Most studies on buoyant microplastics in the marine environment rely on sea surface sampling. Consequently, microplastic amounts can be underestimated, as turbulence leads to vertical mixing. Models that correct for vertical mixing are based on limited data. In this study we report measurements of the depth profile of buoyant microplastics in the North Atlantic subtropical gyre, from 0 to 5 m depth. Microplastics were separated into size classes (0.5–1.5 and 1.5–5.0 mm) and types (‘fragments’ and ‘lines’), and associated with a sea state. Microplastic concentrations decreased exponentially with depth, with both sea state and particle properties affecting the steepness of the decrease. Concentrations approached zero within 5 m depth, indicating that most buoyant microplastics are present on or near the surface. Plastic rise velocities were also measured, and were found to differ significantly for different sizes and shapes. Our results suggest that (1) surface samplers such as manta trawls underestimate total buoyant microplastic amounts by a factor of 1.04–30.0 and (2) estimations of depth-integrated buoyant plastic concentrations should be done across different particle sizes and types. Our findings can assist with improving buoyant ocean plastic vertical mixing models, mass balance exercises, impact assessments and mitigation strategies. Microplastics are defined as plastic particles smaller than 5 mm in length35731112131112et al.et al.et al.et al.et al.et al.et al.et al.11121112Several studies10161718et al.10161718One of the main determinants of the vertical distribution of buoyant ocean plastics is the speed at which the particles rise to the surface once vertical downward mixing has occurred1112In this study, a large number of depth profiles for microplastics (0.5–5.0 mm) were measured over a range of sea states (Beaufort 1–5), as well as for different plastic types and size classes, using multi-level net tow samples . We demonstrated that the vertical distribution of buoyant microplastics varies not only with environmental conditions, but also with the plastic size and shape. The buoyant terminal rise velocities of microplastics were measured, discriminating between different plastic sizes and types. The comprehensive datasets of microplastic terminal rise velocities and depth profiles reported in this study can be used to validate and improve hydrodynamic models predicting the vertical distribution of buoyant plastics at sea.A total of 47 net tows were carried out using a ‘multi-level trawl’ (MLT)The content of each cod-end was divided into two size classes by consecutively sieving the sample over 5000, 1500 and 500 microns meshes. The two size classes contained 0.5–1.5 mm and 1.5–5.0 mm particles. Since macroplastics (>5 mm) and plastics smaller than 0.5 mm were not the focus of this study, the materials in the 5000 micron sieve and the <0.5 mm particles were not analysed. The collected particles were placed in containers with filtered salt water  to facilitate the separation of buoyant plastics from biomass. Floating particles identified as plastics were manually extracted using forceps, separated into types, and counted. Microplastics were classified as (1) ‘fragments’ - either hard plastic or plastic sheet fragments; (2) ‘lines’ – fibres from ropes, fishing nets or lines; (3) ‘foam’ – particles of expanded polystyrene (Styrofoam) or other types of foam material; and (4) ‘pellets’ – preproduction plastic nurdles in the shape of a cylinder, disk or sphere. Once counted and categorised, the particles were washed with distilled water, transferred to aluminium dishes, dried overnight at 60 °C, and weighed with an analytical balance .et al. (2002), which is included in the rcalcofi packageMicroplastic numerical/mass depth profiles were calculated by dividing the total number/mass of microplastic particles collected in each MLT net by the water volume that passed through the net. Depths sampled by each MLT net were inferred using the median depth recorded at the bottom of the MLT device by a HOBO U20L Water Level Data Logger , as well as separately for each plastic category: fragments 0.5–1.5 mm, fragments 1.5–5.0 mm, lines 0.5–1.5 mm and lines 1.5–5.0 mm . Due to et al.The terminal rise velocities of microplastics were measured individually for the two sizes and types considered in this study, using the method described by Reisser An exponential decrease function was fitted to each normalized microplastic depth profile. In this model, the normalized microplastic concentration (N) was modelled as a function of the decrease coefficient (λ) and depth (z)A lower λ value indicates stronger vertical mixing or a smaller buoyant terminal rise velocity. Due to some plastic concentrations at the surface being zero, resulting in “NA” values for normalized concentrations at greater depths, not all decrease coefficients could be calculated. Of the potential 188 λ values (47 stations *2 types *2 size classes), 181 could be calculated, both for mass and numerical concentrations. The parameter was fitted using the nls function from the nlstools packageet al.b (1.5 u*w k Hs)−1 et al.*w is the frictional velocity of water, k the von Karman constant, Hs the significant wave height and wb the microplastic buoyant terminal rise velocity. The frictional velocity can be approximated as u*w = 0.0012 W10, with W10 the wind speed at 10 m height28We tested whether Beaufort sea state, particle size and particle type affected λ values for mass and numerical profiles using a generalized linear mixed model (GLMM). The lme4 package in combination with the glmer test was usedet al.Different microplastic rise velocities were utilized when Kukulka’s model was applied. In addition to the mean ± standard deviation of the buoyant terminal rise velocity reported by Kukulka et al.The microplastic amount missed by typical ocean surface samplers (i.e. manta and neuston nets) was estimated. For this underestimation (in %), −3 and 1.13 mg m−3 of microplastic fragments at the ocean surface (top two nets) compared to 0.02 fragments m−3 and 6.1*10−3 mg m−3 in the bottom net. Depth integrated (0–5 m) microplastic mass concentrations  were on average 9 times lower than surface concentrations (top two nets): 0.128 and 1.159 g m−3, respectively  for fragments with size 1.5–5.0 mm and 0.5–1.5 mm were 0.019 ± 0.006 m sectively . The buoet al.et al.et al.The exponential decrease model fitted the data well Ret al.. The uncet al.et al.Size and type of microplastics significantly explained the differences in the decrease coefficients (p < 0.05), both for mass and numerical results. Based on our statistical fitted parameter λ, we calculated average, minimum and maximum correction factors, which correct surface concentrations for mixing to 5 m depth. Average values were calculated assuming that Beaufort 1–5 occur equally often. For manta nets (sampling depth is 0–15 cm)et al.et al.et al.1112We studied the vertical mixing of buoyant microplastics (0.5–5 mm), based on extensive data from the North Atlantic accumulation zone. Numerical and mass concentrations decreased exponentially with depth, the strongest decline occurring in calmer oceans and for bigger particles. Similar to previous research121112et al.Other studies report underestimation factors in the same order of magnitude as found in our study. Brunner −2 16−2 found in the present study.Both mass and numerical abundance of microplastics are reported in this study, since measurements of mass are required for mass balance and emission studies, while particle numbers are more relevant for the assessment of exposure risks32et al.Recent studies observed a lower than expected amount of millimetre-sized particles at the surface, when assuming a linear fragmentation model in equilibrium91636163839111216111210161718et al.1016171810161718916The hydrodynamic model of Kukulka Other vertical microplastic mixing models exist, that focus on Langmuir circulations and breaking waves15121520Knowledge of the distribution of plastics, both horizontally and vertically, is essential to understand and mitigate their impact on the marine environmentHow to cite this article: Kooi, M. et al. The effect of particle properties on the depth profile of buoyant plastics in the ocean. Sci. Rep.6, 33882; doi: 10.1038/srep33882 (2016).


