, 11 An. lesteri, 22 An. sineroides, 23 An. belenrae, and 26 An. pullus. Each species was clearly distinguished by electrophoresis as follows: 1,112 bp for An. sinensis; 925 bp for An. koreicus; 650 bp for An. lindesayi; 527 bp for An. kleini; 436 bp for An. lesteri; 315 bp for An. sineroides; 260 bp for An. belenrae; and, 157 bp for An. pullus.DNA from a total of 165 Anopheles spp. distributed in ROK. This method can be used to accurately identify Anopheles species that are difficult to identify morphologically to determine species distributions and malaria infection rates.A multiplex PCR assay was developed to identify  Malaria has a major impact on global public health with more than 200 million people infected and about 4,00,000 deaths annually . Most maAnopheles are vectors of Plasmodium spp., the causative agent of malaria. Plasmodium spp. that are considered human pathogens include: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi, the latter previously considered a monkey malaria [P. vivax, P. falciparum and P. malariae were eradicated in 1979 by the National Malaria Eradication Service (NMES) of the Korean Government [P. vivax is present in ROK and, following its peak of > 4000 cases in 2010, continues to be responsible for 300–500 cases annually [Members of the genus  malaria . In the vernment , and thevernment . Except annually .Anopheles species  [An. lesteri and An. kleini, were proposed to be the primary vectors of malaria in ROK, while An. sinensis is considered a poor vector. Anopheles lesteri showed a large number of P. vivax sporozoites (up to 2105) in the salivary glands when compared to An. sinensis (0–14) in a single microscope field (750 × 560 μM). Also, An. kleini had higher oocyst rates of P. vivax (8.8%) in the midgut than An. sinensis (4.2%) [An. kleini and An. sinensis demonstrated similar numbers of oocysts, An. kleini demonstrated + 1 (1–10 sporozoites) to + 4 (> 1000 sporozoites) salivary gland infections, while An. sinensis only had + 1 salivary glands [An. pullus and An. belenrae are poor to moderate vectors of malaria in ROK . While An. sineroides has been implicated as a malaria vector, its status is unknown. Although there are no records of malaria infections in An. koreicus, several members of the Barbirostris group are primary vectors of malaria in Southeast Asia [An. lindesayi has not been found positive for malaria in ROK, it has been implicated as a vector of P. malariae in Southeast Asia [Anopheles species to determine their distribution and malaria infection rates in order to develop vector control measures is needed in ROK.In ROK there are eight i group) –15. Reces (4.2%) . In anoty glands . Recent ast Asia , 21. Whiast Asia . AccuratAnopheles spp. is necessary to identify their geographic distributions, larval habitats and population dynamics to manage or conduct epidemiological investigations that identify the most likely sites where infections occurred. Although scales on wings (wing patterns) and spots on legs are used as the primary key characters for species identification, it is extremely difficult if the characters are lost during collections [An. sinensis, An. lesteri, An. kleini, An. belenrae, and An. pullus are morphologically very similar and species cannot be identified using current morphological characters [Anopheles species in ROK had not yet been developed. In this study, a new multiplex PCR assay was developed to identify all Anopheles species simultaneously that are present in ROK.Accurate species identification and subsequent monitoring of lections , 23. In aracters , 24–26. aracters , moleculAnopheles mosquitoes used in the study were collected at six sites in/near the DMZ where most malaria infections are contracted: 1) Neutral Nations Supervisory Commission (NNSC) camp adjacent to the Panmunjeom ; 2) Daeseongdong ); 3) South Gate (South gate entrance to the DMZ) ); 4) Camp Bonifas ); 5) Warrior Base (US Army training sites approximately 3 km from the south gate of DMZ), ); and, 6) Dagmar North training area . Mosquitoes were collected using Mosquito Magnets®   was performed using a multiplex PCR assay diagnostic method [An. koreicus and An. lindesayi [Genomic DNA used in this study was extracted using the Chelex protocol . Identifc method . The uniindesayi . Each saAnopheles species.Two pairs of universal primers  were designed between the 5.8 S and 28 S regions of the ribosomal DNA (rDNA) to analyse the entire internal transcribed spacer 2 (ITS2) sequences for the eight 2, and 0.5 units Taq DNA polymerase  with 1.0 μl genomic DNA extracted from an individual specimen. The PCR cycling conditions were as follows: denaturation at 94 °C for 5 min followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 40 s, and extension at 72 °C for 60 s; and final extension at 72 °C for 5 min. Each product was visualized on 1.5% (wt/vol) agarose gels stained with ethidium bromide , and then sequenced in both directions by Macrogen. Sequence data were analysed using Bioedit v7.2.6.1 [PCR amplification of the whole ITS2 region was conducted as follows. Each individual reaction mixture (25 μl) included: 0.2 mM each dNTP, 0.4 μM each primer, 1X PCR buffer, 1.5 mM MgClv7.2.6.1  and depoAn. sinensis—MW546412, MW546421; An. pullus—MW546424, MW546423; An. lesteri—MW546426; An. sineroides—MW546417, MW546414; An. kleini—MW546419, MW546415; An. belenrae—MW546422, MW546418; An. koreicus—MW546413, MW546416; An. lindesayi—MW546425, MW546420.Anopheles present in ROK. Reverse primers for the three species  were designed using the 28 S rDNA region, while primers for the remaining species were designed using the ITS2 region , and 1.0 μl genomic DNA from an individual specimen. PCR amplification was performed under conditions of denaturation at 94 °C for 5 min; 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 2 min; and final extension at 72 °C for 5 min. The PCR products were visualized on ethidium-bromide–stained 2.0% (wt/vol) agarose gels (VWR Life Science). The whole aligned sequences showing positions for the universal primers and the specific reverse primers are described in Fig. Universal forward and species-specific reverse primers were designed for the eight species of on Table . The mulAnopheles species were used: An. sinensis (20), An. koreicus (21), An. lindesayi (17), An. kleini (25), An. lesteri (11), An. sineroides (22), An. belenrae (23), and An. pullus (26). A gel showing the products of multiplex PCR assay separated by agarose gel electrophoresis for the eight species is shown in Fig. An. sinensis; 925 bp for An. koreicus; 650 bp for An. lindesayi; 527 bp for An. kleini; 436 bp for An. lesteri,, 315 bp for An. sineroides; 260 bp for An. belenrae; 157 bp for An. pullus). This method allowed identification of all eight Anopheles spp., including An. koreicus and An. lindesayi, and is comparable to the current molecular diagnosis method applied to identify six Anopheles species belonging to members of the Anopheles Hyrcanus group present in ROK [An. koreicus and An. lindesayi, which were not included in the previous method [A total of 165 DNA samples extracted from individual elenrae 2, and An.An. belenrae and An. kleini by Rueda [Anopheles minimus, a primary malaria vector, was morphologically misidentified as Anopheles fluviatilis, while each species was identified correctly using PCR of the ITS2 regions [Anopheles vaneedeni also was reported as a new malaria vector during a malaria surveillance programme using the ITS2 region for specific identification [Aedes albopictus and Aedes aegypti, to verify morphological identification of specimens, as well as screening for potential new invasive species in Europe [In Africa and Southeast Asia where malaria is widespread, multiplex PCR assays have been developed and used to identify species accurately and to investigate malaria vector distributions and infection rates –40. In aby Rueda ) in ROK by Rueda . Accuratby Rueda . Several regions . In Soutfication . Moleculn Europe . These sAnopheles species present in ROK included in three groups  can be identified based on a new multiplex molecular-based method. Morphological identification of these species is challenging, particularly in cases when legs or wing scales used as the primary identification characters are missing or damaged during collections. The method described here enables simple and accurate identification requiring only PCR of individual specimens followed by electrophoresis. It would also be useful to acquire geographic, habitat and population distributions of An. koreicus and An. lindesayi that are less frequently collected than the other species. Since the re-emergence of vivax malaria in ROK in 1993, most malaria cases have been attributed to exposure near the DMZ. Although the reason for the concentrated outbreak of malaria in/near the DMZ is uncertain, one of the primary vectors, An. kleini, is more prevalent near the DMZ than south of Seoul [An. lesteri and An. kleini) showed higher infection rates in artificial experiments than An. sinensis that was previously considered to be the primary vivax malaria vector in ROK [An. sinensis and An. lesteri were considered the primary malaria vectors [An. lesteri demonstrated more anthropophilic behaviour and 20 times higher sporozoite rates (0.58%) than An. sinensis (0.02%) [P. vivax cases varies with environment factors that impact on mosquito population densities, which may be further impacted by climate change [Anopheles spp. distributions and population densities in ROK.The eight of Seoul . Additioof Seoul –49. Idenr in ROK , 19. In  vectors . However (0.02%) , 52. In e change , 54. ThuAnopheles spp. present in ROK. This multiplex PCR assay is a simple and accurate method to identify Anopheles spp. and can be used as a surveillance tool for monitoring malaria vector population distributions in ROK.In this study, a new molecular diagnostic method was developed for the identification of eight


