The irrigation source plays an important role in the amount of contaminates in soil and cultivated agricultural products. In this study, the concentration and human health risks of heavy metals  were evaluated in soil, water and vegetables collected from two sites  of Bushehr, Iran. The trend of heavy metals concentration in vegetables from either site was in the following order: Mn ˃ Zn ˃ Cu ˃ Pb ˃ Cr ˃ Cd. Except Cd and Pb, the concentration of heavy metals was in the range of European Union (2006) permissible limit. THQ (Target hazard quotient) values were the highest in Mn followed by the Pb, Cd, Cu, Zn and Cr. Mn, Pb and Cd exceeded safe limit of THQ in several cases, while THQ of other heavy metals was in the range of safe limit. Based on the results, it can be concluded that heavy metals contamination in vegetables grown in Bushehr, especially in FWW site, may pose a great health risks to the local inhabitant through consumption of vegetables. Therefore, it is recommended that the discharge of industrial and municipal wastewater into water resources witch used for vegetable irrigation should be the first step in controlling the level of heavy metals in vegetables. Specifications tableValue of the data•Vegetables are one of the main components of the food basket of people in the world, including Iranians. Therefore, considering their high consumption by humans, their quality control is very important. The data of this study are in line with the above goal•So far, a similar study has not been done in the study area (Bushehr city). Therefore, the data from this study can provide a basis for future similar studies.•The data of present study showed that heavy metals contamination in vegetables grown in Bushehr, especially in farms irrigated by wastewater (FWW) site, may pose a great health risks to the local inhabitant through consumption of vegetables.•The obtained data of this study showed that agricultural lands irrigated with wastewater contained higher levels of heavy metals in their soil compared to those irrigated with ground water (FGW).11.1−1 in FWW and FGW, respectively, followed by Cr, Pb, Mn, Cu and Cd with concentrations of 53.25 (23.84), 27.29 (12.83), 23.32 (8.24), 17.66 (9.27) and 7.63 (4.31) µg L−1 in FWW (and FGW), respectively.Concentrations of heavy metals recorded in water that used for irrigation and also soils from FGW and FWW are given in −1 for Zn, Mn, Cu, Cr, Cd and Pb in FWW (and FGW), respectively , 277.62 (196.12), 32.68 (18.31), 42.19 (17.09), 1.76 (0.87) and 12.04 (5.83) mg kgectively . Heavy m1.2Concentrations of heavy metals in the different vegetables collected from two locations of FWW and FGW in Bushehr are summarized in the Concentrations of heavy metals in the edible parts of vegetables collected from two locations of Bushehr are represented in 1.3Transfer factor is defined as metals migration in any form from the soil to the edible part of plants that make them available for consumption. This function is related to several physiochemical factors of soil and plant species that mentioned earlier 1.4−1), Cu (0.0069 mg d−1), Cd (0.00011 mg d−1) and Pb (0.00065 mg d−1) were observed in spinach, and the greatest DIM of Mn (0.01917 mg d−1) and Cr (0.00009 mg d−1) were found in lettuce and potato respectively. Additionally, the general trends for DIMs in vegetables grown in FWW were in the order of Mn > Zn > Cu > Pb > Cd > Cr. In FWW site, the highest DIM of Zn (0.00503 mg d−1), Cu (0.00160 mg d−1), Cd (0.00039 mg d−1) and Pb (0.00079 mg d−1) were observed in spinach, and the greatest DIM of Mn (0.02207 mg d−1) and Cr (0.00013 mg d−1) were found in lettuce and potato respectively.The Daily intakes of metals (DIM) for adults in the Bushehr through consumption of vegetables are given in the The target hazard quotient (THQ) for metals through consumption of vegetables grown in FGW and FWW were calculated for adults and values are presented in 22.1Study area, Bushehr city, is located in south of Iran, on the hot Persian Gulf, has a subtropical desert climate, with a mean annual temperature of 24.8 °C, an annual rainfall of 223.2 mm and an average altitude of 28 m 2.2Soil samples from farm lands were obtained by digging a monolith of 10 × 10 × 10 size using a stainless steel auger. Particles that did not belong to soil such as wooden pieces, stones, organic debris and rocks were removed. All Samples were mixed and dried for 12 h in 100 °C, thereafter grounded to pass 2 mm sieve and kept in poly ethylene bags until analysis. During sampling and storage, it was ensured that samples had not any contact with metal tools for inhibiting cross-contamination 2.3Water samples were collected into pre cleaned bottles that washed earlier with a metal-free soap and eventually rinsed with deionized water. Samples were stored in a 4 °C refrigerator to minimize biodegradation and volatilization prior to analysis 2.4During 2017, a total of 336 samples of harvested fresh vegetables were obtained from different farm lands. Samples were classified into leafy vegetables , tuber vegetables , and fruit vegetables . Vegetable samples from each site were collected in three replicates and transported to laboratory in clean polythene bags. The samples were cleaned with distilled water to remove the extraneous materials, then dried in an oven at 100 °C and grinded into powder form to be prepared for digestion 2.53, H2SO4 and HClO4 at ratio of 5:1:1 at 110 °C until the solution became transparent −1 for Mn, 50 mg L−1 for Pb and Zn, 20 mg L−1 for Cu, 5.0 for Cr and 2.0 mg L−1 for Cd were applied for preparing working standards. The analysis was operated with radiofrequency power of 1250 W. Delay time was 28 s, and the flow rate of nebulizer, plasma, auxiliary and sample gas were 0.9, 15, 0.2 and 1.6 L min−1, respectively. The detection limit (mg kg−1) and wavelength (nm) of heavy metals were: 0.006 and 206.2 for Zn; 0.008 and 267.7 for Cr, 0.05 and 221.4 for Pb, 0.004 and 222.8 for Cd, 0.01 and 257.6 for Mn, and, 0.007 and 321.4 for Cu, respectively.Vegetable samples were digested by a mixture of HNO2.63/H2SO4/HClO4 ratio 5:1:1), and NIST Standard reference Material 1570A for vegetables, SRM 2709 for soil and RM 1643E for water for heavy metals and delicately analysis of samples via ICP–AES. The recovery rate for Mn, Cd, Cu, Cr, Zn and Pb were 97.5%, 94.3%, 96.2%, 94.9%, 93.6% and 97.4%, respectively, showing high performance analysis method of heavy metals.All Chemical reagents which used for the sample preparation were of analytical grade and purchased from MERCK Chemicals Germany. A quality control was carried out using reagent blanks ; Cfactor is conversion factor (0.085); Wfood is the average of vegetables daily intake; Bw represent the body weight. Our survey on 250 males and females revealed that a local inhabitant consumes averagely 167 g of vegetables per day. The age of participants varied from 31 to 64 year-olds, with an average value of 44 year-olds in male inhabitant; the corresponding data were 27–56 and 39 year-olds in female inhabitant, respectively. In addition, the body weight of participants varied from 55–82 to kg year-olds, with an average value of 71 kg in male inhabitant; the corresponding data were 47–69 and 58 kg in female inhabitant, respectively. In this study the average of body weight in all adult were assumed to be 65 kg.The Daily intake of metals (DIM) was calculated via data achieved using a questionnaire. DIM is related to the heavy metals concentrations in vegetables and the rates of vegetables intake. DIM was calculated in adults by the following equation:2.7.3−1); ED is exposure duration (70 years); FI shows food ingestion (g person−1 d−1); MC represents metal concentration in food ; RfD is the oral reference dose (mg kg−1 d−1) which for Pb, Ni, Cd, Cr, Cu, Mn and Zn is 0.004, 0.02, 0.001, 1.5, 0.04, 0.033 and 0.3 mg kg−1 d−1, respectively; BW is the average body weight of average adult (65 kg); AT indicates averaging time for noncarcinogens  Target hazard quotient (THQ) was used to evaluate the local inhabitant health risk of heavy metals from consumption of vegetables. Value less than 1 is considered to be safe for exposed population. The THQ was estimated using method proposed by U.S. EPA Region III risk-based concentration table


Centrostachys aquatica is a perennial emergent macrophyte in marshy places and in rivers. The species was recorded in Senegal and Nigeria, but widespread in tropical Africa, and South and East Asia. Aqueous methanol extracts C. aquatica was found to be toxic to several plant species. However, no phytotoxic substance has been reported in this species. Therefore, we investigated phytotoxic activity and searched for phytotoxic substances with allelopathic activity in C. aquatica.C. aquatica inhibited the growth of roots and hypocotyls of cress (Lepidium sativum). The extract was then purified by several chromatographic runs and a phytotoxic substance with allelopathic activity was isolated and identified by spectral analysis as loliolide. Loliolide inhibited cress root and hypocotyl growth at concentrations greater than 0.03 μM. The concentrations required for 50% growth inhibition of cress roots and hypocotyls was 0.18 and 0.15 μM, respectively.An aqueous methanol extract of C. aquatica.These results suggest that loliolide is a phytotoxic substance and may contribute to the allelopathic effect caused by The online version of this article (doi:10.1186/s40529-014-0059-1) contains supplementary material, which is available to authorized users. Centrostachys aquatica (R.Br.). Wall ex Moq Tand  is a perennial emergent macrophyte with 0.3-1.5 m stem in length and rooting at nodes forming meadows in marshy places and in rivers. The species was recorded from Senegal and Nigeria, but widespread in tropical Africa, and South and East Asia including India .Centaures maculosa releases an allelopathic substance, catechin, which makes its invasion into the new habitats . Thus, schehoug ).C. aquatica were toxic to several plant species and shown allelopathic activity . The fiCenstrotachys aquatica (R.Br.) Wall ex Moq Tand