Alterations in dopamine neurotransmission are generally associated with diseases such as attention-deficit/hyperactivity disorder (ADHD) and obsessive-compulsive disorder (OCD). Such diseases typically feature poor decision making and lack of control on executive functions and have been studied through the years using many animal models. Dopamine transporter (DAT) knockout (KO) and heterozygous (HET) mice, in particular, have been widely used to study ADHD. Recently, a strain of DAT KO rats has been developed . Here, w Dysregulation in forebrain dopaminergic signaling and function is associated with many neurological and neuropsychiatric diseases, such as schizophrenia (SZ), attention-deficit/hyperactivity disorder (ADHD), obsessive-compulsive disorder (OCD), and Parkinson disease (PD) (Brain dopamine (DA) is closely involved in the modulation of several neurobiological and behavioral processes, including ase (PD) –5. Encodase (PD) –8.Psychostimulants such as amphetamine (AMPH) or methylphenidate can, however, have an effect also on trace amine-associated receptor 1 (TAAR1), another protein that has a significant role in DA neurotransmission. TAAR1 is a G protein-coupled receptor that colocalizes with DAT, has close interactions with it, and can modulate its activity when activated , 10. At Several of the main features issuing from an impairment of DAT activity in humans have been studied using the DAT knockout (KO) mouse –17. SincWell known to be comorbid with other diseases such as major depression and bipolar disorder , ADHD haRecently, Gainetdinov et al. developed a novel DAT KO rat model that could provide a good translational model for ADHD as well as other human diseases involving alterations in DA circuitry. Their first work on DAT KO rats  showed iThe IDT is an operant task that involves a choice between either an immediate small reward or a delayed larger reward , extensively used to study behavioral impulsivity in rodents . The twoin vivo techniques, if available.All experimental procedures were approved by the Italian Ministry of Health . Procedures were in close agreement with the European Community Council Directive (2010/63/EEC) and Italian law. All efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to ® III cages with sawdust bedding and with food and water ad libitum. No more than a pair of sibling subjects per genotype was chosen to be shipped, in order to avoid possible genetic biases. All dams being DAT–HET and all three genotypes being represented in each litter, there is no risk of biases due to differential maternal behavior. They were kept in an air-conditioned room , on a 12-h reversed light–dark cycle (lights off at 07:00 a.m.) until the start of the experimental protocols.A total of 39 adult (8-month-old) male rats (with a Wistar Han background), of which 10 were WT (average body weight 530 g), 17 HET (average body weight 530 g), and 12 DAT KO (average body weight 305 g), were used. These animals were obtained from Istituto Italiano di Tecnologia (IIT) , with the purpose of setting a colony at Istituto Superiore di Sanità (ISS) . Animals were born in IIT from the breeding of DAT–HET males and females, weaned on PND 21 and shipped to ISS at adult age, where they were housed in pairs within Makrolon® III cages with sawdust bedding for the duration of these procedures only. Afterward, all 39 rats were tested in an IDT, for a total of 6 weeks of testing. During this experimental protocol, a food restriction, to keep subjects at 95% (±2%) of their body weight, was imposed through a limited quantity of extra food given at the end of each IDT free-feeding experimental session. This strategy was applied to increase the animals’ motivation to work for food delivery at the beginning of each session.During the first week, a cohort of 24 subjects  was randomly selected, and they underwent a test to measure their preference for a sweet solution compared to water and a test to study their behavior when exposed to a highly appetitive food. Rats were housed individually within MakrolonBody weight was measured thrice in a 2-week span before the start of the experimental protocols. Food consumption was measured during the same 2 weeks, by weighing the food pellet present in each home cage tray every 2 days. During the IDT, subjects’ body weight was measured three times per week, for the whole duration of the experimental protocol, in order to monitor and to titrate the level of food restriction.During 4 days of testing, each rat had free access to two different bottles in its home cage, one containing water and one containing a sucrose solution (14%). The starting position of each bottle was counterbalanced, and each day the position of the bottles was swapped. The amount of fluid intake was measured for each subject by weighing each bottle every day at the same hour, with a Kern 440-49 N balance .During 4 days of testing, twice per day (10:30 a.m. and 12:00 p.m.), each rat received 4.5 g of mascarpone cheese in its home cage, delivered inside baking paper cups and left on the cage floor for 10 min. After this time, both baking cups and remaining cheese were removed with the quantity of remaining cheese and the condition of the baking cups being measured. The remaining cheese was quantified as “all,” “more than half,” “less than half,” “none.” The condition of the cups was classified as “present” if it was left intact, and “deteriorated” or “absent” if it was destroyed or eaten.® III cage with sawdust bedding: therefore, the apparatus consisted of standard cages identical to the subjects’ home cages. The panels, which occupied one quarter of the living area, were provided with two nose-poking holes; two hole lights, placed inside each nose-poking hole; one feeding magazine where precision food pellets  were dropped, placed centrally between the nose-poking holes; one magazine light; one main chamber light, placed at the top of the panel; one hidden-feeding device that released food pellets into the magazine. Operant panels were connected through an interface to a computer. The whole task procedure was monitored and recorded through software .Eight operant panels  were each placed in a MakrolonSubjects were tested for 5 days per week between 10:00 a.m. and 14:00 p.m., for a total of 15 training and 15 test sessions performed during a 6-week span. Each rat was tested in the same chamber at the same hour, and sessions lasted for 40 min. During each session, subjects could nose-poke into either of the two holes to release pellets in the magazine. Nose-poking in one hole [termed small and soon (SS)] resulted in the immediate delivery of one pellet in the magazine, whereas nose-poking in the other hole [termed large and late (LL)] resulted in the delivery of five pellets in the magazine after a set delay, whose length was 0 s during training, 15 s during test week 1, 30 s during test week 2, and 45 s during test week 3. Lights were used as visual cues: the main light was on to signal whenever the panel could be activated by a nose-poke; the hole lights were switched on just after a nose-poke in the corresponding hole for 1 s until food delivery. After any food delivery, the panel entered a timeout (TO) of 30 s, during which additional nose-poking was recorded but was without any consequence (inadequate nose-pokes); the magazine light was kept switched on for the entire duration of the TO.During a first “2vs2” training phase (on the first week of IDT testing), nose-poking in either hole resulted in the immediate release of two pellets. This phase was used to let subjects familiarize with the apparatus with no reason to prefer any of the two holes. During the second “1vs5” training phase (on the second and third weeks of IDT testing), SS nose-pokes resulted in the release of one pellet, whereas LL nose-pokes resulted in the release of five pellets (but with a delay of 0 s). This phase allowed subjects to reach a clear preference for LL. During this phase, two animals did not reach a preference for LL; thus, they were excluded from data analysis.We used d-AMPH  and RO-5203648 , a partiAmphetamine or saline was injected 15 min before session start, and RO-5203648 or vehicle was injected 10 min before session start. Therefore, both drugs had their peak effect during the middle of sessions. AMPH and RO-5203648 were administered on random days, but never administered on subsequent days; this produced eight different combinations of administration order during a test week, which was randomly assigned to subjects. Each combination was used on average on five subjects (at least one for each genotype), counterbalancing between genotypes to minimize any bias related to a specific administration order.The WT, DAT–HET, and DAT KO genotypes were checked on purified DNA by restriction fragment length polymorphisms (RFLPs): indeed, while the WT allele has one BtsIMutI restriction site, this was specifically canceled in the KO allele . The DNAPCR amplification was performed in a 50-µl reaction volume. After the initial denaturation at 94°C for 15 min, there were 35 cycles at 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s, and then a final extension step of 72°C for 5 min. The DNA was substituted with sterile deionized water for the negative control. The PCR products were resolved on 2% agarose gel and identified by Novel Juice (GeneDirex) staining. For the genotypes analysis, 10 µl of the PCR products was digested for 1 h at 55°C with 1 µl of BtsIMutI (10 U/μl) in separate tubes. The cleaved products were resolved on 2.5% agarose gel.Two weeks after the last behavioral test, 10 WT and 12 KO rats were sacrificed in a random order through decapitation in two consecutive days between 10:00 a.m. and 12:00 p.m. Brains were quickly removed and dissected on ice with single-use metal scalpels. A 1-mm thick slice of ventral striatum was collected from each brain, with slices immediately stored in 1.5-ml Eppendorf tubes containing 500 µl of RNA stabilization reagent RNAlater (QIAGEN). Samples were taken specifically from the ventral striatum as extensive neurochemical analyses already confirmed the expected changes within the dorsal striatum  according to manufacturer’s recommended protocol. Briefly, samples were solubilized and homogenized in RLT lysis buffer, loaded in columns containing silica membranes and subjected to washing and centrifugation processes. RNA was eluted in 100-µl volume and snap-frozen at −80°C. The concentration and purity of RNA samples were determined using a NANODROP 2000 (Thermo Scientific).2 First Strand Kit (QIAGEN) following producer’s recommended protocol. The RNA quantity for each sample was adjusted to 5 ng/µl. Afterward, any genomic contamination was removed by mixing 8 µl from each sample  with 2 µl of genomic DNA elimination buffer (QIAGEN) and incubating at 42°C for 5 min. Finally, the retrotranscription was performed on each sample: 10 µl of each sample was mixed with 10 µl of Oligo(dT) retrotranscription mix and incubated at 42°C for 15 min and at 95° for 5 min in a T100 (BIO-RAD) thermocycler. In the same way, negative retrotranscription controls (without retrotranscriptase enzyme) were performed. cDNA samples were then loaded in a total of six custom 96-well RT-PCR plates (QIAGEN). Each of the first six rows of each plate was used for a different assay: TH, TAAR1, Slc6a4 serotonin transporter (SERT), DRD2 (D2), plus the two housekeeping genes 18SrRNA and β-actin. The last two rows were used as positive and negative controls. Each sample was assayed in triplicate; thus, a total of four samples were assayed on each plate. Each well was loaded with 25 µl of solution containing 11.5 µl of water, 12.5 µl of SYBR Green (QIAGEN) mastermix, and 1 µl of cDNA. Amplification was carried out in an ABI 7500 (Applied Biosystem) thermocycler with the following conditions: 95°C for 15 min, followed by 40 cycles (95°C for 15 s and 60°C for 1 min), after the amplification, a melting curve was obtained.The cDNA for