The influence of various parameters on the responses of the aSPCE/SDS such as pH and concentration of the buffer, SDS concentration, and techniques parameters were investigated. Using optimised conditions , the aSPCE/SDS showed a good linear response in the concentration ranges of 5.0 × 10−8–2.0 × 10−5 for PA, 1.0 × 10−9–2.0 × 10−7 for DF, and 1.0 × 10−8–2.0 × 10−7 and 2.0 × 10−7–2.0 × 10−6 mol L−1 for TR. The limits of detection obtained during the simultaneous determination of PA, DF, and TR are 1.49 × 10−8 mol L−1, 2.10 × 10−10 mol L−1, and 1.71 × 10−9 mol L−1, respectively. The selectivity of the aSPCE/SDS was evaluated by examination of the impact of some inorganic and organic substances that are commonly present in environmental and biological samples on the responses of PA, DF, and TR. Finally, the differential pulse adsorptive stripping voltammetric (DPAdSV) procedure using the aSPCE/SDS was successfully applied for the determination of PA, DF, and TR in river water and serum samples as well as pharmaceuticals. In this work, an electrochemically activated screen-printed carbon electrode modified with sodium dodecyl sulfate (aSPCE/SDS) was proposed for the simultaneous determination of paracetamol (PA), diclofenac (DF), and tramadol (TR). Changes of surface morphology and electrochemical behaviour of the electrode after the electrochemical activation with H Paracetamol (PA), also called acetaminophen, is a widely used pain reliever and antipyretic drug. However, it has no anti-inflammatory effect. Paracetamol is the main ingredient in many cold and flu medications. It is usually used to relieve headaches, toothaches, backaches, muscle aches, and other minor aches. An overdose of paracetamol may result in the accumulation of toxic metabolites that can cause acute and sometimes fatal nephro- and hepatotoxicity ,2,3.Diclofenac (DF) is a well-known, non-steroidal anti-inflammatory drug (NSAID) for the treatment of post-traumatic pain and pain in chronic diseases. It exhibits activities characteristic of this group of drugs, i.e., anti-inflammatory, antipyretic, analgesic, and inhibiting platelet aggregation. Despite its undoubted advantages, it can be very dangerous for living organisms. It causes an increase in blood pressure and thus strokes and worsens the functioning of the liver ,4.Tramadol (TR) is an opioid drug, a synthetic analog of codeine. It is a centrally acting pain reliever and is used to treat mild to severe pain. Tramadol can be used alone or in combination with NSAIDs to deal with cases associated with severe acute or chronic pain, lower back pain, and postoperative pain management. Overdosing on tramadol can cause slow or ceased breaths because this substance can accumulate in the body, causing critical levels of poisoning ,6.The presented pharmaceuticals may be present in the environment and have a negative impact, e.g., diclofenac affects the quality of water and is harmful to fish.Due to the possible side effects for humans as well as the negative impact on the environment, it is extremely important to develop sensitive and accurate methods for the determination of the presented drugs in samples of biological fluids and environmental samples. There are many analytical methods such as high-performance liquid chromatography (HPLC) ,8,9, liqIn this work, for the first time, an electrochemically activated screen-printed carbon sensor (aSPCE) modified with an anionic surfactant  was prepared and applied for the simultaneous determination of paracetamol, diclofenac, and tramadol. The activation can functionalize the electrode surface, increase the active surface or remove surface contamination ,35. Due Voltammetric measurements were performed using a µAutolab analyzer  controlled by GPES 4.9 software. All experiments were carried out in a 10 mL quartz electrochemical cell using commercially available screen-printed sensors , which were activated electrochemically prior to measurements. These sensors consisted of a screen-printed carbon working electrode (SPCE), a platinum screen-printed auxiliary electrode, and a silver screen-printed pseudo-reference electrode. The µAutolab analyzer , controlled in this case by FRA 4.9 software, was also used to record the differential capacity curves and Nyquist plots by the electrochemical impedance spectroscopy (EIS) method.Microscopic images of the SPCE surface were obtained with a high-resolution scanning electron microscope Quanta 3D FEG . The experiments were carried out under required conditions .HPLC analyses were performed on a VWR Hitachi Elite LaChrom HPLC  with PDA detector using an XB-C18 reversed phase core-shell column  .−1 solutions of PA and TR and 0.001 mol L−1 solution of DF. These solutions were diluted with deionized water as needed. A SDS (sodium dodecyl sulfate) solution was obtained by dissolving a weighed amount of Sigma-Aldrich reagent in deionized water. When selecting the base electrolyte and examining the effect of pH on the signals of the analytes, 0.1 mol L−1 solutions of sulfuric acid, acetic acid, and acetate buffers with pH values of 3.5 ± 0.1, 4.0 ± 0.1, 4.5 ± 0.1, 5.0 ± 0.1, 5.5 ± 0.1, and 6.0 ± 0.1 were used. DTPA (diethylenetriaminepentaacetic acid) solution was prepared in deionized water using Sigma-Aldrich reagent. The influence of interferents such as Ca(II), Mg(II), Fe(III), Ni(II), Cd(II), Pb(II), Cu(II), V(V), Mo(VI), and Cl- was checked using Merck standard solutions. The effect of organic substances was checked using Sigma-Aldrich reagents: glucose, ascorbic acid, Triton X-100, and cetyltrimethylammonium bromide (CTAB). The solutions were prepared using ultrapurified water . HPLC-grade acetonitrile and trifluoroacetic acid (TFA) were from Merck. All solutions were prepared with Sigma-Aldrich or Merck reagents purchased from Merck KGaA company . Appropriate amounts of paracetamol sulfate potassium salt (PA), diclofenac sodium salt (DF), and tramadol hydrochloride (TR)  were dissolved in deionized water to obtain 0.01 mol L−1 in 0.1 mol L−1 acetate buffer of pH = 4.0 ± 0.1 containing 10 mmol L−1 H2O2. After activation, the sensor was rinsed with deionized water and allowed to air dry. Then, the electrode surface was modified with SDS during analysis of PA, DF, and TR by immersing the SPCE in a supporting electrolyte solution (acetate buffer of pH = 4.0 ± 0.1) containing 15 mg L−1 SDS. The screen-printed carbon electrode surface was electrochemically activated before the measurements . Activat−1 acetate buffer (pH of 4.0 ± 0.1), 1.0 × 10−5 mol L−1 DTPA and 15 mg L−1 SDS. The procedure consists of an accumulation step at a potential (Eacc.) of −0.4 V for a time (tacc.) of 120 s. Differential pulse adsorptive stripping voltammetric (DPAdSV) curves were recorded from 0 to 2 V with an amplitude (ΔEA) of 150 mV, a scan rate (ν) of 250 mV s−1, and a modulation time (tm) of 10 ms. The background curve was subtracted from each voltammogram. The average values of peak current (Ip) are shown with the standard deviation of n = 3.Voltammetric measurements of PA, DF, and TR in optimized conditions were carried out in a solution composed of 0.075 mol L−1 and temperature was set at 25°C. The injection volume was 20 µL. All samples were analyzed in triplicate, at a wavelength of 248 nm, 273 nm, and 277 nm for paracetamol, tramadol, and diclofenac, respectively.For high-performance liquid chromatography photodiode array detection (HPLC/PDA), a mixture of acetonitrile and water with 0.025% of trifluoroacetic acid was used as mobile phase. The acetonitrile concentration in the eluent increased constantly from 12 to 80% during 0–30 min. The flow rate was 1.0 mL minw/v) trichloroacetic acid solution (Sigma-Aldrich) for protein precipitation, centrifuged at 4000× g for 10 min, and filtered through a 0.45 µm Millipore filter. The collected supernatant was analyzed in triplicates by the optimized voltammetric procedure and HPLC/PDA methods.Bystrzyca river water , pharmaceuticals (first tablets containing PA (325 mg) and TR (37.5 mg)  and second tablets containing DF (25 mg) ), and normal human serum from Merck  were tested. River water samples were spiked with appropriate concentrations of the analytes and filtered through a 0.45 µm Millipore filter. Pharmaceuticals were prepared as follows. Three tablets of each drug were weighed and the average tablet weights were determined. Then, the three tablets of each drug were powdered in a mortar and the samples with corresponding average weight of one tablet were dissolved in 100 mL of deionized water. The samples were subsequently placed in an ultrasonic bath for 10 min and filtered through a 0.45 µm Millipore filter. Frozen human serum was thawed at room temperature. Then, 100 µL of the human serum sample 100 times diluted in deionized water spiked with appropriate concentrations of the analytes was transferred to a centrifugal tube, mixed with 50 µL of 7.5% (2O2 [−5 mol L−1), with a statistically insignificant change in the peak current of PA and DF. The surface morphology of the bare SPCE and the electrochemically activated screen-printed carbon electrode (aSPCE) was examined by SEM. It was found that electrochemical activation leads to visible changes on the surface of the working electrode as the number and size of the pores increase , with a statistically insignificant change in the peak current of PA and DF  and polar groups (‘heads’) of the SDS molecules [Further modification of the activated electrode surface with sodium dodecyl sulfate during the analysis of PA, DF, and TR in a supporting electrolyte solution containing SDS allows for a significant increase in the TR peak  (red curve) compared to the unactivated electrode (blue curve) (388.7 vs. 950.6 Ω cm2). It has also been shown that the modification of the aSPCE surface with SDS causes a slight increase in Rct (black curve) compared to the activated electrode (388.7 vs. 487.6 Ω cm2). In addition, the active surface areas (As) of the SPCE, aSPCE, and aSPCE/SDS were calculated using the Randles–Sevcik equation [p) and the square root of the scan rates (v1/2). For the bare SPCE, aSPCE, and aSPCE/SDS, the As is equal to 0.056, 0.054, and 0.059 cm2, respectively. The As is almost the same for all studied electrodes. In order to characterize the influence of the modifications on the electrochemical properties of the sensor, measurements using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were performed. The impedance spectra (Nyquist plots) were recorded at a potential of 0.25 V in the frequency range from 10 kHz to 0.1 Hz, from a solution of 0.1 mol Lequation . Figure 2O2. This contributes to lowering the charge transfer resistance of the electrode. The SDS modification slightly increases the charge transfer resistance of the activated electrode but does not block the electrode surface, whereas the active surface areas for the bare SPCE, aSPCE, and aSPCE/SDS are almost the same. To sum up, the SPCE morphology surface changed greatly after electrochemical treatment with H−6 mol L−1 PA, 1 × 10−7 mol L−1 DF, and 2 × 10−5 mol L−1 TR was investigated for 0.1 mol L−1 of H2SO4, CH3COOH solutions, and CH3COOH/CH3COONa buffers with pH of 3.5 ± 0.1, 4.0 ± 0.1, 4.5 ± 0.1, 5.0 ± 0.1, 5.5 ± 0.1, and 6.0 ± 0.1. The corresponding data are presented in −1 CH3COOH/CH3COONa buffer of pH of 4.0 ± 0.1, hence it was used for further research.The effect of the type and pH of the supporting electrolyte on the signals of 2 × 10−1 SDS  can be noticed, which may indicate slight adsorption of this analyte on the aSPCE/SDS surface  and time (tacc.), amplitude (ΔEA), scan rate (ν), and modulation time (tm) on the peak currents was investigated. The effect of Eacc