were collected from riversides, in Ô môn district, Cần Thơ City, South of Vietnam , in January of 2012 (dry season), washed with tap water and dried under sunlight. Dry materials were then packed and protected from air humidity by a silica gel desiccant and stored at 3°C until extraction. Cress (Lepidum sativum L.), obtained from Takii Co. , was chosen as a test plant for the bioassays due to their known seedling growth behavior.Whole plants of C. aquatica (300 g) were soaked in 1.5 L of 70% (v/v) aqueous methanol for two days. After filtration using filter paper , the residue was soaked again in 500 mL methanol for two days and filtered, and the two filtrates were combined. An aliquot of the extract  was evaporated to dryness and biological activity of the extracts was determined by a cress bioassay as described latter.Dried plants of C. aquatica were extracted as described above. The two filtrates were combined and concentrated at 40°C in vacuo to produce an aqueous residue. The aqueous residue was then adjusted to pH 7.0 with 1 M phosphate buffer, partitioned three times against an equal volume of ethyl acetate, and separated ethyl acetate and aqueous phase. The biological activity of the aqueous and ethyl acetate fractions was determined using a cress bioassay as described latter.Dried plants of n-hexane (100 mL per step), ethyl acetate (100 mL) and methanol (200 mL). The biological activity of all separated fractions was determined using a cress bioassay as described latter, and the most active fraction was obtained by elution with 70% ethyl acetate in n-hexane.The ethyl acetate fraction was evaporated to dryness and separated on a column of silica gel , eluted with 20, 30, 40, 50, 60, 70 and 80% ethyl acetate in 18 cartridges . The cartridge was eluted with 20, 40, 60, 80 and 90% (v/v) aqueous methanol, and methanol (15 mL per step). The active fraction was eluted by 80% aqueous methanol and evaporated to dryness. The residue was finally purified by reverse-phase HPLC  eluted at flow rate of 1.5 mL min−1 with 40% aqueous methanol and detected at 220 nm. The inhibitory activity was detected at a peak of 162 -172 min, yielding active compound as colorless oil. The compound was characterized by HRESIMS, 1H-NMR spectrum  and the specific rotation.After evaporation of the most active fraction, the residue was purified by a column of Sephadex LH-20 , and eluted with 10, 20, 30, 40, 60 and 80% (v/v) aqueous methanol (100 mL per step), and methanol (200 mL). The active fraction was eluted by 30% aqueous methanol. After evaporation, the residue was dissolved in 20% (v/v) aqueous methanol (2 mL) and loaded onto reverse-phase CC. aquatica extract or isolated compound. The bioassay was repeated three times using a randomized design with 10 plants for each determination. Significant differences between control and treatment were examined by Welch’s t-test. Significant differences among treatments were examined by Duncan’s multiple comparison tests.Test samples (aqueous methanol extracts and a purified compound) were dissolved in a 0.2 mL of methanol and added to a sheet of filter paper (No. 2) in a 3-cm Petri dish. The methanol was evaporated in a fume hood. Then, the filter paper in the Petri dishes was moistened with 0.8 mL of 0.05% (v/v) polyoxyethylene sorbitan monolaurate (Tween 20). Ten seeds of cress were placed in the Petri dishes. The length of roots and hypocotyls of the seedlings were measured after 48 h of incubation in the darkness at 25°C, and compared to control seedlings. Controls were treated exactly as described above, with the exception that 0.2 mL methanol was used instead of C. aquatica inhibited root and hypocotyl growth of cress seedlings. Increasing the extract concentration resulted in an increase in the inhibition . These C. aquatica was then divided into aqueous and ethyl acetate fractions. Both fractions inhibited root and hypocotyl growth of cress seedlings . The 1H spectrum , 4.34 , 2.46 , 1.98 , 1.79 , 1.79 , 1.54 , 1.47 , 1.28 . The specific rotation of the compound was [α]D22 = -41° . The compound was identified as 3-dihydroxy-3,5,5-trimethylcyclohexylidene-4-acetic acid lactone , Undariand Maki ) and Sarg et al. ). The co  and Euca et al. ). Howeve50) in the assay, as determined by a logistic regression analysis, were 0.18 and 0.15 μM for cress roots and hypocotyls, respectively. In addition, loliolide was reported to have anti-algal activity , immunou et al. ) and cytu et al. ).Figure Many of the phytotoxic substances from the invasive plants have been reported to have multiple effects, including anti-herbivore, anti-fungal, anti-microbial and allelopathic activities (Lockwood et al. ; CappuccC. aquatica extract. The chemical structure of the substance was determined as loliolide. IC50 value for cress roots and hypocotyls was 0.18 and 0.15 μM, respectively. These results suggest that loliolide may contribute to the allelopathic effect caused by the C. aquatica extract.A phytotoxic substance with allelopathic activity was isolated from


Eisenia fetida) were used in a 14-day acute toxicity test. Fenoxaprop-ethyl showed low toxicity to earthworms with LC50 value of 322.9 µg g−1. Biochar amendment was non-toxic to earthworms and effectively reduced the toxicity. The results suggested that the application of biochar may reduce the risks of fenoxaprop-ethyl in the soil environment.Biochar, as a soil amendment in agriculture, has attracted considerable attention. In the study, the fate and toxicity of the herbicide fenoxaprop-ethyl were evaluated in soils with and without 5% rice husk biochar amendment. Fenoxaprop-ethyl and metabolite fenoxaprop degradation followed first-order kinetics in the two soils. Fenoxaprop-ethyl decreased fast with half-lives less than 2 days. Large amounts of fenoxaprop formed and remained in the control soil. However, fenoxaprop was much lower in the biochar-amended soil with reduction over 85% on the 35th day. The estimated half-lives of fenoxaprop were 56.9 and 1.5 days in the control and biochar-amended soils, respectively. Biochar restrained the formation and promoted the dissipation of fenoxaprop. Biological indicator earthworms ( Fenoxapet al. [et al. [The fate of fenoxaprop-ethyl in various soils has attracted much attention. It was reported that fenoxaprop-ethyl dissipated in soil with half-lives of 0.5 day . The soiet al.  studied  [et al.  studied Biochar research has progressed considerably in recent years . BiocharIn addition, less attention has been paid to the effects of biochar on toxicity of pesticides to soil biota . EarthwoTo guide the rational application of biochar, the potential risks of returning biochar to soil need to be identified. In this study, the effects of biochar amendment on fenoxaprop-ethyl residues in soil were evaluated. The content of metabolite and the toxicity of fenoxaprop-ethyl to soil organism earthworms were fully considered. Assessing the impacts of biochar on pesticide risk is essential for protecting the environment and human health.2.2.1.Fenoxaprop-ethyl with purity 98.0% was obtained from Institute for the Control of Agrochemicals, Ministry of Agriculture . Fenoxaprop with purity 98.0% was achieved by synthesis according to the literature . ChromatThe biochar was produced from rice husks and subject to pyrolysis at 550°C. The biochar contained 52.2% carbon, 1.6% hydrogen, 1.6% potassium, 0.8% phosphorus, 0.7% nitrogen and 0.2% sulfur.The soil was collected from Baiwang Mountain Forest Park . The soil was air-dried at room temperature and passed through a 2 mm sieve. The soil contained 6.9% clay, 40.8% silt and 52.3% sand. The organic matter was 4.0% and pH 7.1.Eisenia fetida) weighting 0.4 ± 0.05 g were obtained from a farm, Beijing, China. Earthworms were acclimatized to laboratory conditions for 14 days with 20 ± 2°C under a 12 L : 12 D cycle.Earthworms  were between 85% and 102% in the control soil, and between 83% and 106% in the biochar-amended soil based on RSD below 20% (n = 3).HPLC-MS/MS analysis of fenoxaprop-ethyl and fenoxaprop was performed using Thermo Scientific TSQ Quantum Access Max equipped with an Agilent Eclipse XDB-C18 column . The column temperature was maintained at 25°C. Using an autosampler, 5 µl aliquots were injected. The mobile phase consisted of acetonitrile and water (9 and 1 by volume) at a flow rate of 0.5 ml mintC = A 1+ A2ekt−) was used to fitting the degradation of fenoxaprop-ethyl and fenoxaprop in soils [t is the time (days); Ct is the concentration in soil at time t (μg g−1); k is the degradation rate constant (day−1); A1 and A2 are constants (μg g−1). The statistical difference between control treatment and biochar-amended treatment was assayed using analysis of variance (ANOVA). The differences were considered statistically significant when p was less than 0.05.A one-phase exponential decay equation  in a 1 l glass beaker. Solvent control groups were also conducted. The glass beakers were placed in a lighted incubator with continuous illumination at 20 ± 2°C. On the 14th day of exposure, mortality was recorded. The judgement of death was based on the response of the front end to mechanical stimulation. The acute toxicity bioassays were conducted in soil with and without biochar amendment. Biochar was amended and replaced 5% of the soil in glass beakers to study the impacts of biochar on the toxicity of fenoxaprop-ethyl on earthworms.To evaluate the effects of biochar on the toxicity of fenoxaprop-ethyl, an acute toxicity test was conducted using the earthworms. According to the OECD guideline for testing of chemicals 207, earthworms were exposed to the fenoxaprop-ethyl contaminated soil for 14 days. The maximum exposure concentration was set to 1000 µg g3.3.1.t =C 0.215 + 6.370 × 10t−1.431 with R2 of 0.986 . In short, biochar increased the residual risk of fenoxaprop-ethyl in soil.The effects of biochar on the fate of fenoxaprop-ethyl in soil are presented in of 0.968 . The hal−1 in the biochar-amended and control soils, indicating a reduction over 33%. The degradation of fenoxaprop followed first-order kinetic equation t =C 0.686 + 2.965 × 10t−0.620 with R2 of 0.953 (−1) and low log Kow (1.04) [−1 in the biochar-amended and control soils, respectively. The reduction was of over 85%. Therefore, biochar has the potential to reduce residue risk of fenoxaprop in soil.Metabolite fenoxaprop first increased and then decreased in the biochar-amended soil . Fenoxapof 0.953 . The halw (1.04) , which aw (1.04) ,28. Distw (1.04) . Biochar3.3.E. fetida) was evaluated by a 14-day soil test. All earthworms survived in the solvent control group soil at the end of test. In the experimental group soil, there was a dose–response relationship between concentration and mortality rate of earthworms. The mortality rates were 0%, 10%, 30%, 90% and 100% at concentrations of 62.5, 125, 250, 500 and 1000 µg g−1, respectively. Fenoxaprop-ethyl showed no effects on earthworm survival at concentration below 62.5 µg g−1. However, 1000 µg g−1 of fenoxaprop-ethyl caused 100% mortality. The calculated LC50 value was 322.9 µg g−1 (250.4–429.6 µg g−1), which indicated that fenoxaprop-ethyl was low toxic to earthworms.The toxicity of fenoxaprop-ethyl to soil organism earthworms (−1), indicating that the LC50 value was above 1000 µg g−1. The LC50 values were 322.9 µg g−1 (250.4–429.6 µg g−1) in the experimental group soil and above 1000 µg g−1 in the experimental group biochar-amended soil. Based on LC50 values, biochar significantly reduced the toxicity of fenoxaprop-ethyl to earthworms (p < 0.01). The results may be due to the absorption of biochar. Biochar absorbed pesticides and led to prominent decrease in the bioavailability in soils to earthworms [No death was observed in the solvent control group biochar-amended soil at the end of the test. The results show that the toxicity of biochar to earthworms was limited, which was similar to that of the previous literature . It has rthworms . The red4.−1) compared to that in the control soil (maximum 5.787 µg g−1). The dissipation was much faster in the biochar-amended soil  than in the control soil . Biochar absorption and microorganisms condition were possible causes for the results. Earthworm was used as the representative of soil organism in the acute toxicity test. The amendment of biochar caused reduced toxic effects to earthworms. The LC50 values were 322.9 µg g−1 and above 1000 µg g−1 in control and biochar-amended soils, respectively. In short, biochar posed positive effects on residues and toxicity. The application of biochar to soil has good potential for remediation and may serve as a beneficial farming strategy for the soil environment.The fate of fenoxaprop-ethyl and metabolite fenoxaprop in the control and biochar-amended soils was investigated. The dissipation of fenoxaprop-ethyl was retarded by the biochar-amended soil. The half-lives of fenoxaprop-ethyl were 0.5 and 1.2 days in the control and biochar-amended soils, respectively. Less metabolite fenoxaprop was formed in the biochar-amended soil (maximum 3.865 µg g


Compared to previous traditional method, this improved method shortened the experimental cycle and became more efficient because it was successfully applied to analyze total antioxidant activity and contents of twelve antioxidants of Xuebijing injection under the same condition. The results revealed that the on-line DPPH-CE-DAD method was a reagent-saving, rapid, feasible, and green technique for quality control of Xuebijing injection in terms of pharmacological activity and contents of active ingredients. It also offered new opportunities for the analysis of antioxidant activity of complex matrix.Much attention has been focused on treatment of sepsis which leads to high mortality all over the world in every year. Antioxidant activity seems to play a prominent role in the treatment of sepsis exhibited by Xuebijing injection. The aim of the present research was to develop an on-line 1, 1-diphenyl-2-picrylhydrazyl- (DPPH-) capillary electrophoresis-diode array detector (on-line DPPH-CE-DAD) method for rapidly assessing antioxidant properties and efficacious material basis of antioxidant activity as a way of quality control of Xuebijing injection. Several parameters affecting the separation were investigated, including the pH and concentrations of buffer, SDS,  Carthami flos, Paeoniae Radix Rubra, Chuanxiong Rhizoma, Salviae miltiorrhizae, and Angelicae sinensis Radix, is a traditional Chinese medicine that has been approved for many years by the State Food and Drug Administration (SFDA) of China to clinically treat sepsis . P0 was peak area of DPPH (on-line spiked deionized water) and P1 was peak area of DPPH (on-line spiked Xuebijing injection). In the total antioxidant activity assay of samples, Xuebijing injections with different diluted times were on-line spiked with DPPH, respectively, to carry out the maximal inhibitory concentration at 50% (IC50) which denotes the diluted times of sample required to scavenge 50% of DPPH radicals. 50 values of 10 batches of Xuebijing injections. Results illustrated that Xuebijing injections have antioxidant activity and there was a significant difference in the IC50 values of each batch which could speculate that the discrimination of activity was as a result of different contents of antioxidants.DPPH assay based on the reduction of absorbance at 517 nm of the stable DPPH radical by an antiradical is easy and potentially accurate for measuring the general radical scavenging capabilities of antioxidants . Relyingy) versus the concentration in μg/mL as abscissa (x). They were obtained over the range of 33.3–400 μg/mL, 25–600 μg/mL, 4.2–50 μg/mL, 62.5–2000 μg/mL, 5.8–70 μg/mL, 10–120 μg/mL, 13–156 μg/mL, 2.5–30 μg/mL, 2.5–60 μg/mL, 20–480 μg/mL, 13–156 μg/mL, and 11–132 μg/mL for oxypaeoniflorin, hydroxysafflor yellow A, protocatechuic aldehyde, peoniflorin, rosmarinic acid, salvianolic acid B, sodium danshensu, caffeic acid, ferulic acid, senkyunolide I, rutin, and isoquercitrin, respectively. The straight lines of the twelve compounds obtained from six separate experiments had good correlation coefficients. The regression equations and their correlation coefficients were y = 0.0658x + 0.3049 (R2 = 0.9991), y = 0.3099x − 1.1776 (R2 = 0.9999), y = 0.6055x − 0.2398 (R2 = 0.9996), y = 0.1948x − 0.6145 (R2 = 0.9998), y = 0.3097x − 0.0843 (R2 = 0.9990), y = 0.2089x − 0.526 (R2 = 0.9995), y = 0.1248x − 0.311 (R2 = 0.9982), y = 0.8707x − 0.2758 (R2 = 0.9995), y = 0.8666x − 0.3651 (R2 = 0.9995), y = 0.198x − 0.8145 (R2 = 0.9993), y = 0.2938x − 1.0819 (R2 = 0.9990), y = 0.3888x − 0.0695 (R2 = 0.9991), respectively. The limit of detection (LOD) and the limit of quantitation (LOQ) were described as the sample concentrations gave rise to a signal-to-noise (S/N) ratio of 3 and 10, respectively. The calibration graphs of twelve antioxidants of Xuebijing injection were established with the peak area ratio as ordinate . The intraday and interday precisions at three typical assay concentrations of each constituent and DPPH were evaluated for six replicates within one day and over three successive days, respectively. The intra- and interday accuracies of twelve antioxidants were within the range of 92.1%–105%. The RSDs of mean precision of twelve antioxidants were below 5.1% for intraday and 4.9% for interday, respectively . The RSDThe stability expressed by peak area was evaluated by injecting repetitive quality control samples of twelve compounds at low, medium, and high concentrations and DPPH in the CE equipment over 24 h. All these data indicated that electrophoretic assay method was acceptable and could be applied in determining the total antioxidant activity of the multicomponents of Xuebijing injection.Although the antioxidant activity of Xuebijing injection has been demonstrated, it was not clear as to which component was responsible for the antioxidant activity. Taking the identification of active ingredients into account, the peak area of all of the ingredients of Xuebijing injection was compared successively between sample on-line spiked DPPH  and sample on-line spiked methanol (control group II) . The comμg/mL, 381.2–529.4 μg/mL, 15.4–27.3 μg/mL, 1736.6–1980.9 μg/mL, 10.8–25.4 μg/mL, 6.9–17.4 μg/mL, 13.1–24.7 μg/mL, 9.4–14.6 μg/mL, 32.0–37.0 μg/mL, 266.1–382.4 μg/mL, 18.8–28.0 μg/mL, and 26.7–40.2 μg/mL, respectively, which illustrated major difference in the concentrations of twelve antioxidants of each batch of Xuebijing injection  with above-referred data of determination of total antioxidant activity of samples. The result illustrated that twelve selected antioxidants can serve as key quality control markers to maintain batch-to-batch uniformity and efficacy. It was also validated that the proposed method was feasible and accurate in determining the total antioxidant activity with multiple active ingredients for quality control of TCMs.Given the difference in the ICTo improve the previous DPPH-CE-DAD method, we developed a simple method that made it possible to separate and quantify DPPH and the antioxidants under the same condition. In the course of on-line mixing DPPH and sample, the magnitude of decrease of DPPH peak seen in the electrophoretogram could calculate the total antioxidant activity of Xuebijing injection. Similarly, twelve antioxidant active ingredients containing oxypaeoniflorin, hydroxysafflor yellow A, protocatechuic aldehyde, peoniflorin, rosmarinic acid, salvianolic acid B, sodium danshensu, caffeic acid, ferulic acid, senkyunolide I, rutin, and isoquercitrin were rapidly screened relying on the magnitude of decrease of composition peak seen. Results illustrated that the advantages of this developed method were reagent-saving, rapid, feasible, and green technique which is not harmful to the environment. The improved on-line DPPH-CE-DAD method achieved the goal in analyzing the total antioxidant activity and contents of antioxidants of Xuebijing injection in one step and was applicable to evaluate the quality of TCMs which exerted directly their antioxidant effects on the radical itself. Furthermore, this presented approach can be applied in evaluating the antioxidant activity of unknown molecules.