each sample was obtained using RTP ≤ 0.05, NS = not significant. Multiple post hoc comparisons were performed with Tukey’s HSD test.Data were analyzed using repeated measures analysis of variance. For food consumption, we used a one-way model, where the dependent variable was the total food intake and the factor was three-level genotype (WT vs HET vs KO). For preference for a sweet solution, we used a one-way model where the dependent variable was the percentage of a sweet solution intake over the total fluid intake and the factor was three-level genotype (WT vs HET vs KO). For the exposure to a highly appetitive food, we used a logistic multinomial regression for the quantity of cheese and a logistic regression for the cup condition. For IDT, we used a split-plot model, and the factors were three-level genotype (WT vs HET vs KO) × four-level treatment  × three-level delay (15 vs 30 vs 45 s). Statistical analysis was performed using StatView II . Data are expressed as mean ± SEM. Significance level was set at −ΔΔCt). The statistical analysis was performed using Microsoft Excel on normalized expression levels of WT vs KO samples. Significance was assessed using a two-tails Student’s t-test. Significance level was set at P ≤ 0.05, NS = not significant.RT-PCR curves were analyzed using ABI 7500 software v2.0.6. Quantification was carried out measuring the number of amplification cycles needed to cross a 0.04 fluorescence threshold (Ct). Melting curve analysis was then carried out to validate the purity of amplification products, and any replica with multiple or abnormal melting temperature was excluded from analysis. For TH, SERT, and D2 genes the cutoff was fixed at 37 cycles. Because of the extremely weak expression of TAAR1, the cutoff was fixed at 39 cycles for such gene. Data for TH, TAAR1, Slc6a4, and Drd2 genes were normalized to the mean of 18SrRNA and β-actin genes for each sample. The relative expression of each gene transcript was obtained using ΔΔCt and converted in the relative expression ratio  is significantly lower, when compared to both WT (535 ± 9 g) and DAT–HET (532 ± 6 g) rats: ad libitum food intake: F = 0.283, P = NS.Nevertheless, there were no significant differences between genotypes when considering F = 0.129, P = NS, KO rats showed a markedly lower preference for the bottle containing a sweet solution, compared to WT and DAT–HET: F = 24.216, P < 0.0001  and a higher chance to leave “more than a half” compared to leave “none” (P < 0.05), when compared to both WT and DAT–HET. Logistic regression analysis showed that KO rats have a higher chance to destroy the paper cups containing the cheese, when compared to both WT and DAT–HET. Considering the categories for the state of paper cups after 10 min (“present” or “deteriorated”/“absent”), KO had a higher chance to destroy the cups (P < 0.05), when compared to both WT and DAT–HET.Multinomial logistic regression analysis showed that KO rats have a higher chance to eat less cheese when compared to both WT and DAT–HET. Considering the four categories for the quantification of the remaining cheese after 10 min , KO had a higher chance to leave “all” the cheese compared to leave “none”  and the other two groups : F = 12.351, P < 0.0001. This difference was not present when considering latency to approach LL. There was a significant effect of delay on latency: for all three genotypes, at higher delays, there was a trend toward reduction of the latency to approach SS: F = 2.834, 0.05 < P < 0.1 and a significant increase of the latency to approach LL: F = 5.783, P < 0.05. Treatment with both AMPH and RO-5203648 caused a trend toward reduction to approach LL: F = 2.577, 0.05 < P < 0.1.Knockout rats showed a higher latency to approach SS in each session, when compared to WT and DAT–HET: F = 44.597, P < 0.0001. This reduction was, however, less pronounced when considering only KO rats: F = 2.133, 0.05 < P < 0.1. In other terms, KO rats showed a higher preference for LL throughout the whole testing phase: F = 6.644, P < 0.05 . Therefore, WT rats seem to show some tolerance to AMPH effects at a 45-s delay, which can well be interpreted as a floor effect, whereas DAT–HET rats seemed conversely to display sensitization at a 45-s delay, which can well be interpreted as a subchronic effect.As expected, there was a strong effect of delay on LL preference for all three genotypes. At higher delays, there was a significant reduction in LL preference: F = 27.599, P < 0.0001. There was also a significant effect of delay on the number of total trials: while KO rats showed a significant decrease of trials at higher delays, WT rats conversely showed an increase of trials: F = 12.758, P < 0.0001  when compared to both WT and DAT–HET: F2, 3 = 27.599F2, 3 = 27.599F2, 3 = 27.599F = 10.096, P < 0.0001 , while it caused an increase of SS inadequate nose-pokes at higher delays (30 and 45 s): F = 3.972, P < 0.05. No significant genotype by treatment interaction was shown for SS inadequate: F = 1.495, P = NS. There was a significant decrease in LL inadequate nose-pokes at higher delays: F = 14.914, P < 0.0001, and this decrease was much more pronounced in KO rats when compared to both WT and DAT–HET: F = 4.203, P < 0.05. In other words, KO rats reacted to a delay by showing a choice for LL in the fewer trials with no persistence onto LL hole during TO. Treatment, especially AMPH, caused a significant decrease in LL inadequate nose-pokes, for all the three genotypes: F = 10.601, P < 0.0001.As expected, there was a significant increase in the total inadequate nose-pokes in both WT and DAT–HET at higher delays, while KO showed an opposite trend, with a significant decrease of inadequate nose-pokes: F, 68 = 10First of all, we underline that an extensive proof about the effectiveness and consequences of DAT-abolished allele(s) can be found elsewhere . Just byIn our RFLP analysis, the WT genotype undergoes cutting on both alleles and produces two low digested bands (104 and 71 bp); in the DAT–HET genotype, only one of the alleles is cut, resulting in one undigested and two digested bands ; the KO genotype loses all cutting sites on both alleles and displays only one undigested band (175 bp). The genotypes of all rats used in this study were confirmed twice, from samples collected at weaning and at sacrifice.P = 0.52, TAAR1: P = 0.51, SERT: P = 0.25, D2: P = 0.48. ΔΔCt values for the four genes were TH: 1.13 ± 0.08, TAAR1: 1.32 ± 0.06, SERT: 0.81 ± 0.02, and D2: 0.92 ± 0.06  of any of the assayed genes: TH: ad libitum. This result is consistent with that already obtained in DAT KO mice  and neurochemically characterized by the group of Gainetdinov et al. . The wei KO mice . It is s KO mice , by a le KO mice , by someThe IDT yielded several unexpected results regarding behavioral abnormalities of DAT KO rats. Even before the beginning of the test, during the 2vs2 training phase, KO rats tended to show a slight preference  for one of the two nose-poke holes, whereas WT and DAT–HET subjects chose at random level during this training phase. Moreover, during the 1vs5 training phase, some KO individuals developed an extreme attraction toward LL hole, reaching sometimes a 100% preference for such a hole. This is uncommon: preference for the LL hole, when there is no delay yet assigned to the large reward, usually hovers around 85–90%, because rats keep a minimum rate of patrolling at the SS hole during sessions . More suThese behaviors were completely different from the ones usually observed with rats in this task. WT and DAT–HET, for instance, performed as expected: starting from an LL of about 85% at the end of the 1vs5 training phase, both groups reduced their LL preference and eventually switched toward SS, showing a normal curve of impulsivity. Moreover, WT and DAT–HET rats performed an increasing number of SS trials and of SS inadequate nose-pokes at higher delays. These data provide an insight on reactions clearly due to the aversive effects of the delay: normal subjects experience a sort of “impatience” or “frustration” when forced to wait for the LL reward; thus, they start to perform both more SS inadequate nose-pokes during the TO and more SS trials  when the cost of waiting becomes excessive during LL trials with a very long delay. KO rats, instead, seem to react to the increasing delay with conspicuous consummatory motor stereotypies addressed to the feeding magazine, like sniffing or chewing. A reaction of similar fashion happened in the highly appetitive food test with the paper cups, which were destroyed by KO rats that showed little to no interest for the cheese. As delays assigned to LL became higher, KO rats spent increasingly more time performing these stereotypies overall reducing the number of trials, choosing LL in such few trials, and showing almost no inadequate nose-poking. Oral stereotypies are also presumably behind the increased latency to approach the SS nose-poke hole. When put in the experimental cages, at any stages of the task, KO rats often immediately focused on the feeding magazine and performed their stereotypies, then nose-poking for the first time only after several minutes. To sum it up, DAT KO rats did not show an increased impulsivity when compared to WT and DAT–HET: rather, they displayed a reduced sensitivity to rewarding stimuli, an increased behavioral rigidity, and a number of compulsive behaviors.When taking the effect of drugs into account, AMPH reduced LL preference and increased number of SS trials in both WT and DAT–HET, as expected: indeed, this psychostimulant drug makes healthy subjects more impulsive and increases motor activity , 41. TheThese results surprisingly contradict the findings from a previous study in DAT KO mice . As a maThus, KO rats seemed not only less sensitive to rewarding stimuli but also more prone to executing consummatory motor stereotypies. This piece of finding could well point to alterations in the motivational states of KO rats, but also to a different salience assigned to differently meaningful stimuli . Present male DAT KO rats appear somewhat disturbed by objects they find around, a notion that seems confirmed by an increased marble manipulation in a marble-burying test  and by aPresent data can be useful to interpret the many stereotypies that DAT KO rats do have. As far as ADHD is concerned, however, these data point to reward deficiency rather than impairments in attentional processes. Our hypothesis about an increased behavioral rigidity of DAT KO rats seems further supported by results of the Spontaneous Alternation Task . Here, KMany studies , 44 showEven though this study was initially conceived to highlight impulsive behaviors of DAT KO rats, it revealed a compulsive phenotype instead. KO rats are characterized by pervasive consummatory motor stereotypies, noticeable both in their home cages and during tests and operant tasks. Moreover, they seem affected by a strong behavioral rigidity during operant choices. Finally, they show a reduced sensitivity for rewarding properties of food and sweet fluids, although generalization to other natural stimuli like sex or novelty would deserve further work . These bData obtained with this study provided a first phenotypic characterization of DAT KO rats. First evidences obtained so far [present work; Adinolfi et al. ; Leo et in vivo techniques, if available.All experimental procedures were formally approved by the Italian Ministry of Health . Procedures were in close agreement with the European Community Council Directive (2010/63/EEC) and with Italian law. All efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to SC, FZ, and AP realized the experiments; DL and RG provided the animal model; SC, LC, RT, EA, and GL contributed to writing and commented with their expertise; WA supervised all experiments and writing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer FP declared a shared affiliation, with no collaboration, with one of the authors DL to the handling editor.