was tested in the range from 0 to −0.5 V with the tacc. of 30 s. The highest signals were obtained at a potential of −0.4 V , ascorbic acid (up to 10-fold), Fe(III) (up to 10-fold), Ca(II) (up to 40-fold), Cu(II) (up to 2-fold), Mg(II) (up to 100-fold), Cd(II) (up to 10-fold), Pb(II) (up to 1000-fold), Ni(II) ions (up to 10-fold), Mo(VI) (up to 200-fold), and Cl(-I) (up to 20-fold) had negligible effects on the assay of PA. It was observed that an excess of glucose (up to 2500-fold), ascorbic acid (up to 100-fold), Fe(III) (up to 100-fold), Ca(II) (up to 100-fold), Cu(II) (up to 25-fold), Mg(II) (up to 500-fold), Cd(II) (up to 10-fold), Pb(II) (up to 1000-fold), Ni(II) (up to 25-fold), Mo(VI) (up to 500-fold), and Cl(-I) (up to 2500-fold ex) had negligible effects on the assay of DF. Moreover, an excess of glucose (up to 2500-fold), ascorbic acid (up to 5-fold), Fe(III) (up to 100-fold), Ca(II) (up to 500-fold), Cu(II) (up to 10-fold), Mg(II) (up to 250-fold), Cd(II) (up to 25-fold), Pb(II) (up to 1000-fold), Ni(II) (up to 25-fold), Mo(VI) (up to 50-fold), and Cl(-I) (up to 25-fold) had negligible effects on the assay of TR. In the optimized conditions, the influence of potential interferents on the determination of PA, DF, and TRA was investigated. The tolerance limit was defined as the concentration, which gave an error of ≤ 10% in the determination of 5 × 10−1 of Triton X-100 [−1 of Triton X-100 on the voltammetric response of 5 × 10−6 mol L−1 PA, 2 × 10−7 mol L−1 DF, and 2 × 10−6 mol L−1 TR was investigated. Moreover, the influence of cationic surfactant (CTAB) was studied. As can be seen in −1 SDS in the supporting electrolyte, the signals do not fall below 60% of their original values and are well formed and easy to measure.Due to the fact that natural waters contain surfactants with a surface active effect comparable to the effect of 0.2 to 2 mg Lon X-100 , the inf−1, and 49.56, 0.69, and 5.69 nmol L−1, respectively, according to the definitions of LOD = 3SDa/b and LOQ = 10SDa/b (SDa—standard deviation of intercept (n = 3); b—slope of calibration curve) [−10 vs. 1.71 × 10−9 mol L−1), but the first concertation of TR from the calibration graph is higher than the one obtained at the aSPCE/SDS (1.67 × 10−8 vs. 1.0 × 10−8 mol L−1). On the other hand, in the article [Under the optimized conditions, the ability of the aSPCE/SDS for individual and simultaneous determination of PA, DF, and TR was studied. The results are summarized in n curve) ,17,18,47 article , the LOD article  require −6 mol L−1 PA, 2 × 10−7 mol L−1 DF, and 2 × 10−7 mol L−1 TR with ten replicates. The results were 2.7, 1.2, and 1.8%, respectively, indicating the satisfactory repeatability of the signals at the aSPCE/SDS. The reproducibility was assessed based on voltammograms registered in the solution containing 1 × 10−6 mol L−1 PA, 1 × 10−8 mol L−1 DF, and 2 × 10−7 mol L−1 TR at three freshly prepared electrodes. The RSD was calculated as 2.5, 3.1, and 3.5% (n = 6), respectively, confirming the acceptable reproducibility of the aSPCE/SDS.Additionally, the precision was verified for the determination of 5 × 10−7, 5.2 × 10−7, and 2.7 × 10−7 mol L−1, respectively. Moreover, the contents of the analytes, PA and TR in the pharmaceutical tablets 1 and DF in the pharmaceutical tablets 2, were determined. The results are summarized in In order to confirm the usefulness of the developed voltammetric procedure at the aSPCE/SDS, the simultaneous determination of PA, DF, and TR was carried out in Bystrzyca river water samples and human serum samples, the results of which are presented in 2O2 significantly changes the electrode surface morphology and reduces the charge transfer resistance. The modification with SDS allows for the enhancement of the TR signal, while not negatively affecting the PA and DF signals, and greatly minimizes the influence of surfactants (Triton X-100 and CTAB) on the analytical signal of all analyzed substances. The DPAdSV procedure with the aSPCE/SDS allows for selective determination of low PA, DF, and TR concentrations. The LODs and LOQs obtained during simultaneous determination of PA, DF, and TR are 14.87 and 49.56 nmol L−1, 0.21 and 0.69 nmol L−1, and 1.71 and 5.69 nmol L−1, respectively. The developed sensor was successfully used to determine PA, DF, and TR in river water and human serum samples as well as in pharmaceutical preparations. The concentrations of PA, DF, and TR determined by DPAdSV method in river water and human serum samples were below the detection limits of the chromatographic method (HPLC/PDA). The obtained results show that the procedure can be used as a quick, simple, and cheap alternative to other methods. Moreover, it should be highlighted that the further advantage of the aSPCE/SDS sensor is it portability, which is very promising for quick field analysis.In summary, in this study, an electrochemically activated screen-printed carbon electrode modified with sodium dodecyl sulfate (aSPCE/SDS) was introduced for the first time for the simultaneous analysis of paracetamol (PA), diclofenac (DF), and tramadol (TR). The electrochemical activation of the SPCE surface using CV in acetate buffer of pH = 4.0 ± 0.1 containing H


Zospeum, have revealed significant findings concerning its Alpine-Dinaric evolution and taxonomy. Due to the expected discrepancy between the useful, but limited, 1970s’ classification system based on shell data and the results of recent genetic analyses in the latest investigation, a revision of the entire radiation was undertaken, and a new classification system was devised by the present authors in an earlier paper. Concurrent to this work, molecular sequences from two Austrian caves were published independently of our revision by another research group. By incorporating these genetic data within our phylogenetic framework here, we show that the Austrian individuals are genetically most similar to Zospeumamoenum and consequently, classify them within that species. We additionally reveal two new genetic lineages from the largely under-sampled southern extension of Zospeum’s known distributional range. The first lineage, deriving from the region of Dubrovnik, Croatia, is a potential candidate for genetically clarifying Zospeumtroglobalcanicum. The second lineage derives from the municipality of Tomislavgrad, Bosnia-Herzegovina and is herein, described a new species: Zospeumsimplex Inäbnit, Jochum & Neubert, sp. nov.Recent integrative investigations of the terrestrial ellobiid genus,  Zospeum, consists of tiny (0.9–2.6 mm), troglobitic snail species that are distributed in two disjunct areas: a western zone, comprising the western Pyrenees and the Cantabrian mountains of Spain and France , the Z.alpestre group , the Z.obesum group , the Z.pretneri group , and the Z.frauenfeldii group ; five species could not be assigned to any of the five groups due to lack of molecular data.Until recently, the eastern radiation of Zospeum’s eastern distribution has been unevenly sampled throughout its history. Most studies covered almost only Slovenian  Ljubljana, Slovenia;NHMWNaturhistorisches Museum Wien, Wien, Austria;NMBENaturhistorisches Museum der Burgergemeinde Bern, Bern, Switzerland;RSC Rajko Slapnik Collection, Kamnik, Slovenia;SMFSenckenberg Forschungsinstitut und Naturmuseum, Frankfurt am Main, Germany.NMBE 568052-568063). Each specimen was inserted into a 0.2-ml PCR-tube and dried at room temperature. Eight μl ddH2O and 2 μl 5× PCR-buffer (Promega 5× Colorless GoTaq Reaction Buffer) were added and the mixture was heated at 94 °C for 2 min. whereby 1.3 μl proteinase K solution  were then added and the solution was homogenised and then incubated in a PCR-thermocycler at 55 °C for 15 min., afterwards at 70 °C for 10 min. The incubation was repeated once. Ten μl of Gene Releaser (Bioventures Inc.) were then added and the mixture was inserted into a thermocycler with the following protocol: 65 °C for 30 s, 8 °C for 30 s, 65 °C for 1.5 min., 97 °C for 3 min., 8 °C for 1 min., 65 °C for 3 min., 97 °C for 1 min., 65 °C for 1 min., 80 °C for 5 min. The mixture, including the intact shell, was centrifuged for 1 min. using a table centrifuge and the clear phase with the DNA was transferred to another 0.2 mL PCR-tube, where 15 μl of AE-Buffer  was added. The shell was cleaned from the remains of the Gene Releaser chemicals by rinsing with 80% EtOH.In order to preserve the shell from dissolution during the extraction, our DNA extraction protocol was based on a method initially described in We used five markers, two mitochondrial (COI (658 bp), 16S (483 bp)) and three nuclear markers (H3 (330 bp), ITS2 (809 bp), 28S (590 bp)) with a total length of 2870 bp  polymerase, 8.5 μl of nuclease-free water, and 1 μl of both forward and reverse primer (10 μmol) respectively. In cases where the PCR signal was judged too weak, the reaction was repeated using 3 μl template DNA, 3 μl of the previous PCR product, and 5.5 μl of nuclease-free water. The amount of GoTaq and primers remained the same. The amplification was conducted using the following cycling protocols: For COI, the admixture was first heated up to 95 °C for 1 min, followed by 30 cycles of 30 s , and a final extension at 72 °C for 3 min. For 16S, the protocol started with 2:30 min at 90 °C, followed by 10 cycles of 30 s at 92 °C, 30 s at 44 °C, and 40 s at 72 °C, followed again by 30 s at 92 °C, 40 s at 48 °C, and 40 s at 48 °C. The protocol for 28S started with 1 min at 96 °C, then went into 35 cycles of 30 s at 94 °C, 30 s at 50 °C, and 1 min at 72 °C, finishing with 10 min at 72 °C. The ITS2 protocol started with 1 min at 96 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at 44 °C, and 1 min at 72 °C, ending with 10 min at 72 °C. For H3, the admixture was first heated up to 95 °C for 3 min, followed by 40 cycles of 45 s at 94 °C, 45 s at 50 °C, and 2 min at 72 °C, finishing with 10 min at 72 °C. The protocols for COI and H3 could be used for both markers. The PCR products were sequenced at the LGC Genomics GmbH  using their standard protocol.Sequences received from LGC were imported into the Geneious 5.4.7 software . The forML) and Bayesian Inference (BI). The five markers were set as partitions in both of these methods, using a distinct model for the third codon in protein-coding genes . The maximum likelihood (ML) topology was estimated using the RAxML 7.2.8  without H3 and 28S.Topologies were estimated using two different phylogenetic methods: Maximum Likelihood (ML 7.2.8  plugin oMCMC) chain length of 10000000 generations, a subsampling frequency of every 4000 generations, the first 100000 generations were discarded as burn-in, four heated chains and a chain temperature parameter of 0.2. Calculations were performed on the UBELIx (http://www.id.unibe.ch/hpc), the HPC cluster at the University