Chlorella vulgaris biomass and to reduce nutrients of manure. In this study, we focused on investigating the use of flushed manure, produced in a dairy farm for growing C. vulgaris biomass. A series of batch-mode experiments, fed with manure feedstock and synthetic medium, were conducted and corresponding C. vulgaris production was analyzed. Impacts of varying levels of sterilized manure feedstock (SMF) and synthetic culture medium (SCM) (20–100%) on biomass production, and consequential changes in total nitrogen (TN) and total phosphorus (TP) were determined. C. vulgaris production data  was used as a culture medium. On this ratio, TN and TP were reduced by 29.9 and 12.3% on Day 9, and these reductions on Day 30 were 76 and 26.9%, respectively.The utilization of dairy wastewater for producing algal biomass is seen as a two-fold opportunity to treat wastewater and produce algae biomass, which can be potentially used for production of biofuels. In animal agriculture system, one of the major waste streams is dairy manure that contains high levels of nitrogen and phosphorus. Furthermore, it is produced abundantly in California’s dairy industry, as well as many other parts of the world. We hypothesized that flushed manure, wastewater from a dairy farm, can be used as a potential feedstock after pretreatment to grow  Chlorella vulgaris and the biomass production can help in lowering excessive nutrients [total nitrogen (TN) and total phosphorus (TP)] in manure, which poses environmental challenges by increasing nutrients in ambient water. In previous published study , which can lower the cost of algal biomass production, and helps in reducing environmental contamination as well as protecting ambient water , results showed that medium with anaerobic digester effluent from cattle manure produced comparable biomass growth to that in BBM. Biomass productivity was ≈280 mg/L.In addition to municipal waste , animal waste streams generated in livestock industry could be a potential source for algal biomass production  understand the change in TN and TP corresponding to biomass production in a series of experiments using pretreated and untreated flushed manure and (2) calculate TN and TP removal rate, and kinetics of nutrient recovery under C. vulgaris growth in feedstock, where flushed manure was supplemented with various levels of synthetic media.The hypothesis of this study was that the pretreated flushed dairy manure could possibly be used as a suitable medium to grow algal biomass and reduce the nutrients of manure wastewater produced in a dairy farm. This study is built on a previous study , which is a mixture of feces and urine, parlor wash-off, and pre-milking wash pen drainage. Subsequently, the FLM passes through a separator to fractionate liquid and solids. Liquid portions go into lagoons and solid fractions are piled, which is eventually used either as fertilizers or bedding materials. After collections, samples were transported using coolers and subsequently samples stored at 4°C prior to starting the experiment. The FLM samples were centrifuged  at 10,000 rpm for 15 min to remove unwanted solids and fibers. The supernatant was used as manure feedstock medium (MFM). Furthermore, a subset of the MFM was sterilized at 121°C for 15 min to inactivate manure-borne microbial population and this medium was termed as sterilized manure feedstock (SMF).3, 0.075 g of K2HPO4⋅3H2O, 0.075 g of MgSO4⋅7H2O, 0.025 g of CaCl2⋅2H2O, 0.175 g of KH2PO4, 0.025 g of NaCl, 40 mL of soil extract solution, 0.005 g of FeCl3⋅6H2O, 1 mL of Fe-EDTA solution, and 1 mL of A5 solution. The soil extract solution was prepared with 200 g of unfertilized garden soil and 1,000 mL of distilled water. After mixing garden soil with distilled water, the mixture was heated in water bath for 3 h and cooled for 24 h. As a filtering step, the solution was filtered using 0.45 µm filter and supernatant was used as a soil extract solution. The Fe–EDTA solution was prepared by mixing 50 mL distilled water, 1 g of Na2EDTA, 81 mg of FeCl3⋅6H2O, and 50 mL of 0.1 N HCl. The A5 solution includes 2.86 g/L of H3BO3, 1.86 g/L of MnCl2⋅4H2O, 0.22 g/L of ZnSO4⋅7H2O, 0.39 g/L Na2MoO4⋅2H2O, 0.08 g/L of CuSO4⋅5H2O, and 0.05 g/L of Co(NO3)2⋅6H2O. The SCM was autoclaved and stored at 4°C prior to use it.In addition to the manure-based medium (MFM and SMF), synthetic culture medium (SCM) was prepared. The SCM includes 958 mL of distilled water, 0.25 g of NaNOC. vulgaris (UTEX-2714) was assessed in MFM, SMF, and SCM media. Furthermore, the growth was also investigated in mixed environments MFM:SCM and SMF:SCM ratios of 20:80, 40:60, and 70:30. The growth experiment was conducted in 500 mL conical flasks at a constant temperature (25 ± 1°C). The strain of C. vulgaris was obtained from the Culture Collection of Algae, University of Texas, Austin, TX, USA. The pre-cultured C. vulgaris [optical density (OD) 680 ≈ 0.355] was inoculated into 500 mL conical flasks with a proportion of 20% (v/v) under sterile conditions. To avoid the potential ambient contamination, the experiments were conducted in biological controlled environment . Additional details of experiments are described elsewhere .The growth of algae was estimated by measuring the OD of the samples containing tion Eq.  was deveg for 15 min to separate algae. Standard method 10071 and 8190 was applied for the TN and TP analysis. The HACH reagents and a spectrophotometer  were used for the analysis.Total nitrogen and TP concentrations were analyzed for estimating the TN and TP losses in manure due to biomass growth. Samples withdrawn from flasks every 2 days were centrifuged at 10,000 rR represents the substrate removal rate, iS is the initial substrate concentrations as TN or TP, tS is the corresponding substrate concentration at tT. The tT is the time when concentration of the substance did not change. The initial substrate removal rate is the slope of time versus effluent concentration at time tT. The specific rate of substrate removal (Rsr) was estimated as Rsr = rR/(mB)i. The Michaelis–Menten kinetics was used to calculate kinetic coefficients (mK), saturation constant (k), and reaction rate constant (R)  Eq.  (Aslan a (R) Eq. (3)R=RmrRmi = k·iX, which is the maximum initial substrate removal rate. The modified version of Eq. Rsi estimation , iX is the initial biomass concentration for algae. The specific substrate removal (Rsr) rate was calculated as:Considering mK/k) and intercept (1/k) was estimated f is the final biomass concentration (mg/L), (mB)i is the initial biomass concentration (mg/L) , respectively. Slope of plot between [(mB)f − (mB)i] and [TNi − TNf] resulted yield coefficient for TN, i.e., NY (mg mB/mg TN). Similar approach can be implemented for the TP.Yield coefficient for TN and TP removal was calculated as:The change in TN and TP was evaluated for a various levels of sterilized (pretreated) manure feedstock , untreated MFM , and SCM. The experiment lasted for total of 30 days, however, during the first 9 days, the observations were made daily and, subsequently, the measurements were made on Day 30. With regard to algal biomass harvesting, a growth cycle of 10 days is often used for biomass productivity estimation  manure was used as medium, the TN removal for 30 days cultivation period was higher than that for 9 days cultivation period.The dynamics of TN and TP change in SMF and MFM with comparison to SCM is shown in Figure i = 53.6–156.4 mg/L under 9 days cultivation period. For the similar feedstock condition, on 30 days of culture, the removal rates were 92.9, 95.7, 93.1, 90.1, and 47.6%, respectively, for corresponding ratios. Similar to sterilized condition, the TN removal under untreated manure feedstock  condition was higher for 30 days culture period than that for 9 days culture period. However, the TP concentrations remain consistent, i.e., the concentration of TP on Days 9 and 30 was comparable under non-pretreatment condition  manure feedstock condition, the TN removals at ratio of 100% MFM, 70% MFM, 40% MFM, 20% MFM, and 100% SCM were 46.4, 63.0, 25.4, 17.0, and 20.4%, respectively, for TNDuring the first 9 days culture period under SMF condition, the removals of TP for 100% SMF, 70% SMF, 40% SMF, 20% SMF, and 100% SCM were 20.9, 20.0, 12.3, 7.4, and 20.8%, respectively. The removals of TP in 30 days culture for these ratios were 82.4, 47.8, 26.9, 9.6, and 24.7%, respectively. For non-sterilized manure under 9 days culture period, the TP was removed by 22.8, 36.3, 40.6, 16.6, and 20.8% for 100% MFM, 70% MFM, 40% MFM, 20% MFM, and 100% MFM, respectively. Compared to non-sterilized manure condition, the TP removal in sterilized manure condition was slightly more consistent . The reduced biomass on Day 30 for 100% MFM was attributed to the decay of biomass growth. The biomass production for 30 days (under untreated manure) at 70% MFM, 40% MFM, 20% MFM, and 100% SCM were 308.2, 580.2, 612.0, and 627.3 mg/L, respectively. In general, the final biomass content in SMF medium increased with the increase in TNR2 value varied from 0.89 to 0.99). However, the relationships between specific TP removal rate and initial TP concentrations were unpredictable and the linear fit resulted in relatively poor R2 values, which varied from 0.009 to 0.56.The specific TN and TP removal rate for varying TN and TP concentration are shown in Figures Rsr versus 1/TNi are shown in Figure k of 0.39 (mg TN/mg Biomass day) and mK of 60.7 mg/L (R2 = 0.93) were for 9 days cultivation period, k = 0.44 (mg TN/mg Biomass day) and mK = 69.5 mg/L (R2 = 0.85) were for 30 days for SMF experiment . Under untreated manure feedstock condition (for MFM media), kinetic coefficients of TN removal were determined as k = 6.29 (mg TN/mg Biomass day) and mK = 278.6 mg/L (R2 = 0.88) for 9 days cultivation and k = 44.6 (mg TN/mg Biomass day) and mK = 1,538.9 mg/L (R2 = 0.88) for 30 days culture period.The plots between 1/k = 2.0 (mg TP/mg Biomass day) and mK = 190.8 mg/L (R2 = 0.42) for 9 days; and k = 0.92 (mg TP/mg Biomass day) and mK = 0.25 mg/L (R2 = 0.16) for 30 days culture period. For untreated manure feedstock , kinetic coefficients of TP removal were determined as k = 102.0 (mg TP/mg Biomass day) and mK = 0.40 mg/L (R2 = 0.37) for 9 days; and k = 106.4 (mg TP/mg Biomass day) and mK = 0.09 mg/L (R2 = 0.54) for 30 days.Similarly, the coefficients for TP removal in SMF media were found as C. vulgaris use manure nitrogen efficiently. However, over the time (on Day 30), the nitrogen removal was higher for non-sterilized condition , which indicates that the consumption of untreated manure TN by algae was increased over the time.Table C. vulgaris, additional studies are needed to strengthen the results of this study and also to test the feasibility of using manure for producing C. vulgaris at large scale.The TN removal for Day 30 under SMF conditions varied from 69.4 to 90.4% and for MFM condition, it varied from 90.1 to 95.7% (Table NY in 9 days was determined as 25.5 (mg biomass/mg TN) at ratio of 40% SMF and 31.5 (mg biomass/mg TN) at ratio of 20% MFM media. For 30 days cultivation, the maximum yield coefficient for TN  reached 140.1 (mg biomass/mg TN) at 20% SMF and 64.7 (mg biomass/mg TN) at 20% MFM  at 20% SMF and 114.5 (mg biomass/mg TP) at ratio of 100% MFM. For 30 days cultivation, the maximum yield coefficient for TP reached 108.3 (mg biomass/mg TP) at 20% SMF and 78.7 (mg biomass/mg TP) at 20% MFM  for SMF media was 40% SMF > 20% SMF > 70% SCM > 100% SMF > 100% SCM. For MFM media, the TN removal ranking was 20% SMF > 40% SMF > 100% SCM > 70% MFM > 100% MFM. The ranking (higher to lower) for removal of TP in SMF media was found as 100% SMF > 40% SMF > 20% SMF > 70% SMF > 100% SCM. This TP removal ranking for MFM media was found to be 100% MFM > 20% MFM > 40% MFM > 70% MFM > 100% SCM.C. vulgaris is reported 100 and 78%, respectively  , treatment conditions, and algae species. As an example, nitrogen and phosphorus recovery in piggery effluent by C. vulgaris from flushed dairy manure, here we performed a series of preliminary batch experiments for algal biomass production. The biomass production of C. vulgaris in a series of media, which includes synthetic media and manure mix, was used to evaluate nutrient kinetics. Results showed that nitrogen recovery was more consistent compared to phosphorus. The recovery of nitrogen and phosphorus changed with feedstock characteristics. In general, the growth of C. vulgaris in manure-based media resulted in reductions of nitrogen and phosphorus. The removal of TN was more consistent than the removal of TP. For Day 9 cultivation period, the TN removal varied from 29.9 to 74.9% under sterilized (pretreated manure) feedstock conditions. Under untreated manure condition, TN removal varied from 17 to 63%. This indicates that for shorter growth period (≈10 days), sterilization of manure (pretreatment) could be a better approach for TN removal as well as biomass production. These preliminary outcomes indicate that dairy manure can be utilized with certain pretreatment such as sterilization as a potential media for growing algal biomass. Additional studies are needed further to improve the findings of this study and understand the applicability of manure-based feedstock for algal biomass production at large scale, and its effects on dairy manure nutrient removal. The proposed method can reduce the cost of algal biomass production and lower the demand of chemical-based growth media. As manure is abundantly available, the process can result in a higher amount algal biomass production at low cost. This method can further assist in reducing the nutrients of manure, which otherwise would cause environmental pollution such as excessive nutrient concentrations in ambient water.To improve the understanding of nutrient recovery using Experiment for use of animal waste for algal biomass production was conceptualize by PP and experiment setup was designed by PP with the help of JS. Manure feedstock from dairy farm was collected by PP and SCM media was prepared by JS. Experiment was conducted by JS and data were generated by JS. The observation and modeling results were analyzed by PP with the help JS. Manuscript was written by PP with the help from JS and manuscript revision was done by PP.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, YB, and handling editor declared their shared affiliation, and the handling editor states that the process nevertheless met the standards of a fair and objective review.