Allolobophora chlorotica was recorded in Standard OECD Artificial Soil (AS) and also in Kettering Loam (KL). In a second experiment, avoidance behaviour was recorded in a linear gradient with varying proportions of AS and KL  with either A. chlorotica or Octolasion cyaneum. Results showed a significant decrease in A. chlorotica biomass in AS relative to KL, and in the linear gradient, both earthworm species preferentially occupied sections containing higher proportions of KL over AS. Soil texture and specifically % composition and particle size of sand are proposed as key factors that influenced observed results. This research suggests that more suitable substrates are required for ecotoxicology tests with soil dwelling earthworms.Soil dwelling earthworms are now adopted more widely in ecotoxicology, so it is vital to establish if standardised test parameters remain applicable. The main aim of this study was to determine the influence of OECD artificial soil on selected soil-dwelling, endogeic earthworm species. In an initial experiment, biomass change in mature  Eisenia fetida proposed in a number of standardised tests , OECD artificial soil (AS) is recommended. However, the use of OECD AS may not always be appropriate. Hofman et al.  refer toE. fetida and E. andrei. This standard details the methods for a two-section and also a six-section avoidance test, with the latter difficult to set up and rarely used. Lowe et al.  in terms of survival, change in biomass and avoidance behaviour (utilising a linear gradient rather than a two-section methodology).As soil dwelling earthworms are adopted more widely in ecotoxicology, it is important to establish if standardised test parameters remain applicable. Two-section avoidance tests have been used to study the influence of soil properties  was established; with a composition of: Sphagnum Peat 10%, Quartz sand 69.5%, Kaolinite Clay 20%, Calcium Carbonate 0.5%. For comparison, Kettering loam (KL) a natural soil that is widely used in earthworm-related studies  = 0.249 ± 0.013 and 0.225 ± 0.013 g in KL and AS respectively) with two individuals placed into each container. Thereafter, containers were maintained in 24 h darkness at 15 °C in a temperature-controlled incubator (considered by Lowe and Butt (Six replicates of each soil treatment were set up in opaque plastic containers (0.07 m × 0.05 m × 0.07 m), with lids pierced with a mounted needle to allow ventilation. Dried (at 105 °C) and sieved horse manure (2 g per 100 g of soil) was incorporated into each soil as a feed source and the substrate rewetted to a moisture content of approximately 25% (Lowe and Butt and Butt  to be opA. chlorotica and O. cyaneum (mean biomass 0.31 and 0.44 g respectively) was investigated utilising the methodology of Lowe et al.  on avoidance behaviour of e et al. . A gradin = 5 earthworms per mesocosm) and after introduction, the spacers were removed and the containers covered with plastic (cling) film, pierced (with a mounted needle) to allow ventilation and kept in 24 h darkness at 15 °C. Five replicates were established for each earthworm species. After 14 days, containers were carefully removed from the incubators, spacers re-inserted and earthworm positions within soil gradients determined by destructive sampling.A single earthworm was placed on the surface of each soil treatment . In the initial experiment, differences in A. chlorotica survival after 28 days in the initial experiment was 100 and 91.7% in KL and AS treatments respectively. At day 0, there was no significant difference (p = 0.206) in the mean biomass of A. chlorotica in the AS and KL treatments. After 14 and 28 days, individuals in AS had a mean biomass of 0.209 and 0.189 g respectively, significantly lower (p < 0.05) than individuals present in KL (0.270 and 0.315 g respectively) .In the avoidance experiment, 100 and 95% survival of A. chlorotica, significantly more (p < 0.05) earthworms were retrieved from both 100% KL and 25% AS + 75% KL than from the three other soil treatments  were negatively correlated with the sand and gravel content of soil. Sand texture may also influence earthworm behaviour. As soft bodied organisms, earthworms are particularly sensitive to coarse particles within the substrate and this may elicit a behavioural response. As an example, Kretzschmar . While Kettering loam also has a relatively high sand composition (58%), only 1% of sand has a particle size between 1–2 mm, while 53% has a particle size under 0.5 mm (Grundy Soil texture is known to affect soil properties such as cation exchange capacity, nutrient status and soil moisture so can have an important influence on earthworm populations (Edwards and Bohlen tzschmar  advocatem Grundy . As soilHofman et al.  posed thFinally, our work has demonstrated the effectiveness and potential sensitivity of the linear gradient methodology in assessing earthworm avoidance behaviour. This may have specific applications in measuring the potential effects of emerging soil contaminants, such as nanomaterials, that are currently present at low concentrations within the soil matrix.


Mackie, W.A. TomeD.X. Wang,University of Wisconsin‐Madison, Madison, WisconsinTo describe a method of generating a proton path probability map used for proton computed tomography (pCT) reconstruction.Using the framework of Bayesian inference, a semi‐analytical model based on Fermi's multiple Coulomb scattering (MCS) theory is derived to calculate the conditional probability of a proton passing through each point within an imaging object, given the proton's entrance and exit position and direction. The conditional probabilities at all points form a proton path probability density map (PDM). The most likely path (MLP) is extracted from the PDM, and the PDM is also directly used for image reconstruction. A path ‘envelope’ enclosing 90% of the MLP probability (named MLP90) is extracted for reconstruction to account for the statistical uncertainty. Reconstruction using cubic spline path (CSP) is included for comparison. Data are obtained from Geant4 Monte Carlo simulation.The extracted MLP and MLP90 agree well with the real proton path recorded during simulation. Reconstruction using MLP and MLP90 are not significantly different from that using CSP. Reconstruction using the full probability map yields a smoother image than that using MLP or CSP, although it contains artifacts.A proton path probability map can be generated using the method described in this abstract. The map can be used directly for pCT reconstruction. MLP and path ‘envelope’ can also be extracted from the map. The reconstructed image using the map, MLP and MLP90 compare well with that using CSP. This method can be used to benchmark different path estimation methods.su‐f‐grandoaksn/o‐21a C. Shi,1 C. Buckey,1 H. Alkhatib,2 N. Papanikolaou2X. Chen,1 San Antonio, TX; South Carolina Radiation Oncology Associates,2 Columbia, SCUniversity of Texas Health Science Center,Increasing interest in electronic portal imaging devices (EPIDs) for radiation dosimetry and other applications such as adaptive radiotherapy has developed over the past decades, particularly with the recent technique advance in high‐resolution amorphous silicon EPID system. For a clinical quality assurance (QA) program, EPIDs may provide a useful QA tool with potential benefits of reduced cost and added convenience such as reduced setup time. This study is to explore the potential clinical application of EPID as a routine machine QA tool.An integrated EPID‐based QA system was designed, which target to replace conventional machine QA methods. Recommendations by TG‐40 and TG‐45 reports were followed. EPID images were acquired using Varian aS1000 EPID system under integration acquisition mode . Signal intensity of EPID images were calibrated to dose maps using a linear signal‐dose relationship. Specific EPID‐based QA procedures and image analysis algorithms were designed and developed for some basic QA tasks, such as beam flatness/symmetry test and Winston‐Lutz isocenter test.flatness=0.9–1.45%;symmetry=0.48–1.31%;penumbra=3.2–4 mm at SSD=150cm. Star‐shot images were well reconstructed for isocenter check from EPID images acquired at different gantry or collimator angles.EPID‐measured results were comparable to those measured monthly using films in our institute. For example, EPID‐measured beam quality parameters were: The results show that EPID may be properly used as a tool for clinical routine machine QA tasks. su‐f‐grandoaksn/o‐31a C. Shi,1 C. Cheng,2 C. Esquivel,1 T. Eng,1 N. Papanikolaou1B. Guo,1 San Antonio, Texas; University of Oklahoma Health Science Center,2 Oklahoma City, OklahomaUniversity of Texas Health Science Center at San Antonio,To apply 3‐dimensional (3D) intensity‐modulated brachytherapy (IMBT) in accelerated partial breast irradiation (APBI).A dosimetry algorithm for 3D IMBT based on modified TG 43 formulism and Monte Carlo method was proposed and a MATLAB‐based inverse treatment planning system was developed. The system was applied to study the feasibility of 3D IMBT to improve plan quality. Ten intracavitary APBI cases were studied. For each case, an IMBT plan was developed and compared with the original plans used for treatment considering the plan quality, planning and delivery time.IMBT plans showed better plan quality compared to the original plans for all cases. For a patient with a small breast and little sparing to ribs and skin, with similar coverage to the target, IMBT plan decreased the high dose region V200 by 16.1%. Maximum doses to skin and ribs were reduced by 56 cGy and 104 cGy in one fraction, respectively. Mean dose to ipsilateral and contralateral breasts and lungs were also slightly reduced by IMBT. The drawback of IMBT is the longer planning and delivery time. IMBT plans take around 2 hours to optimize; the delivery time of IMBT is typical 4–6 times of an isotropic plan.Inducing source intensity modulation improves the plan quality of APBI brachytherapy treatment plans, increasing dose uniformity in target and reducing the dose to critical structures. Faster computers and higher output of the source could be solutions to compensate for the increase of plan optimization and delivery time.su‐f‐grandoaksn/o‐41a J. Lee,2 K. Choi,3 W. Jung,1 T. Suh1J. Park,1 Seoul, KR; Konkuk University Medical Center,2 Seoul, KR; SAM Anyang Medical Center,3 Anyang, KRThe Catholic University of Korea,The software for GafChromic EBT2 film dosimetry was developed. It provides film calibration functions based on each color channel, and evaluations are available about the effects according to the correction methods for light scattering of flat‐bed scanner and thickness differences of active layer.The developed software using MATLAB is composed of two primary frames with multi‐tabs which display imported images and resultant images at each processed step. Dose verification using EBT2 films is implemented following procedures: file import, noise filtering, background and active layer correction, dose calculation and evaluation. Relative and absolute background correction is selectively applied. Calibration results and the fitting equation of sensitometric curve are exported to files. After two dose matrixes from different types of measurement devices or treatment planning system (RTP) are aligned, profiles and isodose curves are compared. In addition, gamma index and gamma histogram are analyzed according to the determined criteria of distance–to–agreement and dose difference.Dosimetric results from EBT2 films could be compared with those from RTP, ECLIPSE or 2‐dimensional ionization chamber array, MatriXX. As the performance evaluation was achieved by dose verification in the 60° enhanced dynamic wedged field, absolute dose discrepancy between EBT2 film and ECLIPSE was satisfied within 3% error. In analysis using gamma index applied criteria of 3 mm 3%, 99% pass ratio represented, except for the field edges.The software would contribute to recommendation of optimal procedures and the practical QA using EBT2 films.su‐f‐grandoaksn/o‐51a L. Vazquez Q,1 A. Gutiérrez,1 S. Stathakis,2 H. Alkhatib,3 C. Shi,1 N. Papanikolaou1W. He,1 San Antonio, TX; CTRC at UTHSCSA,2 San Antonio, TX; South Carolina Radiation Oncology Associates,3 Columbia, SCUniversity of Texas Health Science Center at San Antonio,MU‐Tomo, has been developed to execute independent point dose calculation within seconds for helical tomotherapy modality.A second check software platform, MU‐Tomo has been designed to verify the planned dose independently. Input parameters required for the software are: archived tomotherapy patient files, initial image coordinates, tomotherapy‐calculated point dose, and machine‐specific dosimetric parameters such as the off‐axis ratios (OARx and OARy), tissue phantom ratios (TPR), and output functions (Scp). The software was validated on four phantom models and 50 tomotherapy patient IMRT plans. The four different phantom studies were: (1) a phantom composed of a 20.0 cm thick rectangular slab of Virtual Water, (2) a step valley phantom in a similar, previously‐mentioned Virtual Water phantom, (3) a monthly output check procedure in a cylindrical cheese phantom (radius 15 cm and length 18 cm), and (4) seven patient quality assurance (DQA) treatment plans in the cheese phantom. Patient plans were selected from various cancer treatment sites.MU‐Tomo were within 5.0% for all cases evaluated; 49 of 50 were within 3.3%.Validations of dose differences for four phantom studies were all within 3%. For the 50 patients, examinations show that point dose differences between TomoTherapy TPS and MU‐Tomo can perform an independent dose calculation accurately and quickly, and provide a secondary point dose validation of helical tomotherapy plans.su‐f‐grandoaksn/o‐6a H. MalhotraJ. Steinman,Roswell Park Cancer Institute, Buffalo, NYExternal beam treatment for patients who have previously had implanted seeds is not standard procedure but an option reserved in salvaging failed prostate brachytherapy treatment. Though small in size, the seeds are many and consist of high‐Z metals capable of causing significant dose perturbation in their immediate vicinity.I‐125 seed placed in a grooved Lucite plate. Five cm buildup and 10 cm backscatter were added at 95 cm SSD. The phantom was irradiated with and without seed for 1×1cm2 field with 6 MV photons. DOSXYZnrc simulations were done utilizing same parameters for 1 billion histories. Other measurements using XV films were done comparing the dose perturbation with 18 MV photons and varying depth. Study was further extended to include other nonradioactive seeds (Pd‐103 and Cs‐131), and other metals of various Z. Films were analyzed obtaining cross‐axial profiles of seed and no‐seed determining percent dose perturbation.Measurements were done with Kodak XV2 and Gafchromic EBT2 films layered above and below a nonradioactive +15% upstream and −20% downstream of the I‐125 seed surface. XV film showed +6% and −11%. Effect was observed up to ~2 mm upstream and ~5 mm downstream from seed. Dose perturbation was found to decrease with increasing energy but increase with Z and depth of seed in tissue.DOSXYZnrc and EBT2 film verified maximum dose enhancement of The perturbation of the seeds is highly localized and, as with other heterogeneities, depends on factors such as energy, depth, and material. Future work seeks to evaluate clinical impact of this effect.su‐f‐grandoaksn/o‐7a N. Ruiz, M. HatabR. Al‐Senan,University of Texas Health Science Center at San Antonio, San Antonio, TexasTo evaluate the angular dependence and linearity of OSL dosimeters in diagnostic X‐ray energy ranges.15×15 cm collimation field and positioned in one of the eight angles. For the linearity, two kVps were tested with a range of mAs from 32 to 400. The stability of the tube output was monitored with a calibrated detector.A diagnostic X‐ray unit was used to evaluate InLight/OSL nanoDot dosimeters. Eight angles were chosen  reflecting the position of an OSL dosimeter with respect to the X‐ray table. The angular dependence was investigated at four techniques: 81 kVp at 50 mAs and 200 mAs, and 117 kVp at 50 and 200 mAs. Each dosimeter was irradiated at 100 cm SID, centered on a < 0.1%). Linearity results at both kVps showed good linearity (R > 0.99).Considering the 0° dosimeter reading as the reference, preliminary results showed a significant reduction in dose at 81 kVp, more pronounced with 50 mAs. A decrease of about 50% was shown in the 90° and slightly less with 270°. Considerable reduction was also observed at 45°, 135°, and 225°. Results for 117 kVp will also be presented. Tube output was highly reproducible throughout the experiment  measurements to represent SNR in partially parallel imaging (PPI) protocols. SNR measurement methods were selected based on their practicality in a standard MRI evaluation by a medical physicist.R = 2, R = 3 and R = 4. Two sets of images were taken and one set with no signal (RF voltage set to 0). Additionally, all protocols were taken with the phase encode oriented in both directions. Four methods of SNR analysis were investigated: N1) Image subtraction method; N2) No signal image method; N4) SD of background method; ACR) ACR method. Image results were analyzed to determine the average g‐factor for each method of SNR calculation and PPI reconstruction method.A spherical phantom provided by the manufacturer was used in all of the scans, which were performed using a 3T Siemens MRI system. Images using EPI, FSE and TruFISP sequences were acquired. Image data for each sequence were taken without PPI and then with PPI method GRAPPA and PPI method mSENSE using acceleration factors: The only method that followed theory in maintaining a g‐factor of greater than one in each one of our protocols was method N1 (image subtraction method). The average g‐factors were as follows (mSENSE/GRAPPA) N1:4.85/4, N2:0.63/‐0.36, N4:0.88/0.09, ACR:0.42/‐0.1.The current method used in SNR evaluation by the ACR does not produce results consistent with theory in the investigated PPI protocols. It is recommended when evaluating PPI protocols that the image subtraction method be used.


Obstetrical management was undertaken by an obstetrician.A 26‐year‐old woman  at 25 weeks’ gestation was brought to the emergency department because of anaphylactic symptoms. She reported eating Japanese soba and developed symptoms of dyspnea, generalized itchy rash, abdominal pain, and severe uterine contractions within 15–30 min of eating. She was immediately treated by normal saline infusion, two injections of epinephrine (intramuscularly), and a nebulized short‐acting βThe patient recovered rapidly without a biphasic reaction of anaphylaxis. After 11 weeks, a healthy, neurologically intact baby was born.Management of anaphylaxis in pregnant patients is basically the same of that in non‐pregnant ones. Treatment should commence immediately to prevent further development of the anaphylaxis reaction and fetal neurological deficiency. Anaphylaxis is a rare event during pregnancy, but its onset may trigger maternal hypotension leading to intrapartum asphyxia and, eventually, the risk of severe fetal brain damage. Furthermore, the risk of cesarean delivery in anaphylactic patients in pregnancy is high (74%).2‐receptor agonist. We manually displaced her uterus to the left to prevent inferior vena cava compression. The treatment was effective, but her generalized itchy rash and abdominal pain persisted. She received an additional 0.4 mg epinephrine i.m. as well as an H1‐antihistamine and methylprednisolone. Serum allergen‐specific immunoglobulin E (IgE) testing  was carried out in the emergency department; total serum tryptase was increased on day 1 (15.5 μg/mL) but decreased by day 2 (3.9 μg/mL). Obstetrical management was undertaken by an obstetrician for threatened preterm labor, but the fetal heart rate was almost normal (140–160 beats/min) with variability  at 25 weeks’ gestation was brought to the emergency department because of severe uterine contractions, dyspnea, generalized itchy rash, abdominal pain, and peripheral cyanosis. She reported eating buckwheat noodles (soba) and tempura  and developed the symptoms within 15–30 min of eating. She had a medical history of atopic dermatitis, but no history of food allergies or anaphylaxis. She was lucid. On examination, her blood pressure was 104/72 mmHg, heart rate was 112 b.p.m., respiratory rate was 40 breaths/min, oxygen saturation was 100% , and body weight was 43 kg. The patient was diagnosed with anaphylaxis because of a suspected food allergy, and she was immediately treated with normal saline infusion, an i.m. injection of 0.4 mg epinephrine, and a nebulized short‐acting βThe cause of the allergy was investigated after birth, because skin prick and challenge test are contraindicated in pregnancy considering the risk of inducing anaphylaxis. The skin prick test was positive for dust mite (4.4 mm × 1.1 mm), buckwheat (3.7 mm × 4.2 mm), and wheat (4.7 mm × 3 mm) and was negative for shrimp (0 mm). An oral food challenge for wheat and buckwheat revealed the anaphylaxis was caused by a buckwheat allergy. She eliminated buckwheat from her diet and has experienced no further allergy symptoms.Anaphylaxis is defined as a serious, generalized or systemic, allergic or hypersensitivity reaction that can be life‐threatening or fatal.We present a case of maternal anaphylaxis caused by food allergy, with details of patient management. Allergic and obstetric management should be commenced immediately to prevent further development of the anaphylactic reaction and fetal brain damage. Drugs for anaphylaxis, such as epinephrine, antihistamines, glucocorticoids, and vasopressors, can be used safely without major side‐effects in pregnancy. Certainly, potential side‐effects of any drugs should be considered.None declared.