of Bern.The Bayesian tree was reconstructed with MrBayes 3.2.6  using thZ.pretneri group  analysis was performed on the COI alignments of the Z.pretneri group and of the Z.alpestre group using the default settings .Additionally, an Automatic Barcode Gap Discovery , and the remaining specimens from Bosnia-Herzegovina . The latter group is not supported in either tree but recovered in both. An additional specimen , from which we were only able to amplify H3, didn’t cluster with any species within the Z.pretneri group. The two groups were also recovered, though here with high node support, in the additional ML tree (Suplementary tree 1) calculated for the Z.pretneri group. The Austrian specimens from Z.amoenum.Both the see Fig.  and coulZ.pretneri group are clearly smaller than the interspecific divergences , which were not included in previous genetic studies  and a second where the alignment was subdivided into seven groups . Both subdivision schemes considered the previously published sequences of Z.pretneri, Z.tholussum, and Z.manitaense as separate groups. The five-group scheme separated the individuals sequenced in this study into a Croatian group (Špilja Jezero) and a Bosnian group , while the seven-group scheme separated those individuals into two Croatian groups (one for each of the two specimens from Špilja Jezero) and two Bosnian groups .The ABGD run on the Z.alpestre-group COI alignment yielded one subdivision scheme with seven groups : Z.isselianum, Z.alpestre, Z.kupitzense, Z.amoenum from Ihanščica, Z.amoenum from Konečka zijalka, Z.amoenum from Potočka zijalka and Zospeumsp. from Austria.The Taxon classificationAnimaliaEllobiidaEllobiidaeInäbnit, Jochum & Neubert, sp. nov.04D6F12E-943D-5710-A586-2DFCA1508456http://zoobank.org/0B924616-1AC1-49B8-BE5F-531286EACE63Holotype:NMBE 568060, Jama Dobravljevac, 25.08.2019, leg. R. Slapnik & J. Valentinčič; Paratypes: NMBE 568061–568063; SMF 349425, 4 shells; RSC 3760, 6 shells; Jama Dobravljevac, 25.08.2019, leg. R. Slapnik & J. Valentinčič.NMBE 568054, Špilja Dahna, 03.09.2009, leg. A. Schoenhoffer; NMBE 568055–568058, Jama u kamenolomu, 24.08.2019, leg. R. Slapnik & J. Valentinčič; NMBE 568059, Vranjača, 24.08.2019, leg. R. Slapnik & J. Valentinčič.Shell usually ca. 1.3 mm in height, transparent, conical, peristome thickened, roundish, with a differentiated parietal shield, lamellae not present.Measurements (n = 9): Shell height: 1.26–1.42 mm (mean: 1.378 ± 0.047 mm); shell width: 0.93–1.04 mm (mean: 0.976 ± 0.035 mm); aperture height: 0.54–0.67 mm (mean: 0.6 ± 0.037); aperture width: 0.54–0.65 mm (mean: 0.601 ± 0.033 mm); number of whorls: 5–5.5.Shell conical, translucent when fresh; suture deep; aperture somewhat roundish to reniform; parietal shield clearly differentiated from the rest of the lip, straight and thin; no lamellae present.Z.pretneri and Z.tholussum by its broader shell and the differentiated parietal shield; differs from Z.manitaense by the absence of a visible parietalis in the aperture; barely differs from Z.aff.troglobalcanicum morphologically, on average with reduced shell broadness and a slightly deeper suture (see Remarks).Differing from Known from four caves  in the municipality of Tomislavgrad in Bosnia-Herzegovina.simplex  due to the lack of any form of shell sculpture or lamellae.Named Z.troglobalcanicum without genetic data  and couldn’t be studied yet). From the photograph in Z.manitaense in shell shape, without any visible lamella in the aperture and with a comparatively large parietal shield. The larger parietal shield might serve as a distinguishing character between Z.simplex and Z.troglobalcanicum, though the illustration of a topotypic specimen in Z.troglobalcanicum ; Z.aff.troglobalcanicum: below 1.3), but that might just be due to the low sample sizes. Investigation of the inner aspects of the shells will be presented in a later work.Difficult to separate from Z.pretneri group and in that of Z.amoenum are now fairly low and the topology is different. This can be explained by the high number of new specimens that sometimes are only represented by one marker . It should also be noted that our current trees resolve Z.robustum, for which we didn’t have any new specimens, with a significant node support as a monophyletic group  The barcode gap of the seven-group scheme is much lower (0.003) than the barcode gap (0.032) that was detected in the Carychiidae alignment in ABGD five-group scheme whereby the between-group divergence between the Croatian and the Bosnian groups  to one of the caves with genetically identified specimens (Jama u kamenolomu), we expect it could well be assignable to Z. simplex as no external morphological inconsistencies separate it from other Z. simplex specimens in our study.A species comprising two specimens from Špilja Jezero in the region of Dubrovnik. This locality is fairly close (around 22 km) to the type locality (Benetina pećina) of Z. troglobalcanicum Absolon, 1916. The sequenced specimens do not show any major external morphological differences from the specimen identified as Z. troglobalcanicum (as figured in Bole 1974: fig. 3h) and from those imaged in Inäbnit et al. 2019: fig. 7u), though the adult specimen clearly has a smaller parietal shield than the specimens figured in Absolon (1916). We propose tentatively classifying those specimens within Z. troglobalcanicum until genetic material from the type locality can clarify its status and the morphological investigation of the singular syntype (NHMW Mol.Coll.Edlauer 32.749) of this species can be taxonomically and nomenclaturally clarified in a separate work.Z.alpestre group, our divergence analysis revealed that the between-group divergence between Z.amoenum and the two Austrian populations is greater than the within-group divergence of either lineage. Our analysis also found that the within-group divergence in Z.amoenum is only slightly increased if the Austrian populations are included within this species. These results agree with the tree reconstruction published in Z.amoenum. Our trees, as mentioned above, lack the resolution to separate the Austrian populations from Z.amoenum and can thus, not confirm this conclusion. The ABGD scheme for the Z.alpestre group recovers the Austrian population as a separate group from Z.amoenum and splits the latter species into three groups. The barcode gap in this scheme is, however, much lower (0.016) than the one proposed for Carychiidae in Z.alpestre group before  that might coincide with the large morphological variation found in this species (Even if it is not as large as the between-group divergence of other species pairs within the e.g., in . We are  species , which wZospeumamoenum described in Z.amoenum (see Z.amoenum assessed in enum see : fig. 6LZ.amoenum, avoiding the now outdated classification of these populations with Z.isselianum (as was done in Our study suggests that a final species assignment for the two Austrian populations is not possible until further supporting information becomes available. Until then, we classify these two Austrian populations as


Evidence indicates that Gα12, Gα13, and its downstream effectors, RhoA and Rac1, regulate neuronal morphology affected by stress. This study was aimed at investigating whether repeated stress influences the expression of proteins related to the Gα12/13 intracellular signaling pathway in selected brain regions sensitive to the effects of stress. Furthermore, the therapeutic impact of β(1)adrenergic receptors (β1AR) blockade was assessed.p.o./8–14 days) in the rat RS model to assess the role of β1AR in stress effects. RT-qPCR and Western Blot were used for mRNA and protein assessments, respectively.Restraint stress (RS) model in mice (2 h/14 days) was used to assess prolonged stress effects on the mRNA expression of Gα12, Gα13, RhoA, Rac1 in the prefrontal cortex (PFC), hippocampus (HIP) and amygdala (AMY). In a separate study, applying RS model in rats (3–4 h/1 day or 14 days), we evaluated stress effects on the expression of Gα12, Gα11, Gαq, RhoA, RhoB, RhoC, Rac1/2/3 in the HIP. Betaxolol (BET), a selective β1AR antagonist, was introduced (5 mg/kg/Chronic RS decreased mRNA expression of Gα12 and increased mRNA for Rac1 in the PFC of mice. In the mice AMY, decreased mRNA expression of Gα12, Gα13 and RhoA was observed. Fourteen days of RS exposure increased RhoA protein level in the rats’ HIP in the manner dependent on β1AR activity.Together, these results suggest that repeated RS affects the expression of genes and proteins known to be engaged in neural plasticity, providing potential targets for further studies aimed at unraveling the molecular mechanisms of stress-related neuropsychiatric diseases.The online version contains supplementary material available at 10.1007/s43440-021-00294-4. Excessive stress is considered an important etiological factor that can disturb the physiological integrity of the organism and contribute to various neuropsychiatric as well as neurodegenerative diseases , 2. The 1 or M3 receptor, or 5-HT2C serotonergic receptor , while Rac1 increased by 83% . The mRNA levels of Gα13 and RhoA remained unchanged in the PFC , sham/BET (p < 0.05) and chronic RS/BET (p < 0.05). Betaxolol did not change RhoA expression in the rat HIP after single  and repeated treatment  . Both processes, the gene transcription and translation to protein, are subject to multi-level and complex regulations. In certain circumstances, the mRNA degradation may be enhanced to maintain mRNA homeostatic turnover. Thus, 24 h after the last stress session, when the brain tissue was isolated, the mRNA changes might be poorly detectable.We are aware that our experimental approach has some limitations regarding the use of different species, i.e. mice and rats. Though both species are commonly used laboratory animals, their genetic diversity should be borne in mind. Furthermore, the regulation of gene expression depends indirectly on the activity of neural circuits. A certain difference between mice and rats may exist in the organization of brain circuits’ connectivity. For instance, Scheel et al.  describeWe have shown for the first time that pharmacotherapy with betaxolol, the β1AR blocker, normalized the stress-evoked increase of RhoA level in the rat HIP. The β1AR are anatomically and functionally predisposed to respond to high noradrenaline concentration released during stress in the HIP. Therefore, these receptors could be implicated in stress-related plasticity. Immunohistochemistry revealed dense expression of β1AR in the HIP . ElectroIn conclusion, both protein families, Gα12/13 and Rho, were previously shown to be associated with brain neuroplasticity during nonpathological conditions , 30. OurSupplementary file1 (DOCX 15 KB)Below is the link to the electronic supplementary material.