Staphylococcus epidermidis is the most common. However, S. epidermidis often displays multidrug resistance (MDR), demanding additional treatment options. The objective was to examine the effectiveness of tedizolid and linezolid against S. epidermidis isolated from PJIs. The standard antibiotic susceptibility pattern of S. epidermidis (n = 183) obtained from PJIs was determined by disc diffusion test, and MIC was determined by Etest for tedizolid, linezolid, and vancomycin. Tedizolid displayed MIC values ranging from 0.094 to 0.5 mg/L , linezolid MIC values ranging from 0.25 to 2 mg/L , and vancomycin MIC values ranging from 0.5 to 3 mg/L (MIC50 and MIC90 both 2 mg/L). According to the disc diffusion test, 153/183 (84%) isolates were resistant to ≥3 antibiotic groups, indicating MDR. In conclusion, S. epidermidis isolates from PJIs were fully susceptible, and the MIC50 and MIC90 values for tedizolid were two- to four-fold dilution steps lower compared with linezolid. Tedizolid is not approved, and there are no reports of long-term treatment, but it may display better tolerability and fewer adverse effects than linezolid; it thus could be a possible treatment option for PJIs, alone or in combination with rifampicin.Prosthetic joint infections (PJIs) are rare but long-lasting and are serious complications without any spontaneous resolution, requiring additional surgery and long-term treatment with antibiotics. Staphylococci are the most important aetiological agents of PJIs, and among the coagulase-negative staphylococci  Joint replacements have significantly improved the quality of life for many patients. Prosthetic joint infections (PJIs) are rare complications, affecting less than 1% of primary joint replacement surgeries –4.S. epidermidis is the most common of the CoNS. This species regularly forms a biofilm, and so the proposed treatment of choice for PJIs is rifampicin, which is efficient against staphylococci in biofilm [S. epidermidis often displays a multidrug resistance (MDR) phenotype [Staphylococci are the most important aetiological agents of PJIs –4, and S biofilm , 4. Rifa biofilm –4. Howevhenotype . Linezolhenotype , and frehenotype . Neverthhenotype , 8.S. epidermidis isolated from PJIs.The aim of this study was to investigate the antibiotic activity of a new oxazolidinone, tedizolid, and to compare its MIC values with those of linezolid, among S. epidermidis isolates (n = 183) were obtained during various surgical procedures due to suspected or verified PJI at the University Hospitals of Örebro and Linköping from 1999 to 2015 and from 1993 to 2015, respectively. The finding of S. epidermidis in multiple tissue samples (≥2) was interpreted as a PJI in accordance with the proposed definition of PJI [n = 126), knee (n = 41), shoulder (n = 12), or elbow (n = 4) joint prostheses, then identified to the species level according to routine laboratory procedures and confirmed by MALDI-TOF MS .es n = 18 were obtn of PJI . IsolateStandard antibiotic susceptibility testing by disc diffusion test (DDT) and MIC determination was performed according to EUCAST guidelines. MIC was determined by Etest for tedizolid, linezolid , and vancomycin . DDT was performed for cefoxitin (30 μg), fusidic acid (10 μg), erythromycin (15 μg), clindamycin (2 μg), trimethoprim/sulfamethoxazole (25 μg), gentamicin (10 μg), norfloxacin (10 μg), and rifampin (5 μg) .50: 0.19 mg/L, MIC90: 0.38 mg/L) and linezolid MIC values ranging from 0.25 to 2 mg/L . All isolates were susceptible according to EUCAST breakpoints . The MIC values of vancomycin ranged from 0.5 to 3 mg/L (MIC50 and MIC90 both 2 mg/L). The breakpoint for susceptibility according to EUCAST is ≤4 mg/L.Results of the antimicrobial susceptibility testing of tedizolid and linezolid are shown in Fig. S. epidermidis isolates were susceptible to cefoxitin, 56% to fusidic acid, 42% to clindamycin, 37% to erythromycin, 72% to rifampicin, 28% to gentamicin, 19% to norfloxacin, and 27% to sulfamethoxazole-trimethoprim. In total, 153/183 (84%) isolates were resistant to ≥3 antibiotic groups, indicating MDR. There were no differences between isolates displaying MDR and non-MDR in MIC50 and MIC90, respectively, neither for tedizolid nor for linezolid.According to the DDT, 22% of the S. epidermidis isolated from PJIs. All isolates were fully susceptible to both antibiotics according to EUCAST breakpoints. The MIC50 and MIC90 values for tedizolid were two- to four-fold dilution steps lower than the corresponding values for linezolid. Our results are in concordance with previous studies [This study investigated the antibiotic activity of a new oxazolidinone, tedizolid, compared to linezolid against  studies .S. epidermidis isolates were also susceptible to vancomycin according to the EUCAST breakpoint. However, 111/183 (61%) showed MIC values ≥2 mg/L, and thus an increased risk of nephrotoxicity may be present if the trough value of the plasma concentration is intended to be approximately 20 mg/L. Heterogeneous glycopeptide intermediate S. epidermidis (hGISE) may also be present among S. epidermidis isolated from PJIs [All rom PJIs , which wrom PJIs . Subsequrom PJIs , 8. Tedirom PJIs , 30 healrom PJIs . The myerom PJIs . Althoug50 and MIC90 for tedizolid, the use of the lower dose of tedizolid, a sixth of the daily dose of linezolid, needs to be evaluated for severe infections and especially during long-term treatment. A study evaluating the pharmacokinetics and response to exposure to tedizolid [A lower dose of tedizolid, 200 mg once daily versus 600 mg twice daily of linezolid, may also contribute to a lower frequency or severity of reported side effects, especially if they are serum concentration dependent , 20. Desedizolid  showed tS. epidermidis in both prosthesis retention following debridement and two-stage exchange surgery.The safety, tolerability, and adverse effects of long-term tedizolid treatment remain to be evaluated. However, tedizolid may represent an oxazolidinone that is better accepted by patients for long-term treatment, due to its favourable adverse effect profile and its once-a-day administration regime. Alone or in combination with rifampicin, tedizolid could be a possible treatment option in the management of PJIs caused by MDR


Lasiodiplodia theobromae (L. theobromae), to alter its behavior. Nonetheless, there is only limited information regarding the effect of temperature on L. theobromae production of enzymes. The need for new, thermostable enzymes, that are biotechnologically relevant, led us to investigate the effect of temperature on the production of several extracellular enzymatic activities by different L. theobromae strains. Fungi were grown at 25 °C, 30 °C and 37 °C and the enzymatic activities were detected by plate assays, quantified by spectrophotometric methods and characterized by zymography. The thermostability (25–80 °C) of the enzymes produced was also tested. Strains CAA019, CBS339.90, LA-SOL3, LA-SV1 and LA-MA-1 produced amylases, gelatinases, caseinases, cellulases, lipases, laccases, xylanases, pectinases and pectin liases. Temperature modulated the expression of the enzymes, and this effect was more visible when fungi were grown at 37 °C than at lower temperatures. Contrary to proteolytic and endoglucanolytic activities, whose highest activities were detected when fungi were grown at 30 °C, lipolytic activity was not detected at this growth temperature. Profiles of proteases and endoglucanases of fungi grown at different temperatures were characterized by zymography. Enzymes were shown to be more thermostable when fungi were grown at 30 °C. Proteases were active up to 50 °C and endoglucanases up to 70 °C. Lipases were the least stable, with activities detected up to 45 °C. The enzymatic profiles detected for L. theobromae strains tested showed to be temperature and strain-dependent, making this species a good target for biotechnological applications.Phytopathogenic fungi are known to produce several types of enzymes usually involved in plant cell wall degradation and pathogenesis. The increasing of global temperature may induce fungi, such as  Fungal plant pathogens are known to express high amounts of hydrolytic and oxidative enzymes ,2,3,4. TLasiodiplodia theobromae (Pat.) Griff. and Maubl. is a phytopathogenic fungus from the family Botryosphaeriaceae, typically found in tropical and subtropical regions . After 48 h, plates were covered with Congo Red  (1 mg mL−1) during 15 min and destained with 1 M NaCl. Activity was detected as a yellow halo around the mycelium in a red background.Cellulolytic and xylanolytic activity were detected by adding 1.5% agar (etection  to a minw/v), 1% peptone (w/v), 0.5% NaCl (w/v), 0.01% CaCl2 (w/v) and 1% Tween 20 (v/v) . After 48 h, the plates were covered with lugol solution during 10 min and the activity was detected as a clear halo in a dark background  at the desired temperature and 400 μL of this mixture was added to 900 μL of 6% tricholoroacetic acid TCA (w/v) after 0 min, 5 min, 10 min and 20 min of incubation. All the samples were maintained in ice and centrifuged at 13,000 rpm for 10 min. The blank sample was prepared by replacing the enzymatic crude extract by ultrapure water and treated as all the samples, replacing the enzymatic crude extract by ultrapure water. The supernatant was used to measure the absorbance at 280 nm.Proteolytic activity was determined as described by Macchione et al. , with slThe quantification of the proteolytic activity was made at: 25 °C, 30 °C, 37 °C, 45 °C, 50 °C, 70 °C and 80 °C. Data is presented as mean of three replicates, normalized for mycelium dry weight. p-nitrophenyl palmitate  was used as substrate. A stock solution (3 mM) was prepared in 25% dimethyl sulfoxide and 0.5% of Triton X-100 and completed with potassium phosphate buffer (pH 6.8). A volume of 50 µL of buffered pNPP solution was added to 150 µL of crude extract, and incubated at the test temperature for 0 min, 45 min and 90 min. The reaction was stopped by adding 50 µL of 0.1 M sodium carbonate. The released p-nitrophenol (pNP) was measured at 405 nm in 96-well plates using a microplate reader . The blank sample was treated as all the samples, replacing the enzymatic crude extract by ultrapure water. The molar absorption coefficient of pNP (ε = 1.2475 × 104 M−1·cm−1) was estimated from the absorbance of pNP standard solutions measured at 405 nm. One enzymatic unit was defined as the amount of the