Participants had heart rate (HR), blood pressure, resting energy expenditure (REE), 12-lead electrocardiograms (ECG), perceptions about readiness to perform, cognitive function (Stroop Color-Word test), bench and leg press performance (2 sets of 10 repetitions at 70% of 1RM and 1 set to failure), and Wingate anaerobic capacity (WAC) sprint performance determined as well as donated blood samples prior to and/or following exercise/supplementation. Data were analyzed by MANOVA with repeated measures as well as mean changes from baseline with 95% confidence intervals (CI).In a randomized, double-blind, and counterbalanced manner; 25 healthy and recreationally active male and female participants ingested a flavored maltodextrin placebo (PLA), a PWS containing beta-alanine (3 g), creatine nitrate as a salt (2 g), arginine alpha-ketoglutarate (2 g), N-Acetyl-L-Tyrosine (300 mg), caffeine (284 mg), No clinically significant differences were observed among treatments in HR, blood pressure, ECG, or general clinical blood panels. There was evidence that PWS and PWS + S ingestion promoted greater changes in REE responses. Participants reported higher perception of optimism about performance and vigor and energy with PWS and PWS + S ingestion and there was evidence that PWS and PWS + S improved changes in cognitive function scores from baseline to a greater degree than PLA after 1 or 2 h. However, the scores in the PWS + S treatment did not exceed PLA or PWS responses at any data point. No statistically significant differences were observed among treatments in total bench press lifting volume, leg press lifting volume or WAC sprint performance.p-synephrine to the PWS provided limited to no additive benefits.Within the confines of this study, ingestion of PWS and/or PWS + S prior to exercise appears to be well-tolerated when consumed by young, healthy individuals. The primary effects appear to be to increase REE responses and improve perceptions about readiness to perform and cognitive function with limited to no effects on muscular endurance and WAC. The addition of 20 mg of NCT02952014) was retrospectively registered on September 13th 2016.This trial ( Research has shown that ingestion of some ergogenic nutrients and/or caffeinated beverages prior to exercise can improve mental focus and/or exercise capacity , 2. For Mucuna pruriens containing L-Dopa , low density lipoprotein [LDL-c], triglycerides [TG]), and glucose were assayed using a Cobas c 111  prior to and following 2 h after supplementation. This analyzer has been known to be highly valid and reliable in previously published reports [Plasma creatine was assayed prior to and after 1 and 2 h of supplementation as a general indicator of supplement bioavailability using calorimetric assay kits . When a significant treatment, time and/or interaction alpha level was observed, Tukey’s least significant difference post-hoc analysis was performed to determine where significance was obtained. Area under the curve (AUC) was calculated on select variables using GraphPad Prism 6 software . Delta values were calculated from subtracting baseline values with means and 95% Confidence Interval (CI) to determine whether changes from baseline and/or among treatments were significant [p > 0.05 to p < 0.10. All data are presented as mean ± SD unless otherwise noted.Data were analyzed using general linear models (GLM) multivariate analysis of variance (MANOVA) with Wilks’ Lambda and Greenhouse-Geisser adjustments using SPSS version 22.0 software , carbon dioxide production (VCO2), and respiratory exchange ratio (RER) values (VCO2/VO2) observed during the post-supplementation REE test. MANOVA revealed an overall significant Wilks’ Lambda treatments x time effect (p < 0.001) as well as significant univariate ANOVA treatments x time interactions VCO2 (p = 0.008) and RER values (p = 0.001). Post-hoc analysis revealed that PWS and PWS + S ingestion primarily effected VCO2 and RER responses compared to the PLA treatment and that PWS ingestion promoted higher VCO2 and RER values toward the end of the 30 min REE test. Analysis of AUC changes from baseline revealed significant differences among treatments in VO2 , VCO2 , and RER . Post-hoc analysis revealed that VO2 AUC in the PWS + S treatment was significantly greater than the PLA treatment  but not the PWS treatment. VCO2 and RER AUC values were significantly higher in the PWS  and PWS + S  compared to the PLA treatment. However, no significant differences were observed between PWS and PWS + S treatments.Figure p = 0.004) in perceptions of readiness to perform questions. Significant univariate ANOVA treatments x time effects were also observed in response to “I am optimistic about my future performance” and “I feel vigorous and energetic”. Post-hoc analysis revealed that participants in the PWS and PWS + S treatments rated optimism about performance and feelings of vigor and energy higher than the PLA treatment after 1 h of ingestion. However, perceptions about optimism about performance and vigor and energy in the PWS + S treatment were lower than PLA and PWS responses after 2 h. No significant treatments x time interactions were seen in remaining questions.Perceptions about readiness to perform are presented in Table p < 0.001). Significant univariate ANOVA treatment x time interactions were also observed in Word, Color and Word-Color scores. Post-hoc analysis revealed that Word, Color and Word-Color scores significantly increased over time in the PWS and/or PWS + S treatments with values in the PWS and PWS + S treatments significantly greater than the PLA at several data points. Figure Table p = 0.086, partial η2 = 0.08) and total lifting volume . Lifting volume for the 3rd set to failure also tended to be greater in the PWS + S compared to the PLA treatment . No significant differences were observed in leg press or cycling sprint peak power, mean power, or total work.Table Table p-synephrine on resting energy expenditure, perceptions of readiness to perform, cognitive function, and anaerobic exercise performance. Additionally, to examine the safety of acute ingestion of these PWS’s on resting heart rate, blood pressure, 12-lead ECG tracings, and standard clinical blood chemistry panels. Results revealed that ingestion of these PWS’s did not promote clinically significant changes in heart rate, blood pressure, ECG findings, or clinical blood markers. Thus, acute ingestion of these PWS’s prior to exercise appeared to be well-tolerated when consumed by young, healthy individuals. The primary effect of ingesting these PWS’s prior to exercise appeared to be an increase resting energy expenditure, improved perceptions about readiness to perform, and improved cognitive function. There were limited to no effects on bench press and leg press muscular endurance as well as anaerobic sprint capacity. Moreover, adding 20 mg of p-synephrine to the PWS provided little to no additive benefits. The following sections provide a more detailed analysis of results observed and comparison to available literature.The purpose of this study was to examine the effects of acute ingestion of a PWS with and without A number of studies have reported that ingestion of energy drinks and/or thermogenic type supplements primarily containing caffeine can increase resting metabolism –65. For Citrus aurantium  has also been purported to increase resting energy expenditure and/or effect carbohydrate and fat oxidation rates [p-synephrine increased lipolysis primarily at rest as well as post-exercise oxygen uptake, energy expenditure, and fat oxidation. Sale et al. [p-synephrine, 150 mg caffeine, and 150 mg catechin polyphenols increased resting and exercise carbohydrate oxidation rates. Additionally, Gutiérrz-Hellín and Del Coso [p-synephrine ingestion (3 mg/kg) increased fat oxidation rates while exercising at low-to-moderate exercise intensities. Thus, there is support to the rationale that adding p-synephrine to a PWS can increase metabolism and affect substrate utilization.on rates –33. For on rates  reportedDel Coso  reported2 and RER to a greater degree than in the PLA treatment indicating greater carbohydrate oxidation. Analysis of AUC changes from baseline indicated that while PWS had a greater impact on VO2 VCO2 and RER values; the PWS + S observed greater changes from baseline compared to the PLA treatment. These findings are consistent with several reports indicating that ingestion of thermogenic type supplements increases energy metabolism and/or carbohydrate oxidation [2 values than the PWS treatment. Therefore, the addition of p-synephrine to the PWS did not promote greater overall energy expenditure. Whether adding higher levels of p-synephrine would promote added benefits remains to be determined.Results of the present study provide some support to this contention. In this regard, mean oxygen uptake during the 30 min REE test was increased by about 1.4% in the PLA treatment compared to 5.6% in the PWS treatment and 8.6% in the PWS + S treatment. There was also evidence that PWS and PWS + S ingestion significantly increased resting VCOxidation , 66, 67 xidation  or greatxidation . HoweverCitrus aurantium ingestion can affect memory [p-synephrine could affect mental focus and/or cognitive function prior to and/or during exercise. In the present study, participants ingesting the PWS’s indicated they felt more optimistic about performance and more vigorous and energetic after ingestion the PWS’s compared to the PLA treatment. Additionally, there was evidence that ingestion of the PWS and PWS + S consistently increased Word, Color, and Word-Color scores from baseline and that some of these changes were greater than PLA responses after 1 or 2 h. However, it should be noted that participants started the PWS + S treatment with lower scores prior to supplementation and the scores in the PWS + S treatment did not exceed PLA or PWS responses at any data point so these results should be interpreted with caution. Further, the addition of 20 mg of p-synephrine to the PWS supplement used in our study did not appear to provide additive benefit. Results are in agreement with Hoffman et al. [Numerous studies indicate that ingesting caffeine containing energy drinks or supplements can improve mental focus and/or cognition , 68–70. t memory , 41. Then et al.  who repon et al.  who repo2 max. Gonzalez et al. [p-synephrine (100 mg), p-synephrine (100 mg) and caffeine (100 mg) increased the number of repetitions and lifting volume (6 sets of squats for up to 10 repetitions at 80% 1RM) compared to control and placebo treatments. Differences were more noted in the final 3 sets of exercise. Further, mean power and lifting velocity was reported to be higher for the caffeine and p-synephrine treatment compared to control and placebo treatments. Consequently, there is evidence that ingestion of caffeine and/or p-synephrine prior to exercise may affect high intensity exercise performance.Numerous studies have evaluated the effects of ingesting PWS’s and/or energy drinks containing a variety of nutrients on exercise performance . For exaz et al.  reportedz et al.  reportedp-synephrine to the PWS or regular use of these PWS’s during training may yield more definitive ergogenic benefit remains to be unclear.In the present study, no clear ergogenic benefit of PWS or PWS + S ingestion was observed. While participants in the PWS + S treatment tended to perform more total work during the final set of bench press, no overall differences were observed in bench press or leg press lifting volume. Additionally, no differences were observed among treatments in the Wingate Anaerobic Capacity cycling sprint test. Therefore, while participants reported they had more energy and were more optimistic about performance, it did not result in statistically significant differences in upper and lower body muscular endurance or sprint capacity. Whether adding more p-synephrine is that they may increase heart, blood pressure, and/or prevalence of arrhythmias [One of the criticisms of use of energy drinks and/or PWS’s containing caffeine and/or hythmias , 73, 74.hythmias –56 as wehythmias . In thishythmias , 53. Morhythmias . We alsohythmias , 75–78.p-synephrine to the PWS yielded limited to no additive benefits. Whether regular use of the PWS’s used in this study and/or higher amounts of p-synephrine may promote greater benefits remains to be determined. Given the widespread use of PWS’s and energy drinks, additional study to examine the safety and efficacy is warranted.Within the limits of the present study, acute ingestion of PWS and/or PWS + S prior to exercise appears to be well-tolerated when consumed by young, healthy individuals. The primary effects appear to be to increase resting energy expenditure responses and improve perceptions about readiness to perform and cognitive function with limited to no effects on muscular endurance and no effects on anaerobic sprint capacity. The addition of 20 mg of