Veterinary pharmaceuticals can enter the environment when excreted after application and burden terrestrial and aquatic ecosystems. However, knowledge about the basic process of sorption in soils and sediments is limited, complicating regulatory decisions. Therefore, batch equilibrium studies were conducted for the widely used antiparasitics abamectin, doramectin, ivermectin, and moxidectin to add to the assessment of their environmental fate.KD of 38–642 mL/g for abamectin, doramectin, and ivermectin. Moxidectin displayed KD between 166 and 3123 mL/g. Normalized to soil organic carbon, log KOC coefficients were 3.63, 3.93, 4.12, and 4.74 mL/g, respectively, revealing high affinity to organic matter of soils and sediments. Within sediments, distribution resulted in higher log KOC of 4.03, 4.13, 4.61, and 4.97 mL/g for the four substances. This emphasizes the diverse nature of organic matter in both environmental media. The results also confirm a newly reported log KOW for ivermectin which is higher than longstanding assumptions. Linear sorption models facilitate comparison with other studies and help establish universal distribution coefficients for the environmental risk assessment of veterinary antiparasitics.We examined 20 soil samples and six sediments from Germany and Morocco. Analysis was based on HPLC-fluorescence detection after derivatization. For soils, this resulted in distribution coefficients KOW estimates also urge to re-assess this important regulatory parameter with contemporary techniques for all four substances.Since environmental exposure affects soils and sediments, future sorption studies should aim to include both matrices to review these essential pharmaceuticals and mitigate environmental risks from their use. The addition of soils and sediments from the African continent (Morocco) touches upon possible broader applications of ivermectin for human use. Especially for ivermectin and moxidectin, strong sorption further indicates high hydrophobicity and provides initial concern for potential aquatic or terrestrial ecotoxicological effects such as bioaccumulation. Our derived The online version contains supplementary material available at 10.1186/s12302-021-00513-y. Discovery of the anthelmintic, actinomycete-derived macrocyclic lactones in the 1970s and their advancement into widely available antiparasitic agents came as nothing less than a medical and economic revelation . GroupedKOC). However, with sorption as a fundamental process in soil chemistry [The use of pharmaceuticals for animals and humans can be accompanied by the release of drug residues into many environmental compartments. Particularly veterinary medicinal products (VMPs) for livestock, poultry or aquaculture come with the risk of direct drug excretion onto agricultural soils, involuntary application via manure fertilization, and release via runoff or erosion into surface waters. Exposure routes also include drug manufacturing and disposal, and all exposure scenarios raise the question of potential ecotoxicological effects and environmental fate of VMPs . In thishemistry , the datKOW). A routinely assumed order of hydrophobicity (as log KOW) appears to be: IVM  with guideline 123 [KOW of 5.6 (± 0.3) for IVM [KOW of 5.41 for IVM’s main component IVM B1a [Another complication for environmental risk assessments of the four antiparasitics is the lack of reliable and transparent octanol–water partition coefficients , ABA (4IVM (3.2  to 4.4 [IVM (3.2 ), DOR (4IVM (3.2 ), MOX . HoweveIVM  which carry nutrients and biomass to terrestrial habitats [Compelling evidence for this pathway is presented in a field study by Mesa et al.  who trear bodies , 50, enar bodies , ABA [52r bodies . When usr bodies . In wateof  <  6  and 15.9of  <  6  have beehabitats , 58. Thehabitats  and it ihabitats . Diepenshabitats  reiteratWe investigated the sorption of 4 macrocyclic lactones used as antiparasitic VMPs: the avermectins ABA , DOR, IVM, and the milbemycin MOX. Sorbates were used simultaneously in each sorption experiment and could be determined at once within an analytical run. The methodology for sorption studies is standardized in OECD guideline 106  to prediKOW estimates from KOC coefficients.This work promotes linear modeling with constrained intercepts to derive comparable sorption coefficients that enable robust regulatory decisions. To assess the general hydrophobicity of the antiparasitics and validate our sorption results, we derive and review German soil samples (label DE) were provided by the Hessian Agency for Nature Conservation, Environment and Geology (HLNUG). From a pool of samples, 17 were selected for sorption studies. The selection was based on OECD guidance instructions . These s1 derivatives. They contain at least 80% B1a component (C-25 s-butyl group) and less than 20% B1b component (C-25 isopropyl group), while DOR holds a sole cyclohexyl group at C-25. Chemically related, the smaller MOX molecule is a semisynthetic derivative of the milbemycin nemadectin, a fermentation product of Streptomyces cyanogriseus, whereas avermectins are derived from Streptomyces avermitilis [ABA and IVM are mixtures of semisynthetic avermectin Brmitilis , 65. Str® system. We used a CHROMABOND® solid-phase extraction (SPE) system (MACHEREY–NAGEL), custom handblown 45 mL glass centrifuge vials with a PTFE-coated silicon seal inside the screw cap, 500 mg Strata C18-E SPE cartridges , and 0.45 µm PTFE membrane syringe filters .Antiparasitics were purchased as analytical standards  in powder form: ABA , DOR , IVM , and MOX . Acetonitrile (ACN) (20060.320) and propan-2-ol (84881.320P), both  ≥  99.9% purity, came from VWR International; Calcium chloride dihydrate (102382) from Merck. Derivatization chemicals (> 99.0% purity) were bought at Sigma-Aldrich: N-methylimidazole (336092), triethylamine (T0886), trifluoroacetic anhydride (106232), and trifluoroacetic acid (302031). Purified water was prepared with a Milli-Q2 solution in purified water for a 1:30 (w/v) solid/solution ratio. This ratio was elaborated in own preliminary studies and is situated between the ratios of 1:20–:40 (w/v) applied in a comparable study by Rath et al. [2 solution for 24 h. Since these antiparasitics represent highly hydrophobic substances, powdered analytical standards were dissolved in ACN to create stock solutions of 4 × 106 µg/L. These solutions were combined in equal proportions for a mixed solution containing 1 × 106 µg/L of each substance. This was diluted into working solutions to simultaneously spike all drugs in a consistent volume of 30 µL ACN for a 0.1% (v/v) solvent concentration [2 solution or CaCl2 solution spiked with antiparasitics absent of soil/sediment. An experimental duration of 48 h was selected to reach apparent sorption equilibrium between macrocyclic lactone concentrations in soil/sediment and the aqueous phase. This was based on own preliminary kinetic studies and is supported by comparable experiments. [Experiments were conducted according to OECD guideline 106 . We susph et al. . Before ntration . Sorptioriments. , 20. An g for 30 min and 25 mL supernatant were added to a reservoir atop the SPE cartridges along with 8.333 mL propan-2-ol and 25 µL triethylamine. Dried cartridges were eluted with 10 mL propan-2-ol. Eluates were evaporated to dryness under an N2 stream at 60 °C. For reconstitution, 1000 µL ACN were added to each vial. Vials were then sonicated for 15 min, horizontally shaken (250 rpm) for 30 min, and again sonicated for 15 min. Between each step, samples were vortexed for 30 s. Subsequently, samples were derivatized and quantified by HPLC-fluorescence detection on an Agilent 1200 HPLC system as elaborated by Wohde et al. [R2  >  0.998.Analytical procedures were based on Wohde et al. , 66 and KD is defined as the ratio of substance concentration in the solid-phase Cs(eq) and the substance concentration in the aqueous-phase Caq(eq) at equilibrium with the equation:Cs(eq) is expressed in µg/g, Caq(eq) in µg/mL, and the KD in mL/g. The measured Caq(eq) was then used to indirectly estimate the remaining amount of substance in the solid phase, delivering Cs(eq). While Eq.  out of 20. Figures Soil and sediment samples were evaluated alike. Evaluation followed OECD guideline 106 . The disl et al.  who concl et al. . A constl et al.  may appel et al. . Thus, tsediment  expresseentclass1pt{minimam) and standard deviation (σ) of the response expressed as standard error of the y estimate (derived with the STEYX function in Microsoft Excel 2019). LOD is expressed as 3.3 × σ/m and LOQ as 10 × σ/m. These results are given in Table Caq(eq) in µg/L in any replicate was ABA (1.2), DOR (0.49), IVM (0.31). Conversely, the order for highest Caq(eq) was IVM (785), DOR (812), ABA (986). MOX conflicts this trend (0.62–247 µg/L), but was not used in soils with low %OC where a high Caq(eq) is suspected.Limit of detection (LOD) and limit of quantification (LOQ) for the HPLC-method were estimated according to recommendations in guideline Q2(R1) by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use . Based on  =  6). After a slight decrease over time, 72 h average fluorescence recovery remained at 86.5, 85.1, 92.7, and 89.3% for ABA, DOR, IVM, and MOX derivates compared to 20 min. Consistently, samples were measured within 24 h after derivatization. Measurements of up to 72 h after derivatization of calibration standards and samples should not impair overall results.We monitored the stability of fluorescent ABA, DOR, IVM, and MOX derivates for 20 min, 24, 48, and 72 h after derivatization  of purchased standards. IVM and MOX peaks showed no overlap with impurities of other analytes. We found a fluorescence increase for ABA and DOR main peaks of 1.1 and 3.4% and downscaled these accordingly. ABA and IVM sorption results will represent their major (> 96%) B2); CEC 8.5 meq/100 g). Therewith, we performed a mass balance determination [n  =  4) were: ABA 90.8 (4.1), DOR 107 (4.1), IVM 112 (2.1), and MOX 115.9% (1.6). This indicates that there is no relevant degradation of analytes within 48 h of shaking. Test substances can be considered to be stable. Determination with the indirect method [KD  was 532 (12) with only IVM and 471 (26) mL/g with four analytes. Under these conditions only negligible competition in sorption is indicated when all four substances are spiked at once. Additional information on the method validation is provided in the Additional file For additional method validation, a standard soil (LUFA 2.2) was purchased from LUFA Speyer  selected soils with the intercept constrained which creates a narrowing confidence corridor. For soils included in this regression, KD values ranged from 38 to 211 (ABA), 63 to 428 (DOR), 76 to 642 (IVM), and 166 to 3123 mL/g (MOX). This dispersion characterizes the variability of the selected soils. Individual KD values are listed in the Additional file Batch equilibrium studies were evaluated with linear sorption isotherms which served for calculation of KD values for each substance in this