enzyme that releases 1 mmol of pNP per minute.Lipolytic activity was determined according to Kotogán et al. , with slThe quantification of the lipolytic activity was made at 25 °C, 30 °C, 37 °C, 45 °C since higher temperatures lead to spontaneous substrate degradation. Data is presented as mean of three replicates, normalized for mycelium dry weight. For each strain, two 5 mm-diameter plugs from an actively growing culture were inoculated on 50 mL PDB for 4 days. The enzymatic crude extract was obtained by filtration as described earlier .v/v); 62.5 mM Tris (w/v), pH 6.8, 10% SDS (w/v) and 20% glycerol (v/v)] and incubated at room temperature for 10 min. The separation of the proteins was carried out in lab casted gels (10% polyacrylamide with the correspondent substrate) in a Mini-PROTEAN 3   was used. After electrophoresis, the gel was incubated for 1 h at 37 °C, in 1.5 mM Tris, pH 8.8, 1 M NaCl, 1 M CaCl2, 2 mM ZnCl2, pH 7.4. Gels were stained with Coomassie Brilliant Blue R-250  and destained with 25% ethanol (v/v) and 5% acetic acid (v/v). Enzymes with gelatinolytic activity were detected as clear bands against a blue background of non-degraded substrate.Gelatinolytic activity was assessed by zymography, as described previously , with slw/v), 3% of olive oil (v/v) and 2.5% agar (w/v) was prepared in Tris-HCl buffer, pH 8.0 and heated with agitation until dissolution of the agar. After electrophoresis, the gel was overlaid onto the solid medium of rhodamine B/olive oil for 3 h at 37 °C. Lipolytic activity was detected exposing the plate with the gel to 365 nm, where the enzymes with activity appeared as fluorescent bands.Lipolytic activity was accessed by zymography as described before, ,49 with p < 0.05). A Tukey’s multiple comparison test was employed to identify significant differences between the enzymatic profile of a strain grown at different temperatures. All the analyses were performed with GraphPad Prism v.7 .Two-way analysis of variance (ANOVA) was used to examine the extracellular enzymatic activity profiles at different temperatures for each strain  was carried out to infer which factors, temperature or strain, is the most relevant on the modulation of the extracellular enzymatic profiles: detection and quantification of enzymatic activity data and zymography data (molecular weight of the bands detected). Due to the different type of data , MFA was considered the most suitable dimension-reducing method. The MFA was conducted with R Statistical Software , using tL. theobromae. All strains produce an arsenal of enzymes typically involved in the degradation of plant cell walls , whose expression was influenced by the temperature of growth. These alterations show the capacity of adaption of these strains to different environments, which could also result in changes in pathogenicity. The analysis of the enzymatic profile of L. theobromae showed that different strains produce significantly different enzymatic profiles, indicating a strain-dependency.We showed that temperature modulates the extracellular enzymatic profile of the strains of The wide range of temperatures tested (from 25 °C to 80 °C) showed that these strains express thermostable enzymes , which could be very interesting to several types of industries using these kinds of enzymes, such as detergents, starch, food or biomedical industries.


Ganoderma lucidum is a well-known medicinal mushroom. At present, numerous G. lucidum products have emerged in the form of dietary supplements in the United States due to its various benefits. However, the quality consistency of these products based on their label ingredients has seldom been evaluated due to the lack of a suitable toolkit. In this study, 19 batches of products of G. lucidum , herbal/mushroom supplements purchased in the United States, were evaluated based on their bioactive components including triterpenes and polysaccharides by using chromatographic methods and saccharide mapping. The results showed that the measured ingredients of only 5 tested samples (26.3%) were in accordance with their labels, which suggested the quality consistency of G. lucidum dietary supplements in the U.S. market was poor, which should be carefully investigated. However, under Dietary Supplements Health and Education Act of 1994 (DSHEA), quality of dietary supplements is not evaluated by the U.S. Food and Drug Administration (FDA)2. Both industry and FDA acknowledge that many products have been introduced into the market without any safety assessment1. Alarmingly, hundreds of products marketed as supplements have been found spiked with illicit substances not listed on the products’ labels, risking serious injury or even death3. Indeed, within the first three months of 2015, FDA warned about or recalled over 30 tainted sexual enhancement products containing prescription erectile-dysfunction drugs or related synthetic analogues4. In February 2015, the New York State Attorney General compelled major retailers – Walgreens, Walmart, Target, and GNC – to halt sales of certain herbal supplements, after deoxyribonucleic acid (DNA) barcoding results failed to detect DNA from the botanical materials listed on the label of ~80% tested products5. Though the absence of DNA might be an artifact caused by its destruction during the manufacturing process, it still triggered a great concern on the quality of dietary supplements in the United States. Usually, chemical characters of herbal supplements are more stable than their DNA sequences during different manufacturing processes. Therefore, taking chemical insights into the quality of herbal/mushroom supplements should be more reasonable.Dietary supplements are consumed with intent to derive important health benefits. An estimated one hundred millions of Americans spend more than $28 billion on dietary supplements every yearGanoderma lucidum, known as “Lingzhi” in China or “Reishi” in Japan, is a well-known medicinal mushroom and traditional Chinese medicine, which has been used for the prevention and treatment of a variety of diseases such as bronchitis, allergies, hepatitis, immunological disorders, and cancer7. Due to its various benefits in protecting human health, numerous G. lucidum products have emerged in the form of dietary supplements in the United States, and it is also listed in the Dietary Supplements and Herbal Medicines of USP. However, their quality consistency to label ingredients has seldom been evaluated due to the lack of a suitable toolkit to audit ingredient and adulterants. Usually, triterpenes and polysaccharides are considered as the main bioactive components in G. lucidum fruiting body8, which have been used as markers for G. lucidum officially recorded in Chinese Pharmacopoeia (2015) due to their anti-cancer and immunomodulatory activities.Ganoderma lucidum  dietary supplements purchased in the United States was evaluated using a reliable and scientific toolkit including colorimetric assay, high performance thin layer chromatography (HPTLC), gas chromatography coupled with mass spectrometry (GC-MS), saccharide mapping based on polysaccharide analysis using carbohydrate gel electrophoresis (PACE), and high performance size exclusion chromatography coupled with multi angle laser light scattering and refractive index detector (HPSEC-MALLS-RID) method.In this study, the quality consistency to their label of 19 batches of 9. Indeed, HPTLC fingerprints of triterpenes in fruiting body of G. lucidum collected from different regions of China were roughly consistent, which exhibited with pink or red bands under white light after being colorized10. Therefore, an authenticated fruiting body of G. lucidum (GL20) was used as the reference material. Figure G. lucidum dietary supplements under white light and ultraviolet (UV) 365 nm, respectively. The results showed that HPTLC fingerprints of triterpenes  produced by different manufacturers were remarkably different, and triterpenes closely related to G. lucidum (GL20) were only detected in 8 out of 19 (42.1%) tested products based on HPTLC analysis.HPTLC is a simple and rapid technique for the routine quality control of herbal medicines, which provides picture-like chromatograms with special colorsG. lucidum, are frequently employed as excipients, or neutral fillers in dietary supplements. Therefore, in order to identify the presence of starch or malto-dextrin in the dietary supplements, the colorimetric assay with iodine-potassium iodide (I2-KI) reagent was performed. Starch or malto-dextrin should be excluded for the determination of polysaccharides from G. lucidum dietary supplements. As shown in Fig. G. lucidum (GL20). Indeed, after the treatment with α-amylase, the positive response of starch-like polysaccharides in these products to I2-KI reagent was disappeared . However, other tested samples were obviously different from that of GL20. Hierarchical cluster analysis was further performed based on the digital scanning chromatograms of PACE fingerprints of 1,3-β-D-glucanase digested polysaccharides  tested products.Generally, the major bioactive polysaccharide in Ganoderma15, which demonstrated that compositional monosaccharides in fruiting body of G. lucidum collected from different regions of China were similar. Indeed, GC-MS is an excellent technique for the analysis of monosaccharides, which has several advantages such as high resolution and high sensitivity16. Therefore, GC-MS fingerprints based on compositional monosaccharides were also used for the authentication of G. lucidum dietary supplements. Figure G. lucidum dietary supplements. The results showed the compositional monosaccharides of polysaccharides in G. lucidum (GL20) were fucose (Fuc), mannose (Man), glucose (Glc), and galactose  with the molar ratios of 2.3: 4.6: 100.0: 12.2, which were in accordance with the previous study17. Indeed, GC-MS analysis showed the types of compositional monosaccharides of polysaccharides in G. lucidum dietary supplements including GL01, GL09, GL13, GL14, and GL15 were similar to those of GL20  were obviously different from those of G. lucidum  tested products, which were in accordance with the results from saccharide mapping based on PACE analysis.Fingerprints based on compositional monosaccharides have been successfully performed for the quality control of polysaccharides from dn/dc method has been proven as a powerful and efficient technique for the determination of contents and molecular weights of polysaccharides