Regenerated tobacco has the potential to allow cultivation of Cd contaminated farmland to produce animal feed, assist in lowering total Cd content of soil, and allow income generation for farmers.The aim of this study was to estimate the influence of regenerated tobacco on the extraction of Cd from two acidic soils as well as to address the problem of how to deal with contaminated leaves following phytoextraction. Results showed that a coppicing tobacco led to a decline in Cd concentration in regenerated leaves and stalks when plants were grown in pots, but increased concentrations in regenerated lower and middle leaves when plants were grown under field conditions. The highest recorded bioconcentration factors in Chaling and Guanxi soil were 37.53 and 19.21 in lower leaves in the field, respectively. Total Cd extraction efficiency in practice  under field conditions confirmed our theoretical calculations . Use of a 0.5% hydrochloric acid(HCl) solution was sufficient to reduce Cd (98.4%) in tobacco leaves to permissible levels as required by the Hygienic Standard for Feeds in China (≤0.5 mg kg The non-essential element cadmium (Cd) is one of the most dangerous and potentially toxic metals present in the biosphere, highly mobile in soils, water, and the atmosphere, and causing damage even at low concentrations4. Pollution by Cd is especially significant as contaminated soils are used for agriculture; this metal is easily absorbed from soils into crops, inhibiting plant growth and nutrient uptake. The increasing presence of Cd in the environment has led to considerable concerns over the last decade6; contamination also represents a serious risk to human health as Cd is easily transferred within the food chain7.Contamination of agricultural soil is becoming an increasingly serious problem because of long-term exposure to pollutants in irrigation water, sludge, fertilizers, and pesticides7. One promising alternative approach is phytoextraction which is a remediation technique that uses the ability of the plant to uptake heavy metals from soil8. Although this technique has a lower cost and is less harmful to the soil environment, a longer time is required to reduce soil heavy metal concentrations to levels that conform to environment standards10. In reality, because farmland area is being reduced in some densely populated countries like China as population levels increase and cities expand, areas of contaminated soil are already exploited for agriculture. It is thought that 19.4% of farmland in China is currently contaminated11, which leads to serious economic losses for both farmers and local government. Therefore, if generated biomass can be switched to an alternative revenue stream that augments the livelihood of farmers, then the time needed to decontaminate soil may become less critical and slower phytoextraction projects based on the gradual attenuation of heavy metals rather than short-term forced removal may be possible13.It is an urgent requirement to either clean Cd from contaminated soil or control its uptake by crops, as these can be consumed by animals and humans. A number of technological developments aimed at achieving this, including soil washing, excavation, and land filling, are wasteful and expensive, and often destroy soil structure and fertilityNicotiana tabacum L.) is a commercial crop that fits the criteria for phytoextraction. It is fast growing, easily-harvested, generates a high biomass yield, and can accumulate high levels of Cd in tissues even under conditions of low exposure. Cd levels in most plant tissues decrease in the order: root > leaf > fruit > seed14; however, in tobacco, concentrations are higher in leaves than in the other parts of the plant15. Various studies have shown that tobacco has enhanced capabilities to uptake Cd from soil and to accumulate it in high volumes in leaves17. Indeed, the results of pot and hydroponic experiments have shown that, compared to other crops, tobacco is able to accumulate relatively higher concentrations of Cd. One greenhouse experiment showed, for example, that Cd concentrations in tobacco leaves reached 86.9 mg kg−1 and 271.5 mg kg−1, respectively, when nutrient solutions of 0.25 mg L−1 and 1.0 mg L−1 cadmium chloride (CdCl2) were applied18. Tobacco cultivated hydroponically and subjected to a 1.5 mg L−1 CdCl2 solution will grow normally without visible symptoms of toxicity while at the same time accumulating Cd concentrations of up to 226 mg kg−1 in its lower leaves.Tobacco  to determine the absorption and enrichment of Cd in tobacco before and after regeneration in two different contaminated soils on the basis of pot and field experiments; (2) to investigate whether the phytoextraction efficiency of Cd from contaminated soil can be enhanced by regenerated tobacco; and (3) to assess the possibility of using HCl as an extractant for the removal of Cd from contaminated tobacco leaves and to evaluate potential uses of Cd depleted leaves as animal feed.The majority of recent field and experimental studies have focused on the effects of Cd on the growth, development, physiology, and biochemical properties of tobacco, as well as on the differences between the various cultivars in Cd uptake and toleranceP ≤ 0.05). Cd levels in spring growth and regrown tobacco occur in the order, lower leaves > middle leaves > upper leaves > root > stalk . Following the first cutting of plants grown in Chaling soil, the proportions of Cd in upper, middle, and lower leaves were 11.33 mg kg−1, 14.08 mg kg−1, and 24.13 mg kg−1, respectively, while BCF values were high, up to 19.31, 23.99, and 41.11, respectively. In Guanxi pot experiments, total Cd was 1.34 mg kg−1 in soil, while the highest recorded concentration was 31.59 mg kg−1 in lower leaves following the first cut, corresponding with a BCF value of 23.45. With the exception of Cd concentrations in upper leaves, the proportion of this element in other parts of tobacco plants grown in Guanxi soil were higher than in plants grown in the Chaling soil. Results also show that cutting treatment had an effect on pot experiments using both soil types, and that the second cut resulted in a lower Cd concentration in tobacco leaves and stalks.Results of Cd concentrations and BCF (bioconcentration factors) values for tobacco at two cutting times in our pot experiments are presented in Fig. −1 in upper leaves to 16.57 mg kg−1 in lower leaves in the Chaling soil field, and from 15.56 mg kg−1 in upper leaves to 23.55 mg kg−1 in lower leaves in the Guanxi soil field. Following the second cutting in the field experiment, regenerated tobacco leaves (with the exception of upper leaves in the Guanxi soil field) showed a marked difference compared to those in the pot experiment; concentrations of Cd in lower and middle regenerated leaves were elevated to 22.03 mg kg−1 and 16.27 mg kg−1, respectively, in the Chaling soil field, and elevated to 25.88 mg kg−1 and 20.67 mg kg−1, respectively, in the Guanxi soil field. In addition, BCF values of regenerated tobacco plants grown under field conditions were also enhanced and higher than in pot experiments , potentially due to the more complex field environment. Average total leaves weights measured in pot experiments ranged between 194.93 g plant−1 and 197.40 g plant−1, with upper and middle leaves accounting for over 70% of this mass. Stalk weights ranged between 62.16 g plant−1 and 63.30 g plant−1, while under field conditions, the total biomass of tobacco leaves and stalks were in the ranges 4.24 ton ha−1 to 5.03 ton ha−1 and 1.41 ton ha−1 to 1.48 ton ha−1, respectively.None of the tobacco plants considered in this study exhibited symptoms of deficiency and yields recorded were within the normal range for plants in the local area. Variation in tobacco yields between the two cutting times in the field experiment were generally more obvious than those seen in pot experiments , before and after phytoextraction following two cuts under pot and field conditions are shown in Fig. −1 dry weight are shown in Fig. P ≤ 0.05). In addition, Cd concentration in tobacco leaves decreased markedly, from 18.23 mg kg−1 to 11.25 mg kg−1, 3.18 mg kg−1, 2.01 mg kg−1, and 1.28 mg kg−1 following first-extractions with 0.1%, 0.5%, 1%, and 2% HCl. On the basis of these results, Cd extraction efficiency was calculated based on the volume of metal removed from tobacco leaves. After first extraction, 2% HCl exhibited a significant high Cd removal efficiency of 92.98%, followed by the subsequent concentrations of 1%, 0.5%, and 0.1%, which had efficiencies of 88.97%, 82.56%, and 38.29%, respectively. In addition, Cd removal efficiency was significantly increased by the addition of successive extraction steps . In these cases, up to 80.75%, 95.67%, 98.68%, and 98.83% of total Cd was removed from plants via HCl extraction. Indeed, following three successive extractions, the Cd removal efficiency of tobacco leaves subject to 0.1% HCl  was remarkably lower than that seen at other HCl concentrations. Differences in Cd concentration in tobacco leaves treated with 0.5%, 1%, and 2% HCl following third extraction were not obvious, however, decreasing to 0.29 mg kg−1, 0.18 mg kg−1, and 0.16 mg kg−1, respectively, as up to 98.40%, 99.01%, and 99.12% of Cd were extracted in these cases. These results show that it is not necessary to use high HCl concentrations to extract Cd from tobacco leaves; just a 0.5% HCl solution is sufficient to attain satisfactory removal efficiency, the same as a 2% HCl solution following three successive extractions.The effects of different HCl concentrations on the removal of Cd from mixed tobacco leaves with an initial concentration of 18.23 mg kg−1 to 2,896 mg kg−1, from 3,780 mg kg−1 to 333 mg kg−1, from 3,488 mg kg−1 to 872 mg kg−1, and from 18,745 mg kg−1 to 13,697 mg kg−1, respectively. In contrast, extraction with 0.1% and 0.5% HCl solutions had little influence on the concentration of nutrient elements  compared to extraction with 2% HCl. However, a lower reduction was seen in the content of crude protein and Fe remaining in residual tobacco leaves following extraction with 0.1% HCl compared to a 2% solution; these values decreased from 13.19% to12.17%, and from 221 mg kg−1 to 209 mg kg−1, similar to the values seen in untreated tobacco leaves. The level of nicotine also decreased markedly, from 2.56% to 0.26%, following extraction with 0.1% HCl. Indeed, almost all the