region of low fOC. Recommended in OECD guideline 106, the procedure of removing these soils from KOC calculations is corroborated by Krahe et al. [D above the regression. Of the thus five excluded soils, four displayed above average clay or silt concentrations among all 20 soils which could explain their relatively high sorption. Only DE04 with the highest sand content and low cation exchange capacity contradicts this rationale. With the presented parameters, its sorption behavior remains elusive. Both DE15 and DE02 showed relatively low KD values and contradicted the linear trend. This could be attributed to the fact that both samples originated from soil horizons with only moderately decomposed organic material and DE15 displayed stagnic properties. However, quality and detailed composition of organic material and its influence on the pH would have to be considered. Less decomposed organic material can harbor more hydrophilic groups, indicated by a lower elemental H/O ratio [OC calculation which are shown with hollow symbols. In a meta-analysis on atrazine sorption, Ou et al. [The detail area of Fig. e et al.  who also/O ratio . More avu et al.  concludeKOC values using the previously selected 13 soils. We distinguish between the preferable description as slope of a linear regression through the origin (RTO) and an ordinary least squares (OLS) regression with floating intercept. The table further shows KOC values expressed as the mean and median of individual KOC values from soils considered suitable for the linearized approach. This illustrates the ambiguity that comes with the need to define a single value which quantifies a substance’s sorption behavior.Next, Table KOC were 3.63 (ABA), 3.93 (DOR), 4.12 (IVM), and 4.74 mL/g (MOX). For the OLS model, the log KOC were 3.58, 3.87, 4.13, and 4.82 mL/g, respectively. The OLS y-intercepts amounted to 15.1 (ABA), 32.2 (DOR),  − 8.8 (IVM), and  − 349.9 mL/g (MOX). This partially reflects the increasing steepness of sorption from ABA  <  DOR  <  IVM  <<  MOX. However, the negative y-intercepts for IVM and MOX may also illustrate the shortcomings of an OLS regression with a floating intercept since negative sorption at zero or minimal OC would be implausible.Linearized RTO log R2 for the RTO KOC were 0.94 (ABA), 0.85 (DOR), 0.97 (IVM), and 0.97 (MOX). However, since a constrained y-intercept skews R2 calculations it makes it less meaningful and complicates comparison with R2 obtained from OLS. Instead, the standard error (SE) of both regressions can be an alternative measure [KOC appears to provide a more suitable fit. The R2 of the OLS KOC were 0.75 (ABA), 0.53 (DOR), 0.91 (IVM), and 0.93 (MOX). This reflects the wide spread of individual KD values, especially for ABA and DOR. Thus, it is conceivable that sorption of the more hydrophobic IVM and MOX is better explained with the KOC concept than for the slightly less hydrophobic ABA and DOR. This deduction is reiterated by Tolls [D-fOC relation. Lastly, we applied a Box–Cox transformation on all soil KD values to ensure normal distribution and subjected them to a multiple linear regression with OriginPro 2020b to compare them to soil properties from Table α  =  5%, soil OC demonstrated significant influence on ABA, DOR, and IVM KD. C/N and pH were significant predictors for ABA and DOR KD. The complete output is listed in the Additional file The  measure . In thisby Tolls  for hydrLitskas et al.  stressedFor IVM, promising mass drug administrations to livestock to target malaria vectors  may incrKD 10–161 mL/g [KD 57–396 mL/g [KD between 60 and 1953 mL/g and desorption KD between 47 and 431 mL/g. Previous ABA KOC ranged from 1495 to 7893 mL/g [KOC between 4000 and 25,800 mL/g were documented [KD data from linearly modeled sorption experiments with other studies which used Freundlich sorption isotherms to produce a KF. Although nonlinear models can provide a better fit, they lack comparability, especially when the Freundlich exponent differs significantly from 1. Based on Rath et al. [2 solution and shaking for 72 h. Mean percentual desorption  at a single concentration amounted to: ABA 4.6 (0.3), DOR 3.5 (0.1), IVM 2.9 (0.1), and MOX 2.6 (0.1)%. While only a fragmentary approximation for a full desorption study [Lastly, the observed sorption in soils is in line with reported distribution coefficients for ABA of 161 mL/g  and for 396 mL/g . Rath et396 mL/g  describe893 mL/g . For IVMcumented  and for cumented . HoweverKD showed a range from 21 to 296 (ABA), 35 to 376 (DOR), 53 to 915 (IVM), and 87 to 2326 mL/g (MOX). Less indicative, mean sediment KD (mL/g) were higher for ABA (106 vs. 98) and IVM (394 vs. 287) but lower for DOR (137 vs. 197) and MOX (861 vs. 1196). The distribution of all sediment KD results is illustrated in Fig. Compared to soils, sediment KD values for ABA, DOR, and IVM, and the third-highest for MOX among the six sediments. Since it cannot be inferred from the limited sediment sample size if this behavior is an outlier or part of an unknown trend, we removed MA06 from further calculations. However, parameters other than %OC could be more relevant for sorption in MA06. In comparison, MA04 with the highest sand content and similarly low %OC expectedly proved to be least prone for sorption. Because sediments also had a lower average %OC than soils, the resulting log KOC of 4.03 (ABA), 4.13 (DOR), 4.61 (IVM), and 4.97 (MOX) mL/g were higher compared to soils when using an RTO. As was observed in soils, sediment log KOC also ranked MOX  >> IVM  >  DOR  >  ABA, again reflecting the diverging behavior of MOX, presumably due to structural differences to the avermectins. Higher mean KOC in sediments than in soils were also documented by Chiou and Kile [KOC were about 1.7 times higher than soil KOC. Adding to that, we report sediment KOC to soil KOC ratios of 1.8 for ABA, 1.2 for DOR, 2.4 for IVM and 1.4 for MOX when comparing five selected sediments and 13 selected soils. Higher sediment than soil KOC with a factor of about 1.9 was also shown for the antiparasitic drug albendazole by Mutavdžić Pavlović et al. [Although sediment MA06 had the lowest %OC (0.42), it displayed the highest and Kile . For a lć et al. . Chiou ać et al.  reported50  =  127 days) driven by strong sorption in the sediment. Mesocosm experiments by Roberts et al. [KD data.Change of organic matter composition during sedimentation is known to affect sorption especially for nonionic compounds  and couls et al.  with tros et al.  and to ps et al. , 88. This et al.  and orgaKOC and KOW of a substance are inextricably linked since both serve the concept that OC and 1-octanol act as hydrophobic counterparts to a chemical [KOW is also the most frequently used indicator of hydrophobicity of a chemical and an essential parameter in toxicology and environmental sciences [KOW of the studied antiparasitics if the RTO log KOC were the only available variable. For this, we applied well-known concepts [KOC and log KOW based on log KOW data. These predictions are depicted in Table KOC data should allow for an estimate of the antiparasitics’ hydrophobicity when expressed as log KOW. However, these estimations must not be overstated. A KOW is easier to obtain experimentally than performing complex sorption batch studies. Hence, applying the slow-stirring method from OECD guideline 123 [KOW data for the studied VMPs. The log KOW of 5.6 (± 0.3) for IVM [KOC from sediment studies.The chemical . The KOWsciences . Over ticoncepts , 91–93 wline 123  would yi for IVM  defined  for IVM  come cloKOW is a hydrophobicity indicator linked to a molecule itself and it is immaterial whether said molecule would be released into soil, sediment, or other parts of the environment. Thus, an implied distinction between a log KOW based on either soil or sediment sorption coefficients remains theoretical. Still, with the derived log KOC data, all four substances displayed a log KOW  >  4 except for ABA in soils if calculated according to Baker [KOW could underestimate the log KOC. A reverse estimate based solely on sorption coefficients could therefore overestimate the log KOW. However, Tolls [KOC predictions would not deviate to a great extent which bolsters our predictions. The use of these estimations is to provide a general indication of hydrophobicity based on a common dataset of KOC for all four substances.The log to Baker . This mato Baker  with IVMto Baker  describer, Tolls  also conKOC to KOW correlations can be useful if transparent and verifiable KOW data are not available. Benefits are conceivable since the KOW is also an important parameter for environmental risk assessments. Prichard et al. [KOW estimations and used atomic parameters to calculate the following order of coefficients (log KOW): EPR (4.4), IVM (4.8), ABA (5.3), DOR (5.6), MOX (6.0), and selamectin (6.3). Fittingly, selamectin was also assessed by Römbke et al. [KOW of 6.0 (± 0.7). Meanwhile, risk assessments for VMPs rely on robust data. Dissipation of macrocyclic lactone antiparasitics varies depending on climate and field conditions [KOW and their environmental fate properties would be admirable.Although more sophisticated approaches such as quantitative structure‐activity relationships can be employed, d et al.  providede et al.  with thenditions , 66, 94 KOC concept does not account for organic matter composition and may misinterpret substance behavior at particular locations, especially in sediments. If enough data is available, a multiple linear regression with all soil/sediment properties is always advisable. Also, while for IVM low metabolization has been described in animal species [KOW based on log KOC highlight the possible KOW discrepancies and a precarious aspect of regulatory decision-making: while data may appear insufficient, they may be the only data available.A limitation of sorption studies with pharmaceutical compounds is the transferability to the environmental reality. The  species , human m species . Transfo species . Our estKD) and normalized to organic carbon (as KOC) is characterized by the order: ABA  <  DOR  <  IVM  <<  MOX. Exemplary desorption from soils indicates mostly irreversible sorption processes and follows the same rationale with MOX showing the lowest transfer back into the liquid phase. The applied SPE-HPLC method with fluorescence detection is suitable for reliable quantification of all four analytes at once.The investigated antiparasitics show strong sorption to the organic matter of soils and also sediments. Sorption strength in general  when subjected to the presented SPE procedure compared to directly measured standards. Table S2. Output (OriginPro 2020b) of the multiple linear regression with transformed KD values and soil properties. Table S3. Supplemental data for Fig. 2. Individual soil KD values (mL/g). Table S4. Supplemental data for Fig. 3. Individual sediment KD values (mL/g). Figure S1. Chromatogram of standard solution with all 4 analytes . Figure S2. Chromatogram of the extracted aqueous soil solution with all 4 analytes .