and their fractions from herbal medicines18. Indeed, HPSEC chromatograms and molecular weights of polysaccharides in G. lucidum collected from different regions of China were similar15. Therefore, polysaccharides from one batch of G. lucidum could be used as reference standards. In order to exclude the interference from the presence of additives such as starch or malto-dextrin, polysaccharides were treated with α-amylase before HPSEC-MALLS-RID analysis based on dn/dc. Figure 2-KI reagent. Indeed, HPSEC chromatograms of polysaccharides in GL01, GL09, GL13, GL14, and GL15 were similar to that of G. lucidum (GL20)  were determined and calculated. The data showed that the molecular weights of polysaccharide in G. lucidum (GL20) were 1.052 × 106 Da (peak 1) and 5.04 × 104 Da (peak 2), respectively. Indeed, most anti-tumor β-glucans reported in Ganoderma contained the fraction with an average molecular weight of about 1.0 × 106 Da7. Therefore, peak 1 could be considered as a quality marker in G. lucidum, which could be found in tested samples of GL01, GL09, GL13, GL14, and GL15. Their varied molecular weights might be attributed to different preparation processes of manufacturers. However, although peak 1 was also found in tested samples of GL02, GL03, GL12, and GL19, their compositional monosaccharides and PACE fingerprints of 1,3-β-D-glucanase hydrolysates were significantly different from those of G. lucidum (Table HPSEC-MALLS-RID based on 20) Fig. . Due to G. lucidum. Therefore, it is very important to determine whether the products contain triterpenes and polysaccharides derived from G. lucidum. Due to both triterpenes and polysaccharides can be found in G. lucidum, its products should, at least, have chemical characters of either triterpenes or polysaccharides based on their ethanol or water extract and their obvious polarity variations. Quality consistency of the dietary supplements could be considered as desirable if their detected ingredients were in accordance with their labels. Unfortunately, the measured ingredients, only in 5 out of 19 (26.3%) tested samples, were in accordance with their labels according to the results mentioned above . Therefore, this study suggested the quality consistency of G. lucidum dietary supplements collected in USA was extremely poor, which should be carefully investigated. Furthermore, the results also suggested that saccharide mapping based on PACE analysis, and HPSEC-MALLS-RID based on dn/dc had great potential for routine quality evaluation of polysaccharides from dietary supplements.In summary, triterpenes and polysaccharides are officially considered as major active components in G. lucidum dietary supplements were purchased, in February or March 2015, directly from general e-commerce sites such as Amazon.com and eBay Inc. in the United States, and one batch of authenticated fruiting body of G. lucidum (GL20) was collected from Shandong Province of China  of  to GL19 D-glucose, α-amylase, starch (ST), and acetic anhydride were purchased from Sigma . Laminaribiose (DP2), laminaritriose (DP3), and laminaritetraose (DP4), β-1,3-D-glucan (GN), and β-1,3-D-glucanase were purchased from Megazyme , and 8-aminonaphthalene-1,3,6-trisulphonic acid (ANTS) was purchased from Tokyo Chemical Industry . Silica gel 60 F254 TLC plates were obtained from Merck . Polyacrylamide containing acrylamide/N, N-methylenebisacrylamide  was obtained from Bio-Rad . Deionized water was prepared by a Millipore Milli-Q Plus system . All the other reagents were of analytical grade.10. Then the extract was centrifuged at 4,000 × g for 10 min . The supernatant was evaporated to dryness under vacuum using rotary evaporator , and the residue was dissolved in 2.0 mL of methanol. After filtration through a 0.22 μm membrane filter, the extract was used for HPTLC analysis. Finally, ethanol extracted residues were dried under vacuum at 45 °C and further used for polysaccharides preparation.Powder of each sample (1.0 g) was immersed in 20.0 mL of ethanol and refluxed in a Syncore parallel reactor  for 30 min at 78 °C according to previous report12. Briefly, dried ethanol extracted residues (~1.0 g) were suspended in 25.0 mL of deionized water and extracted with microwave assisted extraction . The microwave irradiation program was performed at 900 W and 90 °C for 7 min. Then the extract was centrifuged, and the supernatant (~25.0 mL) was evaporated to about 10.0 mL under vacuum using rotary evaporator. Then three times of solution volume of ethanol  were added for the precipitation of crude polysaccharides. After the solution was kept at 4 °C for 12 h, centrifugation  was performed. The supernatant was collected and the powder of the supernatant was obtained by freeze-drying.Microwave assisted extraction was used for polysaccharides extraction according to a previously reported method with modificationG. lucidum and its dietary supplements was performed according to a previously reported method with minor modification10. Briefly, all samples (5 μL) were applied on a 20 × 10 cm silica TLC plate with an AS30 HPTLC Applicator . The bands, at 10 mm from the bottom edge, were 8 mm wide and 13 mm between two bands. The plate was developed to a distance of 90 mm with dichloromethane/ethyl acetate/petroleum ether/formic acid/ethanol, 8:3:9:0.8:0.5 (v/v/v/v/v) as mobile phase at room temperature. Finally, the developed plates were colorized with 10% (v/v) H2SO4 in ethanol, and heated at 110 °C for 10 min on a YOKO-XR plate heater . Then the plate was covered with transparent glass and photographed under white light and UV 365 nm, respectively.Determination of triterpenes in 2-KI reagent, and then photographed under white light. Water and soluble starch were used as negative and positive control, respectively. Furthermore, all samples  were treated with α-amylase at a final concentration of 20 U/mL, and digested overnight (12 h) at 40 °C. Then, colorimetric assay was also performed.All samples  were mixed with 20 μL of I19. Briefly, polysaccharides of each sample (30.0 mg) were dissolved in 5.0 mL of hot water (60 °C). Then the compounds with molecular weights less than 3 kDa were removed by centrifugation  with an ultra centrifugal filter  for seven times.Saccharide mapping based on PACE analysis was performed according to the previous method with minor modificationD-glucanase , and digested overnight (12 h) at 40 °C. The hydrolysates were dried, and then used for the derivatization with ANTS for PACE analysis. Polysaccharide solutions without enzymatic digestion were used as blank control after the treatment as described above. Reference polysaccharides including 1,3-β-D-glucan  and starch  treated with the corresponding enzymes, respectively, were used as positive control.The polysaccharide solution (1.0 mL) of each sample was mixed with α-amylase and 1,3-β-All samples (1–3 μL) were separated using a vertical slab gel electrophoresis apparatus, Mini-Protean Tetra System . Electrophoresis on 30% (w/v) polyacrylamide as the resolving gel with a stacking gel of 8% (w/v) polyacrylamide was used for the separation of enzymatic hydrolytes. Samples were electrophoresed firstly at 200 V for 10 min and then at 700 V for 45 min. All runs were performed at least two times. Gels were imaged using an InGenius LHR CCD camera system  under UV 365 nm.G. lucidum fruiting dietary supplements were investigated by using GC-MS analysis according to previous report with minor modification21. Briefly, the sample (3.0 mg) was hydrolyzed with 2.0 M TFA (1.0 mL) at 95 °C in a sealed tube for 10 h. Then the hydrolysates were washed with methanol and evaporated to dryness before derivation with hydroxylamine hydrochloride and acetic anhydride at 90 °C for 30 min. Furthermore, the derivatives of mixed monosaccharide standards  were prepared as described above. The derivatives were analyzed by using an Agilent 6890 gas chromatography instrument coupled to an Agilent 5973 mass spectrometer . A capillary column  coated with 0.25 μm film 5% phenyl methyl siloxane was used for separation. High purity helium was used as carried gas with a flow rate of 1.0 mL/min. The column temperature was set at 165 °C and held for 7 min for injection, then programmed at 5 °C/min to 185 °C and held for 5 min, then at 4 °C/min to 200 °C, and finally at 20 °C/min to 280 °C, and held for 2 min.Compositional monosaccharides of polysaccharides in G. lucidum dietary supplements were simultaneously determined using HPSEC-MALLS-RID based on dn/dc method according to our previous report with minor modification18. Firstly, polysaccharide solutions (~5.0 mg/mL) of G. lucidum dietary supplement were treated with α-amylase at a final concentration of 20.0 U/mL in a total volume of 1.0 mL for 24 h at 40 °C to completely hydrolyzed additives  into small sugars. Polysaccharide solutions without α-amylase, and α-amylase without polysaccharide solutions treated as described above, were used as blank control. Secondly, HPSEC-MALLS-RID measurements were carried out on a MALLS  with an Agilent 1100 series LC/DAD system  equipped with columns of TSK-Gel G5000PWXL  and TSK-Gel G3000PWXL  in series at 35 °C. The MALLS instrument was equipped with a He-Ne laser (λ = 658 nm). An Optilab rEX refractometer  was simultaneously connected. The Mw was calculated by the Zimm method of static light scattering based on the basic light scattering equation is as follows23,K was an optical constant equal to [4π2n2(dn/dc)2]/(NAλ4); C, polysaccharide concentration; Rθ, Rayleigh ratio; λ, wavelength; n, refractive index of the solvent (0.9% NaCl aqueous solution); dn/dc, refractive index increment of polysaccharides in 0.9% NaCl aqueous solution, which is recommended as 0.15 mL/g according to our previous study24; NA, Avogadro’s number; A2, second virial coefficient.Contents and molecular weights of polysaccharides and their fractions in dn/dc value according to the following equation25,Ci is the concentration of polymers; α is the RID calibration constant (in RI units per volt); Vi and Vi, baseline are the RID voltages of sample and baseline, respectively; dn/dc is the specific refractive index increment.Contents of polysaccharides were calculated based on the refractive index difference with The digital scanning chromatograms of PACE fingerprints were generated using Quantity-One software , and hierarchical cluster analysis of PACE fingerprints was also analyzed using Quantity-One with average linkage method.