nicotine present in tobacco leaves was removed by three successive extractions using HCl solution concentrations between 0.5% and 2% mixed with 70% ethanol.Analytical concentrations of nicotine and nutrients measured in tobacco leaves before and after HCl extraction are presented in Table 21. It is also clear that the metal concentration in tobacco varies along the stalk, higher in older leaves than in younger top leaves21. Early work demonstrated that Cd accumulates at highest levels in the oldest tobacco leaves16. These results are consistent with time-dependent deposition of Cd in leaves; in other words, Cd accumulation increases with leaf age and is likely a permanent mechanism16. The results of our pot and field experiments show that the concentrations of Cd in spring growth and regenerated tobacco occur in the order, lower leaves > middle leaves > upper leaves > root > stalk . Such a difference in Cd concentrations in various plant parts is not unique to tobacco, this characteristic is seen in many plant species22. However, the strong ability of tobacco leaves to accumulate Cd may be due to the absorption of this metal in contaminated soil by roots and subsequent movement through the conductive system23. Vogeli-Lange and Wagner24 have suggested that Cd can be rapidly transported from root to shoot, probably within a few hours, a finding that is in agreement with the results of Rosén et al.25 who noted that the relatively high concentrations of this metal in tobacco leaves are reflected in a high TFp (ratio: shoot Cd/root Cd). Thus, tobacco meets an important condition as a hyperaccumlator, according to the criteria outlined by Brooks26.Tobacco is well-known for its metal accumulating capabilities; several species have been investigated over the years because Cd accumulation in leaves is known to be higher than in the stem and root27. However, although the phytoremediation of Cd contaminated soil using tobacco has been the subject of some preliminary research, this plant has not previously been considered a hyperaccumulator28. Normal concentrations of Cd in tobacco leaves grown in unpolluted soils range between 1 mg kg−1 and 3 mg kg−1 29. However, a high BCF of Cd in tobacco leaves has been reported not only under highly contaminated soil conditions but also under lower conditions of exposure. Cai et al.30, for example, found BCF values higher than 10 in tobacco leaves grown in Dayu county, Jiangxi Province, China, where the concentration of Cd in soil was only about 1 mg kg−1. In this study, highest BCF values were found in lower leaves while values for field and pot experiments were 37.53 (second cutting) and 41.11 (first cutting) for the Chaling soil, and 19.21 (second cutting) and 23.45 (first cutting) for the Guanxi soil  are most important in accounting for the accumulation of this metal in tobacco−1, and just a fraction of this will be absorbed by plants39. As a result, the concentration of Cd in tobacco leaves under field conditions will be lower than that seen in pot or hydroponic experiments. Results in this study show that Cd concentrations in pot experiment tobacco leaves after the first cut were higher than in the field experiment, while concentrations in lower and middle tobacco leaves in the field after the second cut increased to a level slightly higher than the pot experiment, which decreased after the second cut. This clear difference between natural field and controlled pot conditions may be due to different physiological conditions of both the plant and soil environment41. Total Cd in soil is also controlled and limited in the pot experiment, which will affect accumulation of Cd in regenerated leaves. In contrast, abiotic conditions are variable and more realistic in the field; regeneration of tobacco under field conditions not only does not influence the potential of leaves to accumulate Cd, but also improves uptake because the ripening time of regenerated tobacco is longer than that for first growth plants.Levels of Cd in field situations are typically low, usually less than 1 mg kg42; and (2) Cd removal efficiency from top soil in a theoretical case, calculated as % of soil metal removed by one crop = / × 10044. For comparative purposes, these two methods were adopted in this study to evaluate the phytoextraction efficiency of Cd from soil by regenerated tobacco. Results show that both theoretical and actual phytoextraction efficiencies of Cd by tobacco after two cuts in pot experiments are significantly higher than in field experiments. This is because just 15 kg of soil requires remediation by a potted tobacco plant, while almost eight times as much soil in a field will require remediation. In addition, total Cd extraction efficiency in practice  under field conditions is confirmed by theory , indicating that the remediation potential of Cd by regenerated tobacco is significant. Thus, regenerated tobacco plants will be more efficient at removing Cd from light or moderately contaminated acidic soils.Two approaches were used to determine phytoextraction efficiency: (1) Cd removal efficiency from the top soil (0–20 cm) expressed in terms of the decline percentage of overall soil concentration47, and HCl is one of the most commonly applied reagents. This approach works efficiently because metal extraction from soils or solids is more effective under acidic conditions48. Our leaching experiments demonstrate that just a 0.5% solution of HCl can remove 98.4% of Cd from tobacco leaves, a removal efficiency that is not significantly different compared to 2% HCl following three successive extractions. Subsequent concentrations of Cd in tobacco leaves were lower than permissible levels established by the Hygienic Standard for Feeds in China  in soil according to China Standard (GB15618-1995). In addition, this approach presents both an effective treatment and novel utilization for tobacco products.In summary, the findings of our study suggest that tobacco after being cut twice still appears to accumulate high Cd in leaves in moderately contaminated acidic soils. Compared to other crops, regenerated tobacco produces a large amount of biomass and enhances the removal efficiency of Cd from soil. We have also shown that the concentration of Cd in tobacco can meet the limits for feeds in China following three successive extractions with 0.5% HCl. At the same time, these extractions have little influence on the protein content of leaf residues, which means that they could provide a rich source of plant protein for food. This technique proposed here for the phytoremediation of Cd contaminated soil makes effective use of waste farmland in both time and space, could create considerable revenues for local farmers, and efforts to bring Cd concentration down to an acceptable level . At first cut (after 60 days), three pots each were allocated at random for harvesting of whole tobacco plantlets, while plants in another three pots were cut at two-thirds stalk length, fertilized again. After 80 days, the regenerated whole plants along with the top 20 cm of soil were harvested. The concentration of Cd in all parts of the tobacco plant and soil were determined after two successive cuts, and the dry weights of leaves and stalks were recorded.All the pot experiments reported here were carried out in a greenhouse at Hunan Agricultural University, Hunan, China. Polluted soils were sampled at the Chaling and Guanxi sites, were loaded into pots , and each soil treatment was replicated six times. One tobacco seedling was transplanted into each pot; fertilization and cultivation management were the same as in field experiment (N 150 kg ha−1), P (150 kg ha−1), and K (375 kg ha−1) in order to provide the necessary nutrients for plant growth before tobacco seedlings were transplanted to the field at a density of 2.25 seedlings per m2. The first cut was taken after 60 days; mature tobacco plants were cut at two-thirds of stalk length so as to allow them to regenerate into complete plants. At the same time, three randomly selected tobacco samples  were harvested from each plot. Following the first cut, plots were re-fertilized and allowed to re-grow without interruption until 80 days when three further samples  as well as the top 20 cm of soils were harvested from each plot. The concentration of Cd in all parts of the tobacco plant and soil were determined after two successive cuts, and the dry weights of leaves and stalks were recorded.The field experiments at the two sites comprised three plots each 10 m ×10 m planted with tobacco. The plots were fertilized with N (150 kg haw/v). These bottles were agitated on a rotary shaking table at a speed of 40 r min−1 for 12 hours at room temperature. After extraction, the sample was de-watered in a centrifuge at 4,000 rpm for 10 minutes and rinsed three times with 30 ml of deionized water. Leaves remaining in the bottle were drained and dried at 60°C for 72 hours, and all experiments were conducted in triplicate. Concentrations of Cd in tobacco leaves after each extraction, as well as nicotine and nutrients , after three successive extractions were analyzed.Tobacco leaves from both pot and field experiments were dried, ground, and mixed. Two factors, including different concentrations  of HCl in 70% ethanol solutions, and three successive extraction procedures were chosen to evaluate the effect of HCl on the removal of Cd from tobacco leaves. Batch extraction experiments were conducted in 60 ml centrifuge bottles that contained 1.00 g of powdered leaves alongside 20 ml of different concentrations of HCl (1:20 3-HCLO4) digestion57, while analysis of available soil Cd concentration was determined via extraction with 0.1 mol L−1 KCl. Soil pH  was determined using a pH meter (PHS-3C).Soil samples were collected from the top 20 cm of the field surface, air dried, and homogenized in an agitate mortar to pass through a 1mm sieve. Total soil Cd was determined following mixed acid  to determine total Cd and nutrient elements 58. The concentrations of Cd, Ca, Mg, Fe, K, and P in all samples were determined using either Optima 8300 ICP-OES  or graphite furnace flame atomic absorption spectrometry in a SpectrAA-GTA120 . Crude protein contents were determined via Kjeldahl analysis with a factor of 6.2559, while determination of nicotine in tobacco leaves follows the method reported by Saunders and Blume60.Tobacco samples, including roots, stalks, upper leaves (eight pieces), middle leaves (nine pieces), and lower leaves (eight pieces), were rinsed with tap water before being washed with deionized water. All plant samples were then oven dried at 105°C for two hours and then at 60°C until completely dry (48 hours). Subsequently, dried plant samples were then ground into a powder using a knife mill , and this was digested in a mixture of HNOBCF was calculated using Eq. CCd-aboveground (mg kg−1) is the Cd concentration measured in the aboveground parts of the plant, including the stalks and leaves, while CCd-soil (mg kg−1) is the Cd concentration in the soil.In this expression, Theoretical total Cd uptake was calculated using Eq. Field experiment:Pot experiment:41, as follows:Theoretical phytoextraction efficiency (%) of harvested tobacco was calculated using Eq. Field experiment:Pot experiment:The phytoextraction efficiency (%) of the top soil (0–20 cm) in practice was calculated using Eq. p ≤ 0.05) calculated using the statistical package SPSS V21were applied to compare the differences in heavy metal concentrations in crops, oils, and seed meals, as well as total metal uptake.All dates are presented as means and standard deviations using Origin 8.0 and Microsoft Excel software. One-way ANOVAs and Duncan’s multiple range tests (