The application of AgNPs did not affect the germination efficiency; however, diverse results were reported for the growth and biochemical activity of the seedlings, depending on the species tested and the AgNPs concentration. Tomato seedlings treated with nanoparticles, particularly at 100 mg·L−1, had shorter shoots with lower fresh and dry weights and produced roots with lower fresh weight. Simultaneously, at the biochemical level, a decrease in the content of chlorophylls and carotenoids and an increase in the anthocyanins content and guaiacol peroxidase (GPOX) activity were reported. AgNPs-treated radish plants had shorter shoots of higher fresh and dry weight and longer roots with lower fresh weight. Treatment with 50 mg·L−1 and 100 mg·L−1 resulted in the highest and lowest accumulation of chlorophylls and carotenoids in the leaves, respectively; however, seedlings treated with 100 mg·L−1 produced less anthocyanins and polyphenols and exhibited lower GPOX activity. In kale, AgNPs-derived seedlings had a lower content of chlorophylls, carotenoids, and anthocyanins but higher GPOX activity of and were characterized by higher fresh and dry shoot weights and higher heterogeneous biometric parameters of the roots. The results of these experiments may be of great significance for broadening the scope of knowledge on the influence of AgNPs on plant cells and the micropropagation of the vegetable species. Future studies should be aimed at testing lower or even higher concentrations of AgNPs and other NPs and to evaluate the genetic stability of NPs-treated vegetable crops and their yielding efficiency.The interactions between nanoparticles and plant cells are still not sufficiently understood, and studies related to this subject are of scientific and practical importance. Silver nanoparticles (AgNPs) are one of the most commonly produced and used nanomaterials. This study aimed to investigate the influence of AgNPs applied at the concentrations of 0, 50, and 100 mg·L Engineered nanomaterials (ENMs) are atomic or molecular aggregates, which, due to their small size between 1 and 100 nm, present unique and different chemical and physical characteristics from those at the micrometric scale, such as a large surface-area-to-volume ratio, the ability to engineer electron exchange, high surface reactive capabilities, high reactivity, and easy absorption by cells. Over the past few decades, ENMs have been used in industrial sectors and in daily life, in biotechnology, and in agriculture, which has led to their inevitable release into the environment and ecosystems and has created possible hazards for all living organisms, including plants. Understanding the influence of ENMs on plants is crucial, not only from the scientific point of view but also for the assessment of environmental risks on food safety and human health ,3,4,5.A variety of nanomaterials has been developed, including carbon nanotubes, iron, aluminum, copper, gold, silver, silica, zinc, zinc oxide, titanium oxide, among others . Silver The influence of ENMs on living organisms is complex, and interestingly, the responses of plants to nanomaterials are often contradictory. The same type of nanomaterial may have various biological impacts depending on the plant genotype, organ, age, and developmental stage; physicochemical properties, concentration, size, shape, surface area, surface coatings, and the stability of the particles or solvent applied as well as the experimental methodology  ,5,8. Pla−1 positively affected the germination in Allium cepa L. with no negative effects on the further growth and development of seedlings being observed [2O3 nanocubes, Fe2O3 short nanorods, Fe2O3 long nanorods, and TiO2 NPs tested at concentrations 5–150 mg·L−1 inhibited the germination of rice (Oryza sativa L.) seeds but promoted the growth of roots and shoots and had no effect on the fresh weight [Pennisetum glaucum L. (50 mg·L−1) [Alnus subcordata L. (10 and 20 mg·L−1) [Zea mays L. (0.005–2.5 mg·L−1) [Seed germination, beginning with dry mature seed imbibition and ending with a radical protrusion, is the first step of plant growth and is not only crucial for seedling establishment but is also important for crop yield . The gerobserved . Interesh weight . Convers mg·L−1) ; decreas mg·L−1) ; and had mg·L−1) .−1 on the in vitro seed germination, growth, and biochemical activity of developed seedlings in three vegetable species that are known and commonly consumed worldwide—Solanum lycopersicum L. (Solanaceae), Raphanus sativus L. var. sativus (Brassicaceae), and Brassica oleracea var. sabellica L. (Brassicaceae). The simultaneous testing of species belonging to the same or different botanical family and differing in terms of traits such as seed size, germination dynamics and growth of vegetative organs, and the type of organ used as a crop allows the multifaceted influence of silver nanoparticles on plants to a high extent to be determined.Despite the fact that different studies have been performed on the regulation of seed germination, the interactions between ENMs and plant cells are not yet fully understood, and require further investigation and elucidation, especially due to the hazards of using NPs treatments in plants. Therefore, the present research aimed to broaden the scope of knowledge on the influence of AgNPs applied at the concentrations of 50 and 100 mg·L−1 CaCl2·2H2O, 41.7 mg·L−1 FeSO4·7H2O; 55.8 mg·L−1 Na2EDTA·2H2O)  increased by half. The medium contained 30 g·L−1 sucrose and was solidified with 0.8% (w/v) Plant Propagation LAB-AGARTM . No plant growth regulators (PGR) were added. The medium pH was set to 5.8 after adding all of the nutrients. Next, 40 mL of the medium was poured into 350-mL glass jars sealed with plastic and was autoclaved at 121 °C for 20 min.The modified Murashige and Skoog (MS) basal medium was used  with the−2·s−1.In vitro cultures were kept in the growth room at a temperature of 23 ± 1 °C with a 16/8-h day/night regime. Philips TLD 36W/54 fluorescent lamps emitting cool daylight  were used as the light source. The photosynthetic photon flux density was set at 35 μmol·mIn the experiments, silver nanoparticles (AgNPs) manufactured by Nanoparticles Innovation NPIN s.c.  were used. The AgNPs were produced by the seeded-mediated growth method ,26. The Solanum lycopersicum L. ‘Poranek’, radish Raphanus sativus L. var. sativus ‘Ramona’, and kale Brassica oleracea var. sabellica L. ‘Nero di Toscana’ were used. Seeds were purchased at PlantiCo—Hodowla i Nasiennictwo Ogrodnicze Zielonki Sp. z o.o. . Before establishing the in vitro culture, the seeds were surface disinfected. First, they were rinsed in running tap water. Next, the seeds were incubated in sequence in a 5% (v/v) detergent solution for 5 min; a 70% (v/v) ethanol solution for 1 min; and a 1.5% (v/v) NaClO solution for 10 min . Afterward, the seeds were rinsed twice in sterile bi-distilled water for 5 min. Then, the seeds were inoculated on the modified MS medium, with five seeds in each jar. At concentrations of 50 and 100 mg·L−1, the colloid of the AgNPs was poured with a sterile pipette tip and was gently mixed to cover the entirety of the medium and seed surfaces (1 mL per each culture jar). Seeds poured with sterile bi-distilled water were the control object. For each experimental treatment, 20 repetitions were used with 5 seeds in each (1 jar). During the first week of the experiment, the jars were covered with aluminum foil to limit access to light.In the experiments, seeds of tomato Observations of seed germination and seedling growth were performed weekly for three successive weeks. The dynamics of seed germination were determined based on the number of germinating seeds. The biometric data of all of the developed three-week-old seedlings, such as the shoot length (cm), fresh weight of the shoot (mg), dry weight of the shoot (mg), the root length (cm), fresh weight of the root (mg), and dry weight of the root (mg), were collected.(w/v) polyvinylpyrrolidone (PVP) , according to the protocols of Homaee and Ehsanpour [g for 20 min at 4 °C . Supernatants were used to determine the total protein content and the activities of the antioxidant enzymes. The protein content was measured based on the Bradford method [The leaves of three-week-old seedlings were used as fresh tissue samples. Chlorophylls and carotenoids were extracted according to Lichtenthaler  using 10hsanpour . The homd method  with bovd method  by measud method  methodold method .max): for chlorophyll a and b at 645 and 662 nm, for carotenoids at 470 nm, for phenolics at 765 nm, for proteins at 595 nm, for SOD at 560 nm, and for GPOX at 470. The contents of the plant pigments and the phenolic compounds were calculated per 1 g of sample fresh weight (FW). The enzymatic activity U (μmol·min−1) was calculated per 1 mg of protein. All of the performed biochemical analyses were repeated six times.The spectrophotometric analysis of the extracts was performed in the SmartSpec PlusTM spectrophotometer  at specific wavelengths (λp ≤ 0.05. Data were presented as mean ± standard deviation (SD). For data expressed as a percentage, the Freeman–Tukey double-arcsine transformation was used. Tables with results provide numerical data, with the alphabet indicating the homogeneous groups.The experiments were set up in a completely randomized design. Data were statistically verified using Statistica 13.3  software. The analysis of variance (ANOVA) was performed, and means were compared with the Tukey post hoc test at the significance level of The appearance of the first germinating seeds was observed already in the first week of in vitro culture and proceeded to the third week, but with different intensities depending on the tested vegetable species .−1 AgNPs, only 7 and 3 germinating seeds were reported, respectively. The highest increase in germination was found in the second week. Finally, in the third week, germination reached 67 seeds in the control object, 52 seeds in the 50 mg·L−1 AgNPs treatment and 53 seeds in the 100 mg·L−1 AgNPs treatment, without statistically significant differences. Similarly, the addition of silver nanoparticles  to the MS medium did not affect the germination rate in Physalis peruviana L. (Solanaceae) [−1) seeds of seven S. lycopersicum cultivars germinated faster, germinating within the first three days, whereas the control seeds germinated later. Interestingly, the AgNPs at concentrations of 75 and 100 mg·L−1 caused a significant decrease in the germination percentage in two of the tested cultivars, while no differences were observed in the germination percentage in the other five cultivars. As suggested by the authors, the NPs-accelerated seed germination in the first days after treatment may result from better water uptake by the seeds, whereas the later decrease in germination efficiency can be attributed to the accelerated breakdown of the stored nutrients and/or alternations in the permeability properties of the cell membrane resulting from AgNPs application. According to Yasur and Rani [During the first week, the germination of 15 control tomato seeds was observed, whereas in the experimental combinations with 50 and 100 mg·Lanaceae) , the AgN−1 and 100 mg·L−1 AgNPs treatment, respectively. In the following weeks, the number of germinated seeds increased slightly (from 7 to 11). No significant difference was found between the number of germinated seeds depending on the AgNPs treatment at the end of the observation period .As for radish, most of the seeds had already germinated in the first week of culture, with 92 seeds germinating in the control object and 86 and 88 seeds germinating after 50 mg·L−1 AgNPs and 100 mg·L−1 AgNPs treatments, respectively, and 58 seeds in the control object;). The number of germinating seeds increased significantly in the second week and finally amounted to 94 and 96 after the 50 mg·L−1 AgNPs and 100 mg·L−1 AgNPs applications, respectively, and 89 in the control object. These values were not significantly different.The vast majority of kale seeds also started to germinate during the first week of culture  [Brassica rapa ssp. rapa L. decreased when the AgNPs concentration increased although the NPs were applied at low concentrations . In Ricinus communis L., treatment with AgNPs (70 nm) at a very high concentration of 500–4000 mg·L−1 did not disturb the germination process [−1) were characterized by the lowest length (3.64 cm), fresh weight (63.34 mg), and dry weight (3.31 mg)  and root dry weight (1.45–3.84 mg). On the other hand, the control and 100 mg·L−1 AgNPs-derived plants had the highest and lowest root fresh weights .Silver nanoparticles that were 29 nm in size at concentrations 25–50 mg·L mg·L−1) . Accordi mg·L−1) , the ger process . Differe process . Seed co process . Most li process . The inf process . Tomato 3.31 mg) . The hig−1 AgNPs had the longest roots (12.07 cm), whereas the control plants had the shortest roots (8.87 cm). However, the root FW of the seedlings treated with 50 and 100 mg·L−1 AgNPs was lower  than in the control seedlings (53.47 mg). No differences were found between the tested concentrations of silver nanoparticles in terms of the root dry weight.As for radish, the treatment with AgNPs caused a significant decrease in the length of the shoots and a simultaneous increase in fresh and dry weight . The con−1 silver nanoparticles, but the 50 mg·L−1 AgNPs-derived shoots were significantly longer (11.36 cm) than those after the 100 mg·L−1 AgNPs-treatment (9.78 cm) and the controls (9.26 cm) (−1 AgNPs-derived plants produced the longest roots (9.89 cm), which were also characterized by low fresh (9.22 mg) and dry (2.19 mg) weights. In contrast, the 100 mg·L−1 AgNPs-derived seedlings had the shortest roots (8.32 cm) but had the highest fresh (19.64 mg) and dry (2.97 mg) weights.In kale, the highest values for shoot fresh (196.07–245.39 mg) and dry weight (15.26–17.98 mg) were found in the plants produced from seeds treated with 50 and 100 mg·L9.26 cm) . The 50 −1 AgNPs promoted an increase in the fresh and dry weights of the P. peruviana seedlings, whereas a higher concentration (15.4 mg·L−1) led to a growth reduction of the shoots and roots [Brassica juncea seedlings increased with concentrations of up to 200 mg·L−1 AgNPs and decreased with higher nanoparticle concentrations, indicating that this phenomenon is dose-dependent [−1 was deleterious for the growth of B. rapa ssp. Rapa seedlings. A 10% increase in the fresh weight of seedlings was recorded at 1 mg·L−1, while exposure to higher concentrations resulted in a gradual decrease in the fresh weight. Root length was increased at 1 mg·L−1 concentrations and decreased at higher concentrations. Seedlings treated with 10 mg·L−1 concentrations had the shortest shoots [−1 did not disturb the growth of R. communis seedlings [S. lycopersicum, root elongation was inhibited in the seven tested cultivars after using 25, 50, 75, and 100 mg·L−1 AgNPs concentrations compared to the control. As for the shoot length, a drastic decrease in the value of this parameter was usually reported, with the exception of one cultivar treated with 25 mg·L−1 AgNPs and another cultivar treated with 25 and 75 mg·L−1 concentrations. The decrease in root length was stronger than in the shoots of all of the cultivars that were studied [The low concentration of 0.385 mg·Lnd roots . Similarependent . Treatmet shoots . On the ntration .4 mg·L−1 studied . In the −1, the decrease of the root fresh weight and shoot parameters in tomato, and the decrease of root fresh weight and shoot length in radish. At the molecular level, nanoparticles cause the upregulation of the genes involved in cell division and carbon/nitrogen metabolism. The negative effects observed in seedling growth are probably due to chromosomal aberrations and mitotic abnormalities, leading to reduced cell division in the root meristem, hormonal imbalance, ROS overproduction, and an increased level of lipid peroxidation [Triticum aestivum L. [Thymus daenensis Celak seedlings.Roots are the primary tissues through which AgNPs enter plants and are the organ that is the most responsive to nanoparticle effects. AgNPs accumulate in the initial cells of the root columella, preventing cell division and hence the growth of roots. The presence of AgNPs in plasmodesmata, precluding the transport of nutrients, could lead to the reduction of plant biomass. Moreover, AgNPs can additionally modify the absorption of metal ions important for plant development . These mxidation . On the tivum L. . As repotivum L. , treatme−1 AgNPs-treated radish seedlings might be associated with the avoidance mechanism of roots to AgNPs as a stress factor. Moreover, when nanoparticles are used at a lower concentration, the hormesis effect is often observed, i.e., a generally favorable biological response to low exposures to a toxin/stressor. This phenomenon could also explain the increase in shoot and root length in kale plants after using 50 mg·L−1 AgNPs [The stimulation of root elongation in 50 and 100 mg·L−1 AgNPs . Nonethea (1.30 mg·g−1 FW), chlorophyll b (0.54 mg·g−1 FW), total chlorophyll (1.84 mg·g−1 FW), and carotenoids (0.36 mg·g−1 FW) than seedlings exposed to AgNPs  than in the untreated control plants (2.45), whereas the highest and the lowest chlorophylls/carotenoids ratio was reported in the control object (5.05) and in the 50 mg·L−1 AgNPs-treatment plants (4.42), respectively. The kale seedlings did not differ in terms of chlorophyll a/b (2.37–2.52) and chlorophylls/carotenoids (5.19–5.60) ratios, regardless of the AgNPs treatment. In contrast, in radish, the highest content of chlorophyll a (1.25 mg·g−1 FW), chlorophyll b (0.50 mg·g−1 FW), total chlorophyll (1.75 mg·g−1 FW), and carotenoids (0.30 mg·g−1 FW) were detected in seedlings after the application of 50 mg·L−1 AgNPs. The highest ratio of chlorophyll a/b (2.90) and the lowest ratio of chlorophylls/carotenoids (5.60) were found in the radish control plants.The leaves of the control tomato seedlings produced significantly more chlorophyll to AgNPs . A simil−1 FW) and kale (1.02–1.08 mg·g−1 FW) seedlings compared to the control objects—1.50 and 1.05 mg·g−1 FW, respectively (−1 AgNPs-treatment plants had the lowest phenolics content (1.51 mg·g−1 FW). Varied results were obtained regarding the anthocyanins accumulation in the leaves of the tested vegetable species. In tomato, the highest content of these compounds was reported in the 100 mg·L−1 AgNPs-derived seedlings (0.23 mg·g−1 FW), while the lowest concentration was detected in the control seedlings (0.17 mg·g−1 FW). Conversely, the radish control seedlings produced more pigments from this group (0.30 mg·g−1 FW) than seedlings from the 100 mg·L−1 AgNPs experimental object (0.22 mg·g−1 FW). The exposure to silver nanoparticles at the concentrations of 50 and 100 mg·L−1 significantly decreased the anthocyanins synthesis in kale.Treatment with silver nanoparticles did not affect the content of phenolic compounds in tomato (1.52–1.57 mg·gectively . In cont−1 FW) and kale (13.21–13.35 mg·g−1 FW) seedlings (−1 AgNPs-derived radish seedlings contained significantly less protein (12.26 mg·g−1 FW) than seedlings from the 50 mg·L−1 treatment (13.41 mg·g−1 FW) and the control (13.77 mg·g−1 FW). In each species, none of the tested experimental objects differed in terms of SOD activity. In contrast, in tomato, the application of 100 mg·L−1 AgNPs resulted in an almost two-fold higher GPOX activity (10.85 U) than in the control seedlings (5.54 U). Radish seeds exposed to 100 mg·L−1 AgNPs produced seedlings with lower guaiacol peroxidase activity (2.18 U) than the control seedlings (5.16 U) and the seedlings in the 50 mg·L−1 treatment (4.12 U). As for kale, a significant increase in GPOX activity was reported in the seedlings treated with silver nanoparticles at the concentration of 50 mg·L−1 (3.09 U) and 100 mg·L−1 (4.11 U) compared to the nontreated control seedlings (1.28 U).The treatment of seeds with AgNPs did not influence the total protein content in the leaves of the three-week-old tomato  resulted in a significantly lower total chlorophyll content in 2-week-old B. rapa ssp. rapa seedlings [−1 AgNPs increased the chlorophyll and carotenoid content in the radish ‘Ramona’ seedlings, whereas the 100 mg·L−1-treatment decreased the values of these parameters. Similarly, all AgNPs-treated tomato ‘Poranek’ and kale ‘Nero di Toscana’ seedlings were characterized by lower chlorophyll and carotenoid contents. The reduction in the chlorophyll content was significant at the higher AgNPs concentrations (1.54 and 15.4 mg·L−1) in the P.s peruviana seedlings [Seed priming with AgNPs significantly affected photosynthetic parameters such as the chlorophyll and carotenoid content, photosynthetic rate, stomatal conductance, and transpiration rate in nd yield . An increatments . Intereseedlings . The treeedlings . The loseedlings  and, fin−1-treated radish seedlings, whereas the 100 mg·L−1-treated tomato seedlings had a higher content of these compounds. Anthocyanin content was significantly increased with higher concentrations of AgNPs in B. rapa ssp. rapa seedlings. The accumulation of anthocyanins resulting from AgNPs exposure might be due to higher oxidative stress caused by the AgNPs. Moreover, the 5 and 10 mg·L−1 AgNPs induced the expression of genes related to phenolics biosynthesis and down-regulated the carotenoids gene expression [R. communis shoots increased at 500 and 1000 mg·L−1 treatments and decreased gradually at the 2000 and 4000 mg·L−1 AgNPs treatments [In the conducted experiments, the anthocyanin content was significantly lower in all of the AgNPs-treated kale seedlings and in the 100 mg·Lpression . The poleatments . TherefoP. peruviana [−1 AgNPs treatments in R. communis [P. glaucum, silver nanoparticles improved superoxide dismutase and catalase activity but decreased peroxide activity [B. juncea, GPOX activity increased continuously with an increasing concentration of AgNPs, reaching the maximum at 400 mg·L−1. No differences were reported in the activity of CAT in the AgNPs concentration range of 25–100 mg·L−1. The activity of this enzyme was significantly higher at 200 and 400 mg·L−1. The lowest APX activity was reported at 50 mg·L−1, whereas the highest activity was observed at 200 and 400 mg·L−1 [−1) in 4-week-old Solanum tuberosum L. plants. Interestingly, the activities of these enzymes were found at the maximum level at 10 mg·L−1 AgNPs but declined at 20 mg·L−1 [With respect to the influence of silver nanoparticles on the activity of enzymes associated with oxidative stress, no differences were reported in the SOD and APX activities in eruviana . On the communis . As for activity . In B. j0 mg·L−1 . The SOD0 mg·L−1 . How can0 mg·L−1 . Interes0 mg·L−1 . The infP. glaucum [Zea mays L. [Lactuca sativa L. treated with silica, palladium, gold, and copper nanoparticles [Triticum aestivum L. treated with copper and zinc oxide [Capsicum annuum L. treated with zinc oxide nanoparticles [Interestingly, contrary results regarding the germination efficiency, growth, and biochemical activity of seedlings, depending on the species tested and type of nanoparticles used, were reported by other authors, e.g., in  glaucum  and Zea  mays L.  treated articles ; in Tritnc oxide ; or in Carticles . These rThe further development of nanotechnology is inevitable, and the emission of various ENMs to the environment will dynamically increase. The spectrum of possible interactions between nanomaterials and living organisms, including plants, is very diverse. Contradictions in plant responses to ENMs depend on several factors, i.e., plant genotype, ENMs type and properties, method and time of ENMs treatment, or interactions with different environmental factors. Each subsequent experiment broadening the scope of knowledge on the influence of nanomaterials on plants seems to be fully reasonable. The results presented in this paper indicate that silver nanoparticles of the same size, at the same concentration, and applied with a uniform methodology affect different biometric and biochemical responses depending on whether the tested plant species belongs to the same or different botanical family. Future studies should aim to test different types and concentrations of ENMs to evaluate their possible utility in the micropropagation of vegetable plant species and to verify the genetic and phenotype stability of the treated plants for breeding purposes.