W´) across repeated tests using ecologically valid maximal effort time-trials (TT) under laboratory conditions. After an initial incremental exercise test, ten well-trained male triathletes  performed three testing sessions  each comprising three TT . CP and W´ were determined using a linear regression of power vs. the inverse of time (1/t) (P = W´ ∙ 1/t + CP). A repeated-measures ANOVA was used to detect differences in CP and W´ and reliability was assessed using the intra-class correlation coefficient (ICC) and the coefficient of variation (CoV). CP and W´ values were not significantly different between repeated tests . The ICC between Familiarization and Test I was r = 0.86 (CP) and r = 0.58 (W´) and between Tests I and II it was r = 0.94 (CP) and r = 0.95 (W´). The CoV notably decreased from 4.1% to 2.6% and from 25.3% to 8.2% for CP and W´, respectively. Despite the non-significant differences for both parameter estimates between Familiarization, Test I, and Test II, ICC and CoV values improved notably after the familiarization trial. Our novel findings indicate that for both, CP and W´ a familiarization trial increased reliability. It is therefore advisable to familiarize well-trained athletes when determining the power-duration relationship using TT under laboratory conditions.The purpose of this study was to assess the reliability of critical power (CP) and the total amount of work accomplished above CP ( W´) has long been a question of interest. Whilst CP represents a work rate that can be sustained for a long time without a continuous loss of metabolic  and systemic  on a cycle ergometer, leading to exhaustion within 2–15 min . However, TTE have no predefined endpoints and therefore are not comparable to the tasks athletes are confronted with during competition.A reliable determination of critical power (CP) and the total amount of work accomplished above CP until task failure   to return to baseline values in order to minimize any effect of prior exercise on nt trial , 27. Teslast is the last fully completed work stage (W) and t is the duration of the incomplete work stage (s).A GXT was performed to determine MAP. After an unloaded cycling phase for 3 min, resistance was set to 100 W and was increased by 20 W every 3 min until volitional exhaustion. If the last work stage could not be fully completed, MAP was calculated using the following equation of Kuipers et al. :MAP=PlaFamiliarization, and the second and third test Test I and Test II, respectively. During the TT participants were advised to produce the highest mean power output for 12, 7 and 3 min in that order [Participants performed three identical tests to determine the power-duration relationship. The first test was used as a familiarization session and it was included in the analysis. The first test is consequently termed at order  and wereat order . ParticiW´ is the total amount of work accomplished above CP until task failure (J) and CP is the critical power (W):Mean PO for each TT was plotted against the inverse-of-time using a linear regression where PO is the mean power output (W), W´. The individual SEE was calculated for each participant and each parameter estimate in absolute and relative values. Nimmerichter et al. [W´  revealed non-significant differences between the models, neither for CP  nor for W´ . Consequently, we decided to use the model that provides the smallest error of the estimates (SEE) and thus results in most accurate estimates of CP and W´ [Least square modelling procedures were used to fit the parameter estimates. The y-intercept represents CP and the slope represents r et al.  demonstrr et al. . AnalysiP and W´ .P < 0.001) the Greenhouse-Geisser correction has been used [d  [high reliability, values >0.8 indicate moderate reliability, values >0.6 indicate questionable reliability, and values <0.6 indicate poor reliability of repeated tests. The coefficient of variation (CoV) was used to rate intra-individual variation. An upper limit of 5% [W´ [P < 0.05. Before the beginning of the study an a priori power-analysis was conducted and revealed that 10 participants were required to detect a significant difference of 15 W and 3 kJ for CP and W´, respectively with a statistical power of >80% [W´ would result in a calculated TT20min time difference of <5%. That is within the typical day-to-day variation of TT performance [After testing for normality using Shapiro-Wilk procedures, a repeated-measures analysis of variance (ANOVA) was conducted to assess differences between the tests. If the assumption of sphericity had been violated (een used . Signifid = 0.8) . The intd = 0.8) . An ICC it of 5%  or 10% [it of 5%  is propof 5% [W´ . Pearson of >80% . A diffeformance .Familiarization, Tests I and II  and between Familiarization and Test II for CP and W´ (P = 0.008 and d = 1.74 for both parameters). No significant differences were found for the absolute SEE for CP (P = 0.989 and d < 0.01) and the absolute SEE for W´ (P = 0.945 and d < 0.01) between Test I and Test II. Bonferroni post-hoc procedures for the relative SEE revealed significant differences between Familiarization and Test I and between Familiarization and Test II for CP only , but not for W´ . No significant differences were found for the relative SEE for CP (P = 0.850 and d = 0.12) and the relative SEE for W´ (P = 0.841 and d = 0.12) between Test I and Test II.I and II , Table 2I and II  and 2, aW´ values provide reliable results in a cohort of well-trained athletes after a familiarization trial. Importantly, this is the first study, which demonstrates such a high reliability for the estimates of W´ (ICC r = 0.94). Even though participants were familiar with TT efforts in the field, they produced slightly higher CP estimates (~3.5%) and notably lower W´ estimates (~13%) after the familiarization trial. Although non-significant differences in the parameter estimates were revealed, the effect size is of a moderate order for both parameter estimates, small effects were observed between Familiarization and Test I for CP (d = 0.28) and W´ (d = 0.47). The effect sizes for CP and W´ between Tests I and II were trivial . Considering effect sizes seems to be more appropriate when assessing smaller sample sizes and small mean differences [The main novel findings of the present study were that both, CP and ferences .W´ however, remained too high to be used for detecting small training induced changes . Recently, it was demonstrated that the high intra-individual variation in parameter estimates can be reduced when using iso-duration TT compared with TTE efforts [8.7% [W´ , 12 and Familiarization and Test I and between Tests I and II can be interpreted as moderate and highly reliable, respectively. The CoV for CP notably decreased following the familiarization trial (4.1% vs. 2.6%). But both testing trials were within what is currently acknowledged as an accepted range (i.e. <10% for W´ [r = 0.99 and CoV = 2.2%). A recent study by Wright et al. [r = 0.97–0.99 [W´, respectively), when using the three minute all-out test (3MT). However, whilst employing TT for the determination of the parameter estimates is a valid method [W´, respectively) [ICC values for CP between % for W´  and <5% % for W´ ). Our CP% for W´  and unde% for W´ . Karsten% for W´  found sit et al.  found co.97–0.99 ) and com.97–0.99  for CP actively) . This suW´ is interpreted as poor between Familiarization and Test I, it changes to be highly reliable between Tests I and II. Furthermore, the CoV was >10% for W´ between Familiarization and Test I, whilst it improved to values that according to Atkinson and Nevill [Tests I and II, confirming W´ to be reliable post familiarization. However, such a high reliability was not present in a field-based study using a similar methodology . It is therefore suggested that standardized and controlled laboratory conditions alleviate influencing effects on W´ and consequently result in a higher reliability of the parameter estimate. When using the 3MT, Wright et al. [W´ . KarsteoV = 46%  speculatt et al.  found co5.4–8.4% ).W´ between Tests I and II was close to zero after a familiarization session  after familiarization, and in the present study a familiarization session resulted in an even greater improvement of the 95% LoA from ± 10,000 J to ± 2,500 J (reduction of ~75%). These results provide evidence of a learning effect even in well-trained cyclists. Similar to the LoA, the SEE became notably smaller for both parameter estimates after a familiarization session  after familiarization, also showing the presence of a learning effect. After a familiarization trial, a high agreement of the regression line and the line of identity for both parameter estimates was evident  are also within day-to-day variations and are lower compared to the recent field-based study by Karsten et al. [W´ is similar [The mean bias of CP and ion Figs  and 2. Fion Figs  and 2 whent Figs  and 2b.  similar . It is i similar  used a dW´. Galbraith et al. [Familiarization and Test I in the present study. Contrary to this, Parker-Simpson and Kordi [P = 0.561–0.836).Moreover, Black et al.  and Karsh et al.  reportednd Kordi  stated tCoakley and Passfield  argued tr = 0.94–0.97 and CoV = 2.0–3.0%)  , lower S.0–3.0%) . Thereaf% for W´ , 39) may% for W´ , 12.Generally, the reasons for the higher reliability in the current study compared to earlier work could have been threefold: (i) controlled laboratory conditions; (ii) same TT durations across visits; (iii) no differences in pacing strategy after a familiarization session.A potential limitation of the study was the use of fixed-duration TT. These, whilst arguably carrying a higher ecological validity compared to constant-power TTE, are limited by competitive races commonly using fixed-distances rather than fixed-times. Yet, fixed-duration TT should be preferred to reduce the level of random error and construct the power-duration relationship reproducibly . More reW´ using TT in the laboratory. Even though highly reliable results for individual mean TT PO across multiple tests were evident, the quality of the model increased in subsequent testing sessions. Therefore, using TT is valid, reliable, and ecologically valid . It is consequently suggested that laboratory TT are preferable over TTE efforts and should be considered as a recommended method of best practice when determining CP and W´.To reduce the error inherent in testing, present results demonstrate that trained athletes experienced in TT and competition require to be familiarized when determining CP and S1 FileW´, and relative and absolute SE for CP and W´, respectively.File contains data for CP, (XLSX